Mast Cell Lineage Research Articles

Midostaurin (PKC412) Demonstrates a High Rate of Durable Responses in Patients with Advanced Systemic Mastocytosis: Results from the Fully Accrued Global Phase 2 CPKC412D2201 Trial

Midostaurin (PKC412) Demonstrates a High Rate of Durable Responses in Patients with Advanced Systemic Mastocytosis: Results from the Fully Accrued Global Phase 2 CPKC412D2201 Trial

Mast Cell Disease and Blood Coagulation Abnormalities: Discussion on 14 Cases and Review of the Literature

▪INTRODUCTIONMast cell disease is a clonal haematological disorder resulting in organ infiltration by mast cells and uncontrolled degranulation (mast cell activation syndrome, MCAS). Although not characteristic and poorly explained, both primary (platelet’s adhesion and aggregation) and secondary (coagulation cascade leading to thrombin formation) clotting abnormalities are observed among some patients.METHODSWe retrospectively identified patients with established diagnosis of mastocytosis and related clotting abnormalities (clinical and/or biological) using the French National Reference Centre for Mastocytosis database.RESULTS AND DISCUSSIONOur cohort included 14 adult patients (median age of 42 [range 26-78] years, sex ratio 3.7, CKIT D816V in 13/14 cases, median tryptase of 200 [4-1240] ng/mL).Four patients had typical symptoms and/or clotting tests (PFA, von Willebrand’s disease [vWD] screening) indicating abnormalities of primary haemostasis [Table, patient 1], with n=1 confirmed and n=2 highly suspicious of vWD. They presented with either nil (n=1) or mild (n=3) mucocutaneous bleeding. This could be due to heparin binding to von Willebrand’s factor, therefore preventing platelet aggregation.Ten patients had abnormalities of coagulation cascade (prolonged PT, aPTT, reduced clotting factors II, V, VII, X), usually transient but with bleeding in 7 cases (severe/life-threatening in 5 of them ) [Table, patient 3]. Clotting abnormalities were typically accompanied by anaphylactoid symptoms of MCAS and increased by the presence of hematopoietic organ infiltration [Table, patients 2-4]. Haemostatic management (red blood cells and platelets transfusion, fresh frozen plasma, vitamin K, antifibrinolytic) was unsuccessful, while control of mast cell activity (e.g. with and steroids) would achieve haemostasis, indicating the crucial role of mast cell mediators. These mainly include tryptase and heparin, which inhibit the intrinsic pathway, inducing an anticoagulant state.The exact prevalence of clotting abnormalities in mastocytosis remains unclear and probably underestimated. However, we found a predominance of aggressive types of mastocytosis in the population with coagulation cascade abnormalities: 50% had aggressive systemic mastocytosis associated or not with a clonal haematological non mast cell lineage disease (ASM/AHNMD), 30% had mast cell leukaemia (MCL). All deceased patients (n=5, 36% of the overall population) belonged to this group. In contrast, mastocytosis was indolent in 75% of cases in the group with primary haemostasis abnormalities.Abstract 2856. Table:Description of 4 selected patientsPatient number / gender; DiagnosisAcute phase of MCASH/SMGBleedingBiologyClotting after episodePLT150-300PTr≥70%aPTTr≤1.20#1 / M; CMnonoCutaneousN89%1.23Spontaneously resolved#2 / M; SSM-AHNMD†yesnoGI bleedN37%1.75Resolved after steroidsyesnoGI bleed2062%1.41Resolved after symptomatic control of MCAS.#3 / M; aMCLyesyesAcute SDH8761%1.46Resolved after steroids#4 / M; ASM-AHNMD†noyesNone138 *1462% *43%1.39 *1.97Worsening clotting alongside ASM progression and unrelated to the AHNMD. Response to steroids not assessableAHNMD: associated clonal haematological non mast cell lineage disease; aMCL: acute mast cell leukemia; aPTTr: activated partial thromboplastin time ratio; CM: cutaneous mastocytosis; H/SMG: hepato or splenomegaly; GI: gastrointestinal; ISM: indolent systemic mastocytosis; N: normal; SSM: smouldering systemic mastocytosis; PLT: platelet, in x103/µL; PTr: prothrombin time ratio; SDH: subdural haematoma. Reference values are indicated below PLT, PTr, aPTTr. †: patient deceased from the disease or complications of the latter. *6 months later.CONCLUSIONS AND PERSPECTIVESBlood coagulation abnormalities in mastocytosis appear secondary to a hyperactivity of the clonal mast cell, resulting in an anticoagulant state and typically presenting in the context of MCAS and/or significant mast cell proliferation. They are particularly prevalent in aggressive types of mastocytosis, and are only successfully controlled by mast cell inhibitors, notably steroids. More data are required to better correlate clinical and biological pictures, and to assess whether standard or specific clotting tests could be used as a marker of mast cell activity and potentially of poor prognosis in patients with systemic mastocytosis. DisclosuresNo relevant conflicts of interest to declare.

