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Supplemental Table S2

Mastocytosis is a term used to denote a heterogeneous group of conditions defined by the expansion and accumulation of clonal (neoplastic) tissue mast cells in various organs. The classification of the World Health Organization (WHO) divides the disease into cutaneous mastocytosis, systemic mastocytosis, and localized mast cell tumors. On the basis of histomorphologic criteria, clinical parameters, and organ involvement, systemic mastocytosis is further divided into indolent systemic mastocytosis and advanced systemic mastocytosis variants, including aggressive systemic mastocytosis and mast cell leukemia. The clinical impact and prognostic value of this classification has been confirmed in numerous studies, and its basic concept remains valid. However, refinements have recently been proposed by the consensus group, the WHO, and the European Competence Network on Mastocytosis. In addition, new treatment options are available for patients with advanced systemic mastocytosis, including allogeneic hematopoietic stem cell transplantation and multikinase inhibitors directed against KIT D816V and other key signaling molecules. Our current article provides an overview of recent advances in the field of mastocytosis, with emphasis on classification, prognostication, and emerging new treatment options in advanced systemic mastocytosis. Cancer Res; 77(6); 1261–70. ©2017 AACR.

Providers caring for patients with mastocytosis are tasked with the decision to consider therapeutic options. This can come with some trepidation because information available in the public domain lists numerous mast cell (MC) activators based on data that do not discriminate between primates, rodents, and MC lines; do not consider dosage; and do not take into account previous exposure and resultant clinical findings. This being said, there is support in the literature for an enhanced MC response in some patients with mastocytosis and in cases in which there is a greater incidence of adverse reactions associated with certain antigens, such as venoms and drugs. Thus this report provides a comprehensive guide for those providers who must decide on therapeutic options in the management of patients with clonal MC disease.

// Ivan Alvarez-Twose 1, 5 , Almudena Matito 1, 5 , Jose Mario Morgado 1, 5 , Laura Sanchez-Munoz 1, 5 , Maria Jara-Acevedo 2, 5 , Andres Garcia-Montero 2, 5 , Andrea Mayado 2, 5 , Carolina Caldas 2, 5 , Cristina Teodosio 3 , Javier Ignacio Munoz-Gonzalez 2, 5 , Manuela Mollejo 4, 5 , Luis Escribano 2, 5 and Alberto Orfao 2, 5 1 Instituto de Estudios de Mastocitosis de Castilla La Mancha (CLMast), Hospital Virgen del Valle, Toledo, Spain 2 Centro de Investigacion del Cancer/IBMCC (USAL/CSIC) and IBSAL, Departamento de Medicina and Servicio General de Citometria, University of Salamanca, Salamanca, Spain 3 Department of Immunology, Erasmus Medical Center, University of Rotterdam, Rotterdam, The Netherlands 4 Department of Pathology, Hospital Virgen de la Salud, Toledo, Spain 5 Spanish Network on Mastocytosis (REMA), Toledo and Salamanca, Spain Correspondence to: Ivan Alvarez-Twose, email: ivana@sescam.jccm.es Keywords: mast cell, mastocytosis, well-differentiated systemic mastocytosis, imatinib, KIT Received: March 01, 2016 Accepted: May 29, 2016 Published: July 19, 2016 ABSTRACT Resistance to imatinib has been recurrently reported in systemic mastocytosis (SM) carrying exon 17 KIT mutations. We evaluated the efficacy and safety of imatinib therapy in 10 adult SM patients lacking exon 17 KIT mutations, 9 of which fulfilled criteria for well-differentiated SM (WDSM). The World Health Organization 2008 disease categories among WDSM patients were mast cell (MC) leukemia ( n = 3), indolent SM ( n = 3) and cutaneous mastocytosis ( n = 3); the remainder case had SM associated with a clonal haematological non-MC disease. Patients were given imatinib for 12 months –400 or 300 mg daily depending on the presence vs. absence of > 30% bone marrow (BM) MCs and/or signs of advanced disease–. Absence of exon 17 KIT mutations was confirmed in highly-purified BM MCs by peptide nucleic acid-mediated PCR, while mutations involving other exons were investigated by direct sequencing of purified BM MC DNA. Complete response (CR) was defined as resolution of BM MC infiltration, skin lesions, organomegalies and MC-mediator release-associated symptoms, plus normalization of serum tryptase. Criteria for partial response (PR) included ≥ 50% reduction in BM MC infiltration and improvement of skin lesions and/or organomegalies. Treatment was well-tolerated with an overall response rate of 50%, including early and sustained CR in four patients, three of whom had extracellular mutations of KIT , and PR in one case. This later patient and all non-responders ( n = 5) showed wild-type KIT . These results together with previous data from the literature support the relevance of the KIT mutational status in selecting SM patients who are candidates for imatinib therapy.

