scholarly journals Characterization of human mast cells in long-term culture

Blood ◽  
1983 ◽  
Vol 62 (6) ◽  
pp. 1251-1260 ◽  
Author(s):  
MA Horton ◽  
HA O'Brien
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Systemic Mastocytosis ◽  
Human Mast Cell ◽  
Lymphoid Tissues ◽  
Stromal Fibroblasts ◽  
Human Mast Cells ◽  
Hemopoietic Tissue

Abstract Recent studies in rodents have demonstrated that mast cells derived from lymphoid tissues can be grown in longterm culture, provided that supportive growth factors or stromal fibroblasts are added; such findings have not been reported in man. Furthermore, although a hemopoietic origin for mast cells is supported by transplantation studies in mice, the exact origin of the human mast cell or its relationship to the circulating basophil and other hemopoietic cell lineages is unknown. We have investigated the requirements for in vitro growth of human mast cells derived from the infiltrated bone marrow of a patient with systemic mastocytosis, and have characterized both the mast cells proliferating in these cultures and those obtained from splenic infiltrates. Our data approached two questions: (1) Is there any evidence for the origin of mast cells from a bone-marrow-derived stem cell, and, if so, (2) what lineage relationship is there between mast cells and granulopoietic cells, including basophils? First, we have shown the expression of hemopoietic tissue-specific antigens by mast cells, strongly supporting a bone marrow origin for the mast cell in man (at least for those mast cells analyzed here). Second, the complete lack of granulocyte-monocyte markers contrasts with the phenotype of the basophil and suggests that mast cells diverge considerably from other granulopoietic cells during the acquisition of their differentiated specialized functions.

scholarly journals Characterization of human mast cells in long-term culture

Blood ◽  
1983 ◽  
Vol 62 (6) ◽  
pp. 1251-1260
Author(s):  
MA Horton ◽  
HA O'Brien
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Systemic Mastocytosis ◽  
Human Mast Cell ◽  
Lymphoid Tissues ◽  
Stromal Fibroblasts ◽  
Human Mast Cells ◽  
Hemopoietic Tissue

Recent studies in rodents have demonstrated that mast cells derived from lymphoid tissues can be grown in longterm culture, provided that supportive growth factors or stromal fibroblasts are added; such findings have not been reported in man. Furthermore, although a hemopoietic origin for mast cells is supported by transplantation studies in mice, the exact origin of the human mast cell or its relationship to the circulating basophil and other hemopoietic cell lineages is unknown. We have investigated the requirements for in vitro growth of human mast cells derived from the infiltrated bone marrow of a patient with systemic mastocytosis, and have characterized both the mast cells proliferating in these cultures and those obtained from splenic infiltrates. Our data approached two questions: (1) Is there any evidence for the origin of mast cells from a bone-marrow-derived stem cell, and, if so, (2) what lineage relationship is there between mast cells and granulopoietic cells, including basophils? First, we have shown the expression of hemopoietic tissue-specific antigens by mast cells, strongly supporting a bone marrow origin for the mast cell in man (at least for those mast cells analyzed here). Second, the complete lack of granulocyte-monocyte markers contrasts with the phenotype of the basophil and suggests that mast cells diverge considerably from other granulopoietic cells during the acquisition of their differentiated specialized functions.

Ectopic Expression of Lin28 Biases Myeloid Differentiation to Promote an Immature Mast Cell State and Is Implicated in Aggressive Mastocytosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5571-5571
Author(s):  
Leo D. Wang ◽  
Phi Nguyen ◽  
Robert G Rowe ◽  
Tata Nageswara Rao ◽  
George Q. Daley ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Conflicts Of Interest ◽  
Systemic Mastocytosis ◽  
Expression Patterns ◽  
Ectopic Expression ◽  
In Vitro Assays ◽  
Human Mast Cell ◽  
Adult Mice