Molecular Profiling of Myeloid Progenitor Cells in Multi-Mutated Advanced Systemic Mastocytosis Identifies KIT D816V As a Distinct and Late Event

Systemic mastocytosis (SM) is characterized by abnormal proliferation and accumulation of mast cells (MC) in various tissues and organs, predominantly skin, bone marrow (BM) and visceral organs. The extent of organ infiltration and subsequent organ damage is the basis for the classification of SM into indolent SM (ISM), smoldering SM (SSM), SM with associated clonal hematologic non-MC lineage disease (SM-AHNMD), aggressive SM (ASM) and MC leukemia (MCL). Depending on the subtype of SM, cell source (BM or peripheral blood) and assay sensitivity, an acquired mutation in the receptor tyrosine kinase KIT, usually KIT D816V, is detectable in 80-90% of patients. Next-Generation Deep Amplicon Sequencing (NGS) was performed to investigate 18 candidate genes at known mutational hotspot regions as previously described. Additional mutations in genes encoding for signaling molecules (JAK2, CBL, KRAS, NRAS), transcription factors (RUNX1), epigenetic regulators (ASXL1, DNMT3A, EZH2, TET2) or splicing factors (SRSF2, SF3B1, U2AF1) are detectable in the vast majority of patients KIT D816V+ advanced SM. In order to gain more insight into clonal evolution of myeloid progenitors in indolent and advanced SM with or without AHNMD, we explored the mutation profile of single-cell-derived CFU-GM colonies - by using Sanger sequencing - in 19 KIT D816V+ SM patients (investigated colonies, n=285; median per patient, n=15; range 10-30). The study included 7 patients with ISM/SSM/ASM (0 additional mutations), 4 patients with SM-AHNMD (median 1 additional mutation, range 1-4) and 8 patients with ASM-AHNMD (median 3 additional mutations, range 1-4). KIT D816V+ CFU-GM could be identified in all 8 patients with ASM ± AHNMD but in only 20% (1/5) of patients with SM-AHNMD, while all CFU-GM colonies derived from ISM patients were completely KIT D816V negative. On the other hand, CFU-GM colonies from individual ASM ± AHNMD patients were never entirely KIT D816V+ (median 60%, range 0-95). In contrast to KIT D816V, additional mutations were identified in CFU-GM colonies from all 12 multi-mutated (A)SM-AHNMD patients and many of these mutations were present in 100% of the CFU-GM-derived colonies analyzed. In 8 patients, different subclones with variable proportions of the number of mutated genes were identified and allowed to generate putative evolutionary trees. These mutations included TET2 and SRSF2 mutations in 6/6 and 4/4 patients with (A)SM-AHNMD. In contrast, ASXL1 mutations were not identified in all TET2/SRSF2 positive CFU-GM colonies suggesting that they are likely to occur later than TET2 and SRSF2. When additional mutations and KIT D816V were detected concomitantly in individual single-cell-derived CFU-GM colonies, the overall frequency of CFU-GM colonies positive for additional mutations was always higher than those that were KIT D816V positive, indicating that the KIT D816V mutation was a secondary event. In contrast to advanced SM, all ISM patients were negative for additional mutations. CFU-GM colonies and also microdissected CD15+ cells derived from BM biopsies were entirely KIT D816V negative in these patients, highlighting that the KIT D816V mutation may be restricted to other (probably later) stages of stem cell development and possibly only to the MC lineage. In general, the relative proportion of KIT D816V+ progenitors correlated well with established parameters for quantification of disease burden, e.g. BM MC infiltration, serum tryptase levels and KIT D816V allele burden in individual patients. In conclusion, the presence of multi-mutated myeloid non-MC lineage progenitors of the CFU-GM-type suggests an initial clonal expansion at an early stage of hematopoietic development due to mutations other than KIT D816V with a subsequent phenotype modification towards SM due to a later acquisition of KIT D816V. In contrast, ISM/SSM is not affected by mutations at the CFU-GM level which may at least in part explain its excellent prognosis. DisclosuresSchnittger:MLL Munich Leukemia Laboratory: Other. Horny:Novartis: Consultancy, Honoraria. Haferlach:MLL: Equity Ownership. Valent:Novartis: Consultancy. Reiter:Novartis: Consultancy, Honoraria.