This work was supported by grants from the Fundacion Sociedad Espanola Alergologia e Inmunologia Clinica (SEAIC) 2014.

© 2019 by the authors., Despite recent therapeutic advances, systemic mastocytosis (SM) remains an incurable disease due to limited complete remission (CR) rates even after novel therapies. To date, no study has evaluated the expression on SM bone marrow mast cells (BMMC) of large panel of cell surface suitable for antibody-targeted therapy. In this study, we analyzed the expression profile of six cell-surface proteins for which antibody-based therapies are available, on BMMC from 166 SM patients vs. 40 controls. Overall, variable patterns of expression for the markers evaluated were observed among SM BMMC. Thus, CD22, CD30, and CD123, while expressed on BMMC from patients within every subtype of SM, showed highly variable patterns with a significant fraction of negative cases among advanced SM (aggressive SM (ASM), ASM with an associated clonal non-MC lineage disease (ASM-AHN) and MC leukemia (MCL)), 36%, 46%, and 39%, respectively. In turn, CD25 and FcεRI were found to be expressed in most cases (89% and 92%) in virtually all BMMC (median: 92% and 95%) from both indolent and advanced SM, but with lower/absent levels in a significant fraction of MC leukemia (MCL) and both in MCL and well-differentiated SM (WDSM) patients, respectively. In contrast, CD33 was the only marker expressed on all BMMC from every SM patient. Thus, CD33 emerges as the best potentially targetable cell-surface membrane marker in SM, particularly in advanced SM., This work was supported by the Fondo de Investigaciones Sanitarias (FIS) of the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad, Madrid, Spain; grant number PI16/00642, FEDER), the Consejería de Educación (Regional Government of Castilla and León, Spain, grant number SA013U16), Biomedical Research Networking Center on Cancer (CIBERONC CB16/12/00400) of the Instituto de Salud Carlos III (Madrid, Spain) and grants from the Asociación Española de Mastocitosis y Enfermedades Relacionadas (AEDM 2017), Fondos de Investigación para Enfermedades Raras del Ministerio de Sanidad, Servicios Sociales e Igualdad, Ensayo clínico independiente del Ministerio de Salud y Política Social (EC11-287), Hospital Virgen de la Salud Biobank (BioB-HVS, grant PT13/0010/0007; Toledo, Spain). N D-F was supported by a grant from the Consejería de Educación (Junta de Castilla and León, Valladolid, Spain, ORDEN EDU/346/2013). A M was supported by a contract grant from the Biomedical Research Networking Center on Cancer (CIBERONC CB16/12/00400) (contract profile: 170504_CIBERONC_ORFAO_ONCG03/2017) of the Instituto de Salud Carlos III (Madrid, Spain), and JI M-G was supported by a grant from the Consejería de Educación (Junta de Castilla and León, Valladolid, Spain; ORDEN EDU/310/2015).