Abstract Dysregulated mast cell development leads to systemic mastocytosis, a clinically variable but often devastating family of hematologic disorders. Lin28 is a heterochronic gene and pluripotency factor implicated in many types of malignancy, and prior studies suggest that Lin28 expression can restore a fetal hematopoietic program in adult mice. However, the role of Lin28 in hematologic malignancy remains controversial. In our study, we induced expression of Lin28 in adult mice using a doxycycline-responsive transgenic system. Lin28 induction caused marked mast cell accumulation in target organs such as the skin and peritoneal cavity. In vitro assays revealed a skewing of myeloid commitment in Lin28-expressing hematopoietic progenitors, with increased levels of Lin28 in common myeloid progenitors and basophil-mast cell progenitors altering gene expression patterns to favor cell fate choices that enhance mast cell specification. In addition, Lin28-induced mast cells appeared phenotypically and functionally immature, and in vitro assays suggested a slowing of mast cell terminal differentiation in the context of Lin28 upregulation. Finally, interrogation of human mast cell leukemia samples revealed upregulation of LIN28 in abnormal mast cells from patients with aggressive systemic mastocytosis (ASM). This work identifies Lin28 as a novel regulator of innate immune function and a new protein of interest in mast cell disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Human Mast Cell Line HMC1 Expresses Functional Mas-Related G-Protein Coupled Receptor 2

2021 ◽  
Vol 12 ◽  
Author(s):  
Maud A. W. Hermans ◽  
Astrid C. van Stigt ◽  
Sanne van de Meerendonk ◽  
Benjamin Schrijver ◽  
Paul L. A. van Daele ◽  
...  
Keyword(s):  
Growth Rate ◽  
Mast Cell ◽  
Mast Cells ◽  
Cell Line ◽  
G Protein ◽  
Human Mast Cell ◽  
Human Mast Cells ◽  
Mast Cell Line ◽  
G Protein Coupled

The Mas-related G-protein-coupled receptor X2 (MRGPRX2) is prominently expressed by mast cells and induces degranulation upon binding by different ligands. Its activation has been linked to various mast cell-related diseases, such as chronic spontaneous urticaria, atopic dermatitis and asthma. Therefore, inhibition of MRGPRX2 activity represents a therapeutic target for these conditions. However, the exact pathophysiology of this receptor is still unknown. In vitro research with mast cells is often hampered by the technical limitations of available cell lines. The human mast cell types LAD2 and HuMC (human mast cells cultured from CD34+ progenitor cells) most closely resemble mature human mast cells, yet have a very slow growth rate. A fast proliferating alternative is the human mast cell line HMC1, but they are considered unsuitable for degranulation assays due to their immature phenotype. Moreover, the expression and functionality of MRGPRX2 on HMC1 is controversial. Here, we describe the MRGPRX2 expression and functionality in HMC1 cells, and compare these with LAD2 and HuMC. We also propose a model to render HMC1 suitable for degranulation assays by pre-incubating them with latrunculin-B (Lat-B). Expression of MRGPRX2 by HMC1 was proven by RQ-PCR and flowcytometry, although at lower levels compared with LAD2 and HuMC. Pre-incubation of HMC1 cells with Lat-B significantly increased the overall degranulation capacity, without significantly changing their MRGPRX2 expression, phenotype or morphology. The MRGPRX2 specific compound 48/80 (C48/80) effectively induced degranulation of HMC1 as measured by CD63 membrane expression and β-hexosaminidase release, albeit in lower levels than for LAD2 or HuMC. HMC1, LAD2 and HuMC each had different degranulation kinetics upon stimulation with C48/80. Incubation with the MRGPRX2 specific inhibitor QWF inhibited C48/80-induced degranulation, confirming the functionality of MRGPRX2 on HMC1. In conclusion, HMC1 cells have lower levels of MRGPRX2 expression than LAD2 or HuMC, but are attractive for in vitro research because of their high growth rate and stable phenotype. HMC1 can be used to study MRGPRX2-mediated degranulation after pre-incubation with Lat-B, which provides the opportunity to explore MPRGRX2 biology in mast cells in a feasible way.

scholarly journals Expression of Functional TrkA Receptor Tyrosine Kinase in the HMC-1 Human Mast Cell Line and in Human Mast Cells

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 1807-1820 ◽  
Author(s):  
See-Ying Tam ◽  
Mindy Tsai ◽  
Masao Yamaguchi ◽  
Koji Yano ◽  
Joseph H. Butterfield ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Tyrosine Kinase ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Human Mast Cell ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Human Mast Cells