Human mast cell and basophil/eosinophil progenitors.

Mast cell, basophil, and eosinophil lineages all derive from CD34(+) hemopoietic stem cells; however, mast cells are derived from a distinct, nonmyeloid progenitor, while eosinophils and basophils share a common myeloid progenitor. These progenitors likely evolved from an ancestral leukocyte population involved in innate immunity and currently play a central role in the pathology of allergic disease. Advances in isolation and analysis of mast cell and basophil/eosinophil progenitor populations have been critical to understanding lineage commitment, differentiation, function, and transcriptional regulation of these cells and have provided a way of monitoring the effect of novel investigational therapies on these cell populations in samples of blood, bone marrow, and airway secretions.

Mast cell development and function in the zebrafish.

The many advantages of the zebrafish model provide a unique opportunity to integrate the tools of developmental embryology, transgenesis, and functional assays to elucidate the molecular pathways underlying hematopoiesis and for modeling human blood diseases. These methodologies have recently been applied to the zebrafish mast cell lineage and have resulted in a better understanding of vertebrate mast cell biology. By employing whole-mount in situ hybridization alone and in combination with co-localization approaches, fluorescence-activated cell sorting (FACS), and morpholino gene knockdown studies, new insights into early mast cell transcriptional regulation and ontogeny have been exposed in vivo. Transgenic strategies have permitted the modeling of human mast cell diseases, like systemic mastocytosis in zebrafish, which can subsequently be exploited for high-throughput chemical screens to identify potential therapies in these conditions. Mast cell functional assays have been adapted to zebrafish providing the opportunity to utilize this model for interrogating the cellular players in innate and adaptive immunity and as a live animal readout for drug responses in allergic and inflammatory reactions. These techniques are detailed in the following chapter.

Mechanisms controlling mast cell and basophil lineage decisions.

Basophils and mast cells have long been known to play critical roles in allergic disease and host defense against parasitic infections. Recent recognition of these effector cells in immune regulations, host defense against bacteria and virus, and autoimmune diseases entices increased interest in studying these cells. However, origin and molecular regulation of basophil and mast cell differentiation remain incompletely understood. In this review, we focus on recent advances of the understanding the origin and molecular regulation of mouse basophil and mast cell development. We also summarize progress in the understanding of the origin and molecular regulation of human basophil and mast cell development. A more complete understanding of molecular regulation of basophils and mast cells will lead to the development of interventions that are more effective in achieving long-term success.

Analysis of V600E BRAF and D816V KIT mutations in systemic mastocytosis.

Most patients with systemic mastocytosis (SM) carry a D816V KIT mutation causing a ligand-independent activation of the receptor. Down-stream of KIT is several components known to be mutated in different malignancies. RAF is among the most frequently mutated kinases, where BRAF V600E mutation occurs in most hairy cell leukemias (HCL) and half of malignant melanomas. We investigated BRAF mutations in 36 subjects with different forms of SM, but could not detect BRAF mutation in any of the cases, not even in the mast cell lineage of a patient with V600E BRAF-positive HCL. Thus, although BRAF is commonly mutated it appears not to be present in SM.