[Background]: Recent studies show that most systemic mastocytosis (SM) patients, including indolent SM (ISM) with (ISMs+) and without skin lesions (ISMs−), carry the KIT D816V mutation in PB leukocytes. We investigated the potential association between the degree of involvement of BM hematopoiesis by the KIT D816V mutation and the distribution of different maturation-associated compartments of bone marrow (BM) and peripheral blood (PB) CD34+ hematopoietic precursors (HPC) in ISM and identified the specific PB cell compartments that carry this mutation. [Methods]: The distribution of different maturation-associated subsets of BM and PB CD34+ HPC from 64 newly diagnosed (KIT-mutated) ISM patients and 14 healthy controls was analyzed by flow cytometry. In 18 patients, distinct FACS-purified PB cell compartments were also investigated for the KIT mutation. [Results]: ISM patients showed higher percentages of both BM and PB MC-committed CD34+ HPC vs controls, particularly among ISM cases with MC-restricted KIT mutation (ISMMC); this was associated with progressive blockade of maturation of CD34+ HPC to the neutrophil lineage from ISMMC to multilineage KIT-mutated cases (ISMML). Regarding the frequency of KIT-mutated cases and cell populations in PB, variable patterns were observed, the percentage of KIT-mutated PB CD34+ HPC, eosinophils, neutrophils, monocytes and T cells increasing from ISMs−MC and ISMs+MC to ISMML patients. [Conclusion]: The presence of the KIT D816V mutation in PB of ISM patients is associated with (early) involvement of circulating CD34+ HPC and multiple myeloid cell subpopulations, KIT-mutated PB CD34+ HPC potentially contributing to early dissemination of the disease., This work was supported by Fondo de Investigaciones Sanitarias—FIS—of the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain (grant numbers PI11/02399 and PI16/00642, FEDER); Consejería de Educación (Regional Government of Castilla y León, Spain; grant number SA013U16); Biomedical Research Networking Centre Consortium–CIBER-CIBERONC (CB16/12/00400) of the Instituto de Salud Carlos III, Madrid, Spain; and Fundacion Ramon Areces, Madrid, Spain (grant CIVP16A1806). AM was supported by a RTICC (Red Tematica de Investigacion Cooperativa en Cancer) grant (RD12/0036/0048, FIS, FEDER).

Multilineage involvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here, we investigated the potential involvement of BM mesenchymal stem cells (MSCs) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome. This mutation was investigated in highly purified BM MSCs and other BM cell populations from 83 ISM patients followed for a median of 116 months. KIT D816V-mutated MSCs were detected in 22 of 83 cases. All MSC-mutated patients had multilineage KIT mutation (100% vs 30%, P = .0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P5.03) and a polyclonal pattern of inactivation of the X-chromosome of KIT-mutated BM mast cells (64% vs 0%; P = .01) vs other multilineage ISM cases. Moreover, presence of KIT-mutated MSCs was associated with more advanced disease features, a greater rate of disease progression (50% vs 17%; P = .04), and a shorter progression-free survival (P

PMCID: PMC4522520.-- et al., Although acquired mutations in KIT are commonly detected in various categories of mastocytosis, the methodologies applied to detect and quantify the mutant type and allele burden in various cells and tissues are poorly defined. We here propose a consensus on methodologies used to detect KIT mutations in patients with mastocytosis at diagnosis and during follow-up with sufficient precision and sensitivity in daily practice. In addition, we provide recommendations for sampling and storage of diagnostic material as well as a robust diagnostic algorithm. Using highly sensitive assays, KIT D816V can be detected in peripheral blood leukocytes from most patients with systemic mastocytosis (SM) that is a major step forward in screening and SM diagnosis. In addition, the KIT D816V allele burden can be followed quantitatively during the natural course or during therapy. Our recommendations should greatly facilitate diagnostic and follow-up investigations in SM in daily practice as well as in clinical trials. In addition, the new tools and algorithms proposed should lead to a more effective screen, early diagnosis of SM and help to avoid unnecessary referrals., M. Arock is supported by Fondation de France; P. Dubreuil is supported by La Ligue Nationale Contre le Cancer (équipe labellisée) and INCa; A. Garcia-Montero and A. Orfao are Supported by grants from the Instituto de Salud Carlos III, Ministry of Economy and Competitivity, Madrid, Spain (grant numbers RD12/0036/0048 and PI11/02399, FEDER) and from Fundacion Ramon Areces, Madrid, Spain (grant number CIVP16A1806); D.D. Metcalfe is supported in part by the Division of Intramural Research, NIAID; P. Valent is supported by Austrian Science Funds (FWF) Project SFB F4611 and SFB F4704-B20.