Abstract Nerve growth factor (NGF ) can influence mast cell development and function in murine rodents by interacting with its receptors on mast cells. We now report the identification of mRNA transcripts of full-length tyrosine kinase-containing trkA, trkB, and trkC neurotrophin receptor genes in HMC-1 human mast cell leukemia cells. Although HMC-1 cells lacked p75 mRNA, they expressed transcripts for the exon-lacking splice variant of trkA (trkAI), truncated trkB (trkB.T1), and truncated trkC. By flow cytometry, HMC-1 cells exhibited expression of TrkA, TrkB, and TrkC receptor proteins containing full-length tyrosine kinase domains. NGF stimulation of HMC-1 cells induced tyrosine phosphorylation of TrkA protein, increased expression of the early response genes c-fos and NGF1-A, and activation of ERK-mitogen–activated protein (MAP) kinase, results which indicate that TrkA receptors in HMC-1 cells are fully functional. Highly purified populations of human lung mast cells expressed mRNAs for trkA, trkB and trkC, whereas preparations of human umbilical cord blood-derived mast cells expressed mRNAs for trkA and trkC, but not trkB. Moreover, preparations of human umbilical cord blood-derived immature mast cells not only expressed mRNA transcript and protein for TrkA, but exhibited significantly higher numbers of chymase-positive cells after the addition of NGF to their culture medium for 3 weeks. In addition, HMC-1 cells expressed mRNAs for NGF, brain-derived neurotrophic factor (BDNF ), and neurotrophin-3 (NT-3), the cognate ligands for TrkA, TrkB, and TrkC, whereas NGF and BDNF transcripts were detectable in human umbilical cord blood mast cell preparations. Taken together, our findings show that human mast cells express a functional TrkA receptor tyrosine kinase and indicate that NGF may be able to promote certain aspects of mast cell development and/or maturation in humans. Our studies also raise the possibility that human mast cells may represent a potential source for neurotrophins.

Imatinib Mesylate in the Treatment of Systemic Mastocytosis, a Phase I/II Trial.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1516-1516 ◽  
Author(s):  
H.J. Droogendijk ◽  
J.C. Kluin-Nelemans ◽  
P.L.A van Daele
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Mast Cell ◽  
Mast Cells ◽  
Imatinib Mesylate ◽  
Systemic Mastocytosis ◽  
Skin Lesions ◽  
Tissue Response ◽  
Serum Tryptase ◽  
Kit Receptor

Abstract Introduction: mastocytosis comprimes a group of diseases characterized by abnormal proliferation and accumulation of mast cells in one or more organs. A cutaneous and systemic form of mastocytosis is distinguished. Systemic mastocytosis defines the disease process in which mast cell proliferation exceeds the skin. The clinical manifestations of systemic mastocytosis depend on the tissues involved and the tissue response to the accumulation of mast cells. Although in general the disease progresses slowly, it may develop into a malignant disease. Currently there is no cure for systemic mastocytosis. Mast cells develop from pluripotent bone marrow progenitor cells that express CD34 antigen and are dispersed as precursors which undergo proliferation and maturation in different tissues. Normal mast cell development involves the action of stam cell growth factor and c-kit receptors, which are expressed by mast cells at their different developmental stages. Deregulation and/or abnormalities of the c-kit receptor are assumed to play a causal role in disordered mast-cell proliferation. In most patients a mutation in the gene for c-kit exists. One of the mutations is the D816V mutation. Aim of the study:imatinib mesylate, formerly called ST1571, is a potent inhibitor of c-kit receptor tyrosine kinase activity. In this study, we evaluate whether imatinib mesylate is safe and effective in the treatment of patients with systemic mastocytosis. Primary end-points of study are reduction in urinary N-methylhistamine excretion, serum tryptase activity, skin lesions, number of mast cells in sections of bone marrow, hepato-and/or splenomegaly and symptoms.Adverse effects on therapy are also considered. Results: up to now, 10 patients with systemic mastocytosis are treated with 400 mg of imatinib mesylate orally once daily. During the first 2 weeks of the study the patients also received 30 mg of prednisolone daily. In general imatinib mesylate is well tolerated. The first results show a 38–80% reduction in urinary N-methylhistamine excretion and 30–66% reduction in serum tryptase activity. Skin lesions diminish in two of the six patients with cutaneous mastocytosis,. Number of mast cells in sections of bone marrow are reduced in 63% (5/8) of the patients. Hepato-and/or splenomegaly is slightly decreased in two of the three patients with organomegaly. Finally 60 % of all patients experiences relief of symptoms. In eight patients the D816V mutation was found. In contrast with former studies imatinib mesylate is also effective in these patients. Further results are to be awaited. Conclusion: imatinib mesylate is safe and seems effective in the treatment of patients with systemic mastocytosis (including patients with the D816V mutation).