Curine inhibits mast cell-dependent responses in mice

Ethnopharmacological relevanceCurine is a bisbenzylisoquinoline alkaloid and the major constituent isolated from Chondrodendron platyphyllum, a plant that is used to treat inflammatory diseases in Brazilian folk medicine. This study investigates the effectiveness of curine on mast cell-dependent responses in mice. Materials and methodsTo induce mast cell-dependent responses, Swiss mice were subcutaneously sensitized with ovalbumin (OVA—12μg/mouse) and Al(OH)3 in a 0.9% NaCl solution. Fifteen days later, the animals were challenged with OVA through different pathways. Alternatively, the animals were injected with compound 48/80 or histamine, and several parameters, including anaphylaxis, itching, edema and inflammatory mediator production, were analyzed. Promethazine, cromoglycate, and verapamil were used as control drugs, and all of the treatments were performed 1h before the challenges. ResultsCurine pre-treatment significantly inhibited the scratching behavior and the paw edema induced by either compound 48/80 or OVA, and this protective effect was comparable in magnitude with those associated with treatment with either cromoglycate or verapamil. In contrast, curine was a weak inhibitor of histamine-induced paw edema, which was completely inhibited by promethazine. Curine and verapamil significantly inhibited pleural protein extravasations and prostaglandin D2 (PGD2) and cysteinyl leukotrienes (CysLTs) production following allergen-induced pleurisy. Furthermore, like verapamil, curine inhibited the anaphylactic shock caused by either compound 48/80 or an allergen. In in vitro settings, these treatments also inhibited degranulation as well as PGD2 and CysLT production through IgE-dependent activation of the mast cell lineage RBL-2H3. ConclusionCurine significantly inhibited immediate allergic reactions through mechanisms more related to mast cell stabilization and activation inhibition than interference with the pro-inflammatory effects of mast cell products. These findings are in line with the hypothesis that the alkaloid curine may be beneficial for the treatment of allergic disorders.

Systemic Mastocytosis with Associated Acute Myelogenous Leukemia

Systemic mastocytosis (SM) is a condition associated with a clonal neoplastic proliferation of mast cells. Approximately 40% of patients with SM present with an associated clonal hematological non–mast cell lineage disorder. Patients presenting with SM–acute myeloid leukemia (AML) have the worst prognosis. We present a case of a 62-year-old woman who was diagnosed with SM-AML. After initial treatment with a standard regimen of cytosine arabinoside (Ara-C)/idarubicin, her bone marrow showed residual blasts. She was subsequently treated with a second induction regimen of clofarabine and high-dose Ara-C, which resulted in remission of AML, although a residual mast cell infiltrate persisted in her bone marrow. After consolidation therapy with clofarabine/Ara-C, the patient received a stem cell allograft. A follow-up bone marrow showed no residual blasts but persistent mast cells occupying about 5% of the marrow volume.

Functional characterization of a novel FGFR1OP-RET rearrangement in hematopoietic malignancies

The RET (REarranged during Transfection) receptor tyrosine kinase is targeted by oncogenic rearrangements in thyroid and lung adenocarcinoma. Recently, a RET (exon 12) rearrangement with FGFR1OP [fibroblast growth factor receptor 1 (FGFR1) oncogene partner] (exon 12) was identified in one chronic myelomonocytic leukemia (CMML) patient. We report the molecular cloning and functional characterization of a novel FGFR1OP (exon 11)-RET (exon 11) gene fusion event (named FGFR1OP-RET), mediated by a reciprocal translocation t(6; 10)(q27; q11), in a patient affected by primary myelofibrosis (PMF) with secondary acute myeloid leukemia (AML). The FGFR1OP-RET fusion protein displayed constitutive tyrosine kinase and transforming activity in NIH3T3 fibroblasts, and induced IL3-independent growth and activation of PI3K/STAT signaling in hematopoietic Ba/F3 cells. FGFR1OP-RET supported cytokine-independent growth, protection from stress and enhanced self-renewal of primary murine hematopoietic progenitor and stem cells in vitro. In vivo, FGFR1OP-RET caused a spectrum of disease phenotypes, with >50% of mice showing a fatal myeloproliferative disorder (MPD). Other phenotypes were leukemia transplantable in secondary recipients, dramatic expansion of the mast cell lineage, and reduction of repopulating activity upon lethal irradiation. In conclusion, FGFR1OP-RET chimeric oncogenes are endowed with leukemogenic potential and associated to myeloid neoplasms (CMML and PMF/AML).