Systemic mastocytosis (SM) is a highly heterogeneous disease with indolent and aggressive forms, with the mechanisms leading to malignant transformation still remaining to be elucidated. Here, we investigated the presence and frequency of genetic variants in 34 SM patients with multilineal KIT D816V mutations. Initial screening was performed by targeted sequencing of 410 genes in DNA extracted from purified bone marrow cells and hair from 12 patients with nonadvanced SM and 8 patients with advanced SM, followed by whole-genome sequencing (WGS) in 4 cases. Somatic mutations were further investigated in another 14 patients with advanced SM. Despite the fact that no common mutation other than KIT D816V was found in WGS analyses, targeted next-generation sequencing identified 67 nonsynonymous genetic variants involving 39 genes. Half of the mutations were somatic (mostly multilineal), whereas the other half were germline variants. The presence of ≥1 multilineal somatic mutation involving genes other than KIT D816V, ≥3 germline variants, and ≥1 multilineal mutation in the SRSF2, ASXL1, RUNX1, and/or EZH2 genes (S/A/R/E genes), in addition to skin lesions, splenomegaly, thrombocytopenia, low hemoglobin levels, and increased alkaline phosphatase and β2-microglobulin serum levels, were associated with a poorer patient outcome. However, the presence of ≥1 multilineal mutation, particularly involving S/A/R/E genes, was the only independent predictor for progression-free survival and overall survival in our cohort.

Recent studies have found the KIT D816V mutation in peripheral blood of virtually all adult systemic mastocytosis patients once highly sensitive PCR techniques were used; thus, detection of the KIT D816V mutation in peripheral blood has been proposed to be included in the diagnostic work-up of systemic mastocytosis algorithms. However, the precise frequency of the mutation, the biological significance of peripheral blood-mutated cells and their potential association with involvement of bone marrow hematopoietic cells other than mast cells still remain to be investigated. Here, we determined the frequency of peripheral blood involvement by the KIT D816V mutation, as assessed by two highly sensitive PCR methods, and investigated its relationship with multilineage involvement of bone marrow hematopoiesis. Overall, our results confirmed the presence of the KIT D816V mutation in peripheral blood of most systemic mastocytosis cases (161/190; 85%)-with an increasing frequency from indolent systemic mastocytosis without skin lesions (29/44; 66%) to indolent systemic mastocytosis with skin involvement (124/135; 92%), and more aggressive disease subtypes (11/11; 100%)-as assessed by the allele-specific oligonucleotide-qPCR method, which was more sensitive (P, This work was supported in part by grants from the Fondo de Investigaciones Sanitarias (FIS; grant number PI11/02399, FEDER) and Red Temática de Investigación Cooperativa en Cancer (RTICC; grant number RD12/0036/0048, FEDER) of the Instituto deSalud Carlos III (Ministry of Economy and Competitivity, Madrid, Spain), from Fundacion Ramon Areces (Madrid, Spain; grant number CIVP16A1806) and from Ayudas a Proyectos de Investigación en Salud de la Fundación Mutua Madrileña 2014 and Asociación Española de Enfermos de Mastocitosis (AEDM 2014). The National DNA Bank is supported by grants from the Instituto de Salud Carlos III of the Ministerio de Economia y Competitividad of Spain (grand numbers PT13/0001/0037 and PT13/0010/ 0067, FEDER). AM was supported by RTICC.

[Background]: The role of anesthesia as an elicitor of mast cell (MC) mediator release symptoms in mastocytosis is poorly investigated. [Objective]: To determine the frequency and type of MC mediator release symptoms during anesthetic procedures in mastocytosis patients. [Methods]: Medical records were reviewed regarding the anesthetic techniques for 501 mastocytosis patients (459 adults and 42 children; 95 and 5% with systemic involvement, respectively) who were subjected to 676 and 50 anesthetic techniques, respectively. General, sedation, epidural, and local anesthetic techniques were used in 66 (10%), 67 (10%), 76 (11%), and 515 (76%) adult patients and in 24 (48%), 8 (16%), 2 (4%), and 25 (50%) pediatric patients. [Results]: The frequency of perioperative MC mediator-related symptoms and anaphylaxis was 2 and 0.4% in the adult series and 4 and 2% among children. In the adult series, this frequency was significantly higher in patients who previously presented with anaphylaxis (p = 0.03), underwent major surgeries (p < 0.001) and general anesthesia (p = 0.02), and were not given prophylactic antimediator therapy (PAT) 1 h before the anesthesia (H1/H2 antihistamines and benzodiacepines; p = 0.002).Hypersensitivity and/or allergy to the involved drugs and latex allergy were ruled out in all but one symptomatic case; when PAT was given and sedation was added, some cases later tolerated the same anesthetic drugs. [Conclusion]: The frequency of perioperative anaphylaxis appears to be higher in mastocytosis patients than in the general population. Mastocytosis should not be a contraindication for anesthesia since PAT and adequate anesthetic management using the drugs with the safest profile appears to be effective in preventing/controlling MC mediator-associated symptoms.