Thrombopoietin Regulates Proliferation and Maturation of Murine Mast Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1707-1707
Author(s):  
Giovanni Migliaccio ◽  
Barbara Ghinassi ◽  
Lucia Centurione ◽  
Maria Zingariello ◽  
Lucia Bianchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Cell Differentiation ◽  
Growth Factor ◽  
Mast Cells ◽  
Wild Type ◽  
Connective Tissues ◽  
Immature Cells

Abstract Megakaryocytopoiesis is regulated by extrinsic (interaction of the growth factor thrombopoietin, TPO with its receptor Mpl) and intrinsic (interaction between the trascription factors GATA-1 and Fog-1) factors. The observation that mice impaired for GATA-1 expression (i.e. harbouring the GATA-1low mutation) are defective not only in megakaryocyte maturation but also in mast cell differentiation (Migliaccio et al. J Exp Med197:281, 2003), led us to investigate whether TPO might control mast cell differentiation as well. We first observed that mice genetically unable to responde to TPO (Mplnull mice) express in the connective tissues 5 times more mast cells than their normal littermates. Then, we analysed the effects on mast cell differentiation of in vivo treatment with TPO. Normal mice, and their GATA-1low littermates, were injected i.p. with TPO (100 μg/kg/day per 5 days, kindly provided by Kirin Brewery, Japan) and the number of immature (Toluidinepos) and mature (AlcianBlue/Saphraninepos) mast cells present in the connective tissues of the animals, as well as the frequency of GATA-1pos and TUNELpos mast cells, was evaluated 14 days after treatment. In wild-type animals, TPO reduced the presence of GATA-1 in mast cells (by immuno-histochemistry) and increased the number of immature cells (from 320±28 to 852±60) and of those undergoing apoptosis (from 16±1 to 600±43). In contrast, in GATA-1low animals, TPO-treatment induced the expression of GATA-1 in mast cells while decreased the number of immature cells (from 1100±72 to 427±29) as well as that of apoptotic cells (from 600±45 to 60±2). The role of TPO on mast cell differentiation were further confirmed by the analysis of the effects exerted by the growth factor on in vitro differentiation of bone marrow derived mast cells (BMMC). In these experiments, wild type bone marrow and spleen cells were cultured for 21 days with SCF and IL-3 with or without TPO and BMMC differentiation measured on the basis of the number of cells expressing the phenotype c-kithigh/CD34high and FcεRIpos. In cultures stimulated with SCF and IL-3, all the cells expressed the phenotype c-kithigh/CD34high and FcεRIpos. In contrast, in cultures supplemented also with SCF, IL-3 and TPO, only 25% of the cells were c-kithigh/CD34high and none of them was FcεRIpos. These results establish a role for TPO in the control of mast cell differentiation (possibly by modulating the GATA-1 content of the cells) and unveil further similarities between the mechanism(s) controlling megakaryocyte and mast cell differentiation.

Kit Signaling Regulates Mitf Expression in Mastocytosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3601-3601
Author(s):  
Youl-Nam Lee ◽  
Pierre Noel ◽  
Amir Shahlaee ◽  
Melody Carter ◽  
Reuben Kapur ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Systemic Mastocytosis ◽  
Tyrosine Kinase Receptor ◽  
Bone Marrow Biopsies ◽  
Activating Mutations ◽  
Cell Assays ◽  
Mast Cell Disease ◽  
Mitf Expression

Abstract Mastocytosis is a heterogeneous disease arising from abnormal proliferation of mast cells. Activating mutations in codon D816 of the tyrosine kinase receptor, c-kit, are found in the majority of adult patients with systemic mastocytosis, an aggressive form of the disease. Constitutive activation of the Kit signaling pathway is critical to the transformed phenotype, and thus understanding how this pathway regulates downstream events is of great importance. A number of transcription factors are also essential to mast cell development, including the Microphthalmia-associated transcription factor (Mitf). We examined Mitf expression in bone marrow biopsies from nine patients with systemic mastocytosis by immunohistochemistry; we found that Mitf is highly expressed in all cases with the D816V mutation. In contrast, Mitf is not highly expressed in non-malignant mast cells in the bone marrow from patients with aplastic anemia and leukemia, suggesting thatMitf expression is regulated by Kit-dependent signalsMitf may play a role in the transformed phenotype of mastocytosis.We show that in normal mast cells, Kit signaling markedly upregulates Mitf expression. In both normal and malignant mast cells, pharmacologic inhibitors of Kit, and the downstream kinase, PI3K, block Mitf expression. To examine whether Mitf is required for transformed phenotype from constitutive Kit signaling in mast cells, we have used a shRNA-expressing lentivirus to knockdown Mitf expression in mastocytosis cell lines. We found that silencing of Mitf markedly impaired growth in proliferation and colony forming cell assays. This work demonstrates a link between two critical factors, Kit and Mitf, in the development of malignant mast cell disease.