Clinical Features and Outcome Of Clonal Mastocytosis Secondary To Chronic Myelomonocytic Leukemia (SM- AHNMD/CMML) Given Diverse Therapies – A Single Institution Retrospective Study

BackgroundSystemic mastocytosis with associated clonal hematological non mast cell lineage disease (SM-AHNMD), though uncommon, is considered a distinct subcategory of systemic mastocytosis according to the 2008 World Health Organization classification. Prior studies indicate that acute myeloid leukemia with t(8;21) has a strong link to SM-AHNMD. Systematic investigation of systemic mastocytosis associated with chronic myelomonocytic leukemia (SM-CMML) is limited. Bone marrow clonal mast cell proliferation, without overt clinical presentation following CMML often poses a diagnostic challenge and treatment dilemma. The overall survival for SM or SM-AHNMD varies according to subcategories of AHNMD and/or the therapeutic strategies taken. With hypomethylating agents becoming standard therapy for CMML with median overall survival (OS) of 12 to 24 months, acquisition of clonal mast cell proliferation may result in earlier relapse or refractoriness to conventional therapy. We reviewed the clinicopathologic features, cytogenetic and molecular findings, and clinical course of 10 patients with SM-AHNMD specifically chronic myelomonocytic leukemia (SM-CMML). Methods and MaterialsThis retrospective study followed the guidelines of the Institutional Review Board (IRB). Cases diagnosed as SM-AHNMD at Moffitt Cancer Center from 1996 to 2013 were retrieved from the hospital electronic medical record and Cancer Registry. The diagnosis of SM- CMML was confirmed by histomorphology, laboratory data, phenotypic and molecular findings using current WHO criteria. Cases selected were CMML with concurrent or subsequent identification of a clonal mast cell proliferation (SM-CMML). Both solitary SM and SM-AHNMD other than CMML were excluded from this study. ResultsForty patients diagnosed with indolent, cutaneous or systemic mastocytosis were identified, of which 19 were SM- AHNMD. Among the 19 patients, 10 qualified for the study, based on the above criteria, with a median age of 71.4 years (range 43-82) and male: female ratio of 7:3. Nine of 10 patients had no prior evidence of extramedullary mast cell involvement. In one patient systemic lesions were identified 3 months prior to the diagnosis of CMML. Two patients were originally diagnosed with myelodysplastic syndrome [Refractory cytopenia with multilineage dysplasia and Refractory anemia with excess blasts-2], which subsequently were diagnosed as CMML-1 and CMML-2, respectively. The length of time from the original CMML presentation to the diagnosis of SM-AHNMD was widely variable ranging from concurrent to over 80 months. All cases fit the WHO major criterion of aggregates of spindled mast cells. In addition all cases stained positive for CD117 and mast cell tryptase and displayed aberrant staining with either CD2 and/or CD25. Serum tryptase was also elevated (>20ng/ml). Standard treatment protocols with azacitidine were used for CMML. After the diagnosis of SM-AHNMD was rendered the treatment strategies were variable. Key clinical and pathologic features at the time of diagnosis of SM-CMML along with treatment and outcome are summarized in Figure 1. Laboratory values at the time of CMML diagnosis are not shown. The median overall survival was 14.75 months (range 1.0 to 88 months) after CMML was diagnosed and 7.25 months (range 1.0 to 28 months) following diagnosis of SM-CMML. [Display omitted] ConclusionSM- CMML is a rare entity. Clinical presentation is extremely variable. The subtle morphologic features of a developing SM warrants careful histopathologic evaluation, judicious use of immunohistochemical stains and ancilliary techniques. Adherence to the current WHO criteria is important in the identification of these cases. Currently, established treatment guidelines for SM-AHNMD are non-existent. A large scale study is warranted to enrich physician experience in treatment of this rare entity and reduce the associated adverse outcomes. Disclosures:No relevant conflicts of interest to declare.

Committed mast cell progenitors in mouse blood differ in maturity between Th1 and Th2 strains

Mast cell progenitors (MCp) leave the bone marrow and migrate to peripheral tissues where they mature. Although the existence of committed MCp in adult mouse and human blood has been postulated, they have never been found. We have isolated a rare population of cells in adult mouse blood, committed to the mast cell lineage. These were identified as lineage− c-kithi ST2+ integrin β7hi CD16/32hi cells. Moreover, a major difference in maturity of these cells based on FcεRI expression was observed between the Th2-prone BALB/c strain and the Th1-prone C57BL/6 strain (66% vs 25% FcεRI+, respectively). Therefore, the choice of mouse strain is critical when studying disease models such as experimental asthma where mast cells and their progenitors are involved.

T cell dependent induction of novel mast cell-lineage cells in spleen (P6006)

Abstract Mast cells and basophil are known to exert a variety of functions in normal as well as pathological immune responses. Although mutual dependencies between mast cell lineage cells (MCs) and T cells have been recently well documented, how antigen-specific T cell responses leads to differentiate MCs and to MCs-dependent immune responses is not fully understood. When we attempted to generate antigen-specific T cell lines from OVA and KLH immunized mice by repeatedly stimulating their spleen cells or lymph node (LN) cells in vitro, we observed dominant expansion of FcϵR1 and c-kit positive cells in spleen cell culture, not in LN cell culture. Although this surface phenotype has been classified as that of MC, toluidine blue staining and electron microscopic analysis demonstrated that granules found in these cells appeared more like those seen in classic basophils rather than MCs. The functions of these cells were equivalent to those of classical MCs or basophils such as β-hexomidase release in vitro and PCA reaction in vivo. Consistently, expression pattern of MCPs revealed mixed phenotypes of MCs and basophils. These cells produced IFN-γ when they were co-cultured with antigen specific T cells in the presence of relevant antigen, which occurred in mast cell-T cell contact manner. These results suggested the existence of unique mast cell lineage in spleen contributing T cell dependent immune responses. Roles of these cells in vivo are currently under investigation.

Increased interleukin-9 and CD4+IL-9+ T cells in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown origin affecting all the organ systems. Apart from genetic and environmental factors, autoantibody and immune complex deposition as well as cytokine imbalances contribute to immune dysfunction. Interleukin‑9 (IL-9) is a Tcell-derived factor preferentially expressed by CD4+ Tcells and it has been characterized in human and murine systems. IL-9 targets cells of the lymphoid, myeloid and mast cell lineages, and is likely to contribute to the development of allergic and autoimmune diseases such as asthma, arthritis, multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Nevertheless, until recently there have been no studies on its role in SLE in humans. In the present study, the mRNA and serum IL-9 levels in the peripheral blood of SLE patients and healthy controls were assessed using real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Flow cytometry was used to analyze the percentages of CD4+IL-9+ Tcells in SLE patients. Moreover, differences between the groups and the effect of glucocorticoids were analyzed. The results showed that the plasma concentration and mRNA levels of IL-9 were significantly elevated in SLE patients compared with the healthy controls. The percentages of CD4+IL-9+ Tcells were also increased in SLE patients. In addition, serum IL-9 levels and the percentages of CD4+IL-9+ Tcells were correlated with the SLE disease activity index (SLEDAI). Additionally, the percentages of CD4+IL-9+ Tcells and serum IL-9 levels in 8untreated active SLE patients were decreased at 1, 2 and 3weeks after treatment with methylprednisolone. In conclusion, we provide evidence that IL-9 is increased in SLE patients. Moreover, it is described for the first time that high expression of IL-9 levels and the percentages of CD4+IL-9+ Tcells correlate with disease activity and severity. This suggests an important role of IL-9 in the pathogenesis of SLE.

Regulation of GATA Factor Expression Is Distinct between Erythroid and Mast Cell Lineages

The zinc finger transcription factors GATA1 and GATA2 participate in mast cell development. Although the expression of these factors is regulated in a cell lineage-specific and differentiation stage-specific manner, their regulation during mast cell development has not been clarified. Here, we show that the GATA2 mRNA level was significantly increased while GATA1 was maintained at low levels during the differentiation of mast cells derived from mouse bone marrow (BMMCs). Unlike in erythroid cells, forced expression or small interfering RNA (siRNA)-mediated knockdown of GATA1 rarely affected GATA2 expression, and vice versa, in mast cells, indicating the absence of cross-regulation between Gata1 and Gata2 genes. Chromatin immunoprecipitation assays revealed that both GATA factors bound to most of the conserved GATA sites of Gata1 and Gata2 loci in BMMCs. However, the GATA1 hematopoietic enhancer (G1HE) of the Gata1 gene, which is essential for GATA1 expression in erythroid and megakaryocytic lineages, was bound only weakly by both GATA factors in BMMCs. Furthermore, transgenic-mouse reporter assays revealed that the G1HE is not essential for reporter expression in BMMCs and peritoneal mast cells. Collectively, these results demonstrate that the expression of GATA factors in mast cells is regulated in a manner quite distinct from that in erythroid cells.

The zebrafish reveals dependence of the mast cell lineage on Notch signaling in vivo

We used the opportunities afforded by the zebrafish to determine upstream pathways regulating mast cell development in vivo and identify their cellular origin. Colocalization studies demonstrated zebrafish notch receptor expression in cells expressing carboxypeptidase A5 (cpa5), a zebrafish mast cell-specific marker. Inhibition of the Notch pathway resulted in decreased cpa5 expression in mindbomb mutants and wild-type embryos treated with the γ-secretase inhibitor, Compound E. A series of morpholino knockdown studies specifically identified notch1b and gata2 as the critical factors regulating mast cell fate. Moreover, hsp70::GAL4;UAS::nicd1a transgenic embryos overexpressing an activated form of notch1, nicd1a, displayed increased cpa5, gata2, and pu.1 expression. This increase in cpa5 expression could be reversed and reduced below baseline levels in a dose-dependent manner using Compound E. Finally, evidence that cpa5 expression colocalizes with lmo2 in the absence of hematopoietic stem cells revealed that definitive mast cells initially delineate from erythromyeloid progenitors. These studies identify a master role for Notch signaling in vertebrate mast cell development and establish developmental origins of this lineage. Moreover, these findings postulate targeting the Notch pathway as a therapeutic strategy in mast cell diseases.

Myelomastocytic Leukemia With t(8;21) in a 3-year-old Child

Here we report a 3-year-old boy with myelomastocytic leukemia. The patient presented with fatigue and right eye proptosis. Bone marrow revealed acute myeloid leukemia with t(8;21) and trisomy 8. Induction therapy produced marked reduction in marrow myeloblasts with the emergence of 13% atypical mast cells. These cells were subsequently identified in retrospect in the diagnostic marrow consistent with myelomastocytic leukemia. His clinical course was notable for the difficulty in the eradication of the leukemic process and resembled that of adults with systemic mastocytosis with associated hematologic non-mast cell lineage disease. To the best of our knowledge, this is the youngest individual reported. The implications of mast cell lineage involvement in acute myeloid leukemia are reviewed.

Thalidomide in Advanced Mastocytosis. Results from an Open-Label, Multicentric, Phase II Study