Mastocytosis is an emerging differential diagnosis in patients with more or less specific mediator-related symptoms. In some of these patients, typical skin lesions are found and the diagnosis of mastocytosis can be established. In other cases, however, skin lesions are absent, which represents a diagnostic challenge. In the light of this unmet need, we developed a diagnostic algorithm for patients with suspected mastocytosis. In adult patients with typical lesions of mastocytosis in the skin, a bone marrow (BM) biopsy should be considered, regardless of the basal serum tryptase concentration. In adults without skin lesions who suffer from mediator-related or other typical symptoms, the basal tryptase level is an important parameter. In those with a slightly increased tryptase level, additional investigations, including a sensitive KIT mutation analysis of blood leucocytes or measurement of urinary histamine metabolites, may be helpful. In adult patients in whom (i) KIT D816V is detected and/or (ii) the basal serum tryptase level is clearly increased (>25-30 ng/ ml) and/or (iii) other clinical or laboratory features suggest the presence of 'occult' mastocytosis or another haematologic neoplasm, a BM investigation is recommended. In the absence of KIT D816V and other signs or symptoms of mastocytosis or another haematopoietic disease, no BM investigation is required, but the clinical course and tryptase levels are monitored in the follow-up. In paediatric patients, a BM investigation is usually not required, even if the tryptase level is increased. Although validation is required, it can be expected that the algorithm proposed herein will facilitate the management of patients with suspected mastocytosis and help avoid unnecessary referrals and investigations., This work was supported in part by the Austrian Science Funds (FWF) Project SFB F4611 and SFB F4704-B20 and the Division of Intramural Research, NIAID.

This article presents information for the identification and characterization of mast cells from bone marrow and other tissues using multiparametric flow cytometry. In addition, it provides guidelines for the application of this technique in the subclassification of systemic mastocytosis and assessment of the long-term prognosis of patients individually.

Introduction: Mastocytosis comprises a heterogeneous group of disorders characterized by the presence of abnormal mast cells (MCs) in one or more tissues. The great heterogeneity of mastocytosis is determined by the clinical and biological diversity observed in some cases as reflected, for example, by the existence of cases arising in childhood versus those who have an adult-onset disease, patients with mastocytosis restricted to skin versus those with systemic involvement or cases with a chronic, indolent course versus those who suffer from aggressive or leukemic manifestations. This broad spectrum of the disease translates into different therapeutic options, which should be carefully selected in every case based on several factors, such as the patient's age, the frequency, type and severity of MC mediator release symptoms, the overall MC burden, the presence of poor prognosis markers or the detection of specific molecular targets. Areas covered: This comprehensive review is focused on the different ther...

[Aims]: CD30 expression by bone marrow (BM) mast cells (MC) has been reported recently in systemic mastocytosis (SM) patients. The aim of this study was to investigate the potential diagnostic and prognostic value of CD30 expression in SM as assessed by multiparameter flow cytometry. [Methods and results]: A total of 163 consecutive BM samples corresponding to 142 SM patients and 21 non-mastocytosis cases were studied. CD30 was positive in most SM patients (80%), but in only one non-mastocytosis case (4.8%). When combined with CD25, CD30 contributed to an improved accuracy over that of CD25 alone (98% versus 93%) mainly because most (eight of nine) of the well-differentiated SM (WDSM), who lacked CD25, were CD30+. Similar levels of expression of CD30 were observed among all different subgroups of SM except mast cell leukaemia; among indolent SM (ISM) patients, no significant association was observed between the levels of CD30 expression and other clinical and biological features of the disease. [Conclusions]: The increased expression of CD30 associated with absence of CD25 contributes to the diagnosis of WDSM and its distinction from other subtypes of SM. By contrast, CD30 expression did not contribute either to prognostic stratification of ISM or to the differential diagnosis between ISM and aggressive SM cases., This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS) PI11/02399, PS09/00032 and RETICs RD09/0076/00133, RD09/0076/00074 and RD12/0036/0048 (FEDER) from the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain; Fundacion Sociosanitaria de Castilla-La Mancha (2010/008 y G-2010/C-002); Fundación Espanola de Mastocitosis (FEM 2010); and by