Change In Mast Cell Bone Marrow Burden and In Serum Tryptase Level Are Not Accurate Markers of Response In Patients with Systemic Mastocytosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3073-3073
Author(s):  
Alfonso QuintÁs-Cardama, ◽  
Matjaz Sever ◽  
Jorge E. Cortes ◽  
Hagop M. Kantarjian ◽  
Srdan Verstovsek
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Mast Cells ◽  
Systemic Mastocytosis ◽  
Median Number ◽  
Response To Therapy ◽  
Serum Tryptase ◽  
Bone Marrow Biopsies ◽  
Tryptase Level ◽  
Bone Marrow Mast Cell

Abstract Abstract 3073 Background: Bone marrow involvement, with or without cutaneous or visceral involvement, is almost universal in patients with systemic mastocytosis (SM). The KITD816V mutation is present in most patients with SM, thus confirming its clonal nature. Patients with ASM are usually managed with cytoreductive agents such as hydroxyurea (HU), cladribine (2CDA), or interferon-alpha (IFN-α), although the activity of these therapies is limited as they do not target specifically the malignant clone. Response assessment in SM relies on symptom improvement and reduction in serum tryptase levels and visceral and/or bone marrow mast cell burden (percent mast cell involvement). We contend that the later two relatively objective metrics may not be appropriate markers of response because serum tryptase levels may vary significantly at different time-points in the same patient in the absence of intervention, do not correlate accurately with mast cell burden, and bone marrow mast cell burden determination is subject to sampling bias given the patchy infiltration observed in many cases of SM. Objectives: To assess the utility of bone marrow mast cell burden reduction and serum tryptase level reduction as criteria for response in patients with SM. Patients and Therapy: We studied a cohort of 50 patients with SM for whom at least 2 sequential bone marrow biopsies and 2 serum tryptase level determinations were available at our center. The KITD816V mutation was present in 20 (59%) of 34 assessable patients. No patient carried the JAK2V617F mutation or the FIP1L1-PDGFRA rearrangement. Patients had a diagnosis of indolent SM (ISM, n=25), aggressive SM (ASM, n=16), or SM-AHNMD (n=9). All but 1 patient received SM-directed therapy (median number of therapies 2, range 1–5), including: imatinib (n=16), dasatinib (n=23), RAD001 (n=8), denileukin diftitox (n=7). The median number of bone marrow biopsies available per patient was 4 (range, 2–14) and the median number of tryptase measurements was 6 (range, 2–18), which were obtained both on and off SM-directed therapies. Results: Four patients had a bone marrow complete response: 1 with imatinib, 2 with dasatinib, and 1 with decitabine (with SM-MDS). However none of the responders normalized their tryptase levels. We used the coefficient of variation (CV) as a normalized measure of dispersion of a probability distribution for the percentage of mast cells in bone marrow biopsies and serum tryptase levels. In this manner, the CV summarizes/describes the variation in tryptase levels and bone marrow mast cell percentage from the baseline (first recorded value) in the patients evaluated. We found that among the 49 treated patients, the percentage of bone marrow mast cells varied significantly with a CV ranging from 6 – 173% and an average of 65%. Forty-four percent of patients had a CV equal or higher to the average. Similar results were observed regarding tryptase levels, with an average CV of 19% that ranged from 0 to 96%. Thirty-six percent of patients had a CV higher than average. Conclusion: While most patients fail to respond to currently available SM-directed therapies, sequential bone marrow biopsies and tryptase level determinations exhibit remarkable variation both during and in the absence of SM-directed therapy. Therefore, it seems that single time point measurements of these values do not represent proper tools to assess accurately response to therapy. Disclosures: No relevant conflicts of interest to declare.