Abstract 1754▪▪This icon denotes a clinically relevant abstract Background:Mastocytosis is characterized by the abnormal accumulation of mast cells in various tissues responsible for organ failure and/or systemic symptoms that can be disabling and alter the quality of life (QOL). Beside symptomatic treatments, cytoreductive drugs such as a-interferon, purine analogs and to a less extent imatinib mesylate (in case of c-kit mutation other than D816V) and masatinib were shown to be effective but rare complete or long-term remissions were obtained. Thalidomide is an anti-angiogenic immuno-modulatory and anti-inflammatory drug that has preliminary been shown to have an activity in aggressive systemic mastocytosis (ASM) (Damaj et al, BJH 2008). We report the result of a multicentric, open-label, phase II study using thalidomide as a single agent in advanced SM (NCT00769587). Patients and Methods:Twenty patients (pts) were enrolled (1 missing data): smoldering systemic (SSM, n=9), ASM (n=6) and with an associated clonal hematologic non mast cell lineage disease (SM-AHNMD, n=4). Infiltrated organs were bone marrow (n=16), skin (n=14), splenomegaly (n=10), hepatomegaly (n=6), intestinal tract (n=6), lymph nodes (n=1). Median serum tryptase level at inclusion was 83.4 ng/L (range 5.3–775) and all patients except one carried the c-kit D816V mutation. Patients presented with anemia (n=6), thrombocytopenia (n=6) and neutropenia (n=1). Four patients were excluded, 1 for missing data and 3 were not evaluable. Thalidomide was administered orally at a starting dose of 50 mg/day and was progressively increased up to 200 mg/day or appearance of side effects. The duration of treatment was 6 months (1 cycle= 1 month). Responder patients may continue on thalidomide for a maximum of 12 months or progression. Primary objective was to determine the effective response rate by assessing the tumor burden. Secondary objectives were to assess the tolerance of thalidomide and to evaluate the QOL, depression (Hamilton score), pruritis, and handicap (Afirmm V2 score) related to mast cell disease. Results:Sixteen pts [7 males, 9 females; median age 65 years (range 43–76)] who received at least one cycle of thalidomide were analyzed.The median number of thalidomide cycles was 6 (range 1–20) and the median dose received was 100mg/d. Partial response was obtained in 9 pts (56%), 4 pts remained stable (25%) and 3 pts progressed (19%). Pruritis score decreased significantly from 6.5 (95% CI 4.74–12.26) to 4 (95% CI 0–5.25) (p=0.03); the Afirmm score tended to improve from 116.5 (95% CI 55.0–168.3) to 92.5 (95% CI 37.4–131.2) (p=0.38) with no improvement of QoL and Hamilton scores, median tryptase level or cytopenia. Skin infiltration disappeared only in 2 of 14 patients concerned. Treatment discontinuation was due to failure in 4 pts, side effect in 2 pts and patient’s decision in 2. Thalidomide was continued in five pts as a maintenance therapy for a median duration of 6 mo (4–14). The most relevant toxicities (grade 3–4) consisted in peripheral neuropathy (12.5%) and myelosuppression (18.7%). Conclusions:Thalidomide is an acceptable and effective treatment in advanced SM. Disclosures:No relevant conflicts of interest to declare.

Systemic mastocytosis with an associated non mast cell lineage clonal hematological disease in a child.

Systemic mastocytosis with an associated non mast cell lineage clonal hematological disease in a child.

A novel insight to the functional role of Stathmin 1 in IgE-mediated activation of RBL-2H3 cells.

IgE-mediated cell signaling, induced by cross-linking of high affinity receptor for IgE (FcεRI) in the presence of antigen (Ag), is a well known mechanism described for mast cell activation in allergy and hypersensitivity reactions, which induces a spectrum of cellular responses such as secretion and up-regulation of cell surface FcεRI. Although for several years IgE binding to FcεRI was considered to be a passive sensitization process, the outcomes of several recent studies have revealed a variety of different cellular responses to IgE binding compared to IgE plus Antigen binding. The present study applied a functional proteomics-based approach to investigate mast cell signaling events and provided new insights to FcεRI-mediated cell signaling in RBL-2H3.1 cells, and may point to the activation of alternative signaling pathways in response to IgE or IgE plus Ag. Comparative analysis by 2-D PAGE of RBL cells activated with IgE plus Ag for three and four hours compared to non-activated cells was followed by mass spectrometric protein identification and provided evidence for the induction of Stathmin 1 (STMN1) gene expression in response to IgE plus Ag activation.Complementary SDS-PAGE analysis showed a distinct up-regulation of STMN1 induction in response to challenge with IgE plus Ag compared to sensitization with IgE only. Phosphoproteomics analysis gave evidence for significant increase at phosphorylation of STMN1 on ser16 after 1min, though a slight rise at 5 min, and on ser38 after 1 and 5min sensitization with IgE and a similar result was observed for 1min IgE plus Ag-activation. IgE plus Ag-activation was also found to induce the phosphorylation of ser38 to a greater extent than sensitization with IgE. In contrast, IgE alone was more effective than IgE plus Ag at inducing phosphorylation of ser16. Collectively this study provides further insights into the role of stathmin 1 in FcRI-mediated activation of cells of mast cell lineage and might shed light on the diverse response of these cells to IgE or IgE plus Ag.