Familial Mastocytosis: Identification Of KIT K509I Mutation and Its In Vitro Sensitivity To Imatinib, Dasatinib and PKC412

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5267-5267 ◽  
Author(s):  
Paula De Melo Campos ◽  
João Agostinho Machado-Neto ◽  
Adriana Silva Santos Duarte ◽  
Renata Scopim-Ribeiro ◽  
Flavia Fonseca de Carvalho Barra ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Tyrosine Kinase ◽  
Bone Marrow Cells ◽  
Systemic Mastocytosis ◽  
History Of ◽  
Primary Bone ◽  
Primary Bone Marrow ◽  
Apoptosis Ratio

Abstract Background Mast cell diseases are myeloproliferative neoplasms characterized by an abnormal proliferation and accumulation of mast cells in different tissues. The clinical presentation of mastocytosis is heterogeneous, ranging from skin-limited disease to more aggressive variants that may be associated with multiorgan dysfunction/failure and shortened survival. In a relatively high proportion of cases, the clonal nature of the disease can be established on the basis of the demonstration of gain-of-function mutations involving the tyrosine kinase (TK) domain of KIT in skin lesions and BM cells and by the factor-independent proliferation and transforming abilities of these mutations. The tyrosine kinase inhibitor Imatinib is a treatment available for mastocytosis patients; however, some KIT mutations, specially KIT D816V, confer resistance to this drug. Aims To characterize the clinical phenotype and molecular mutations of 2 relatives with diagnosis of systemic mastocytosis (WHO 2008). We also aimed to test the in vitro sensitivity of primary bone marrow (BM) cells from both patients to tyrosine kinase inhibitors. Patients and methods Four individuals were included in the study; two patients (case 1 [mother], and case 2 [daughter]), and the parents of case 1. DNA samples were obtained from total BM cells, CD3+ BM cells and oral mucosa of patients, and from peripheral blood of all individuals. KIT (exons 1 to 21) was submitted for Sanger sequencing analysis. Primary bone marrow cells (5X104) from the 2 patients were cultured and treated with Imatinib (5uM), Dasatinib (80nM) and PKC 412 (100nM) or with vehicle only (control cells) and submitted for proliferation (MTT) and apoptosis assays (Annexin-V/PI) at days 4, 8 and 12 of culture. Results Case 1 was a 33 year-old woman with a chronic history of pruritic skin rash who was referred to our outpatient service for evaluation of massive splenomegaly (25 centimeters in length) and pancytopenia. She had neither comorbidities nor any familial history of hematological malignancies. The patient had no siblings and had only one daughter (case 2). At biopsy, she showed extensive skin and bone marrow infiltration by mast cells. During follow up, the patient presented with spontaneous splenic rupture and had to undergo splenectomy, which led to the resolution of pancytopenia. She was diagnosed with Aggressive Systemic Mastocytosis. Her daughter (case 2), a 17 year-old woman, was also evaluated for an insidious history of diffuse skin rash. Skin and bone marrow biopsies showed massive infiltration by atypical mast cells and a diagnosis of Indolent Systemic Mastocytosis was made. The rare KIT K509I mutation was found in all DNA samples obtained from both patients, but not from the parents of case 1. This suggests that the KIT K509I was a germ line mutation acquired de novo by patient 1 that was subsequently transmitted to her daughter (patient 2). In vitro treatment of primary bone marrow cells harboring the KIT K509I mutation from patients 1 and 2 resulted in variable clinical response rates according to the drug used and the treatment duration. Imatinib treatment resulted in a significant reduction in proliferation (days 4, 8 and 12 of culture) and an increase in apoptosis (days 8 and 12) in cases 1 and 2 (all p≤0.03). Although Dasatinib resulted in decreased proliferation in both patients at day 12 (all p≤0.008), a significantly higher apoptosis ratio was observed only for patient 1 at day 12 of culture (p=0.03). PKC412 had a negative effect over cell growth in patient 1 (days 4 and 8) and in patient 2 (day 4) (all p≤0.03); however, no effect in apoptosis ratio was seen. Conclusions We herein provide a report of a KIT K509I mutation in familial mastocytosis. This mutation has been previously described in the literature in one case of familial mastocytosis. Although rare, the screening for KIT K509I mutation should be considered in all cases of familial mastocytosis. Based on in vitro studies, mastocytosis patients harboring the KIT K509I mutation could benefit from treatment with Imatinib, Dasatinib and PKC 412. However, Imatinib may be more effective in inducing neoplastic mast cells apoptosis. Both patients described were started on Imatinib in June 2013. Disclosures: No relevant conflicts of interest to declare.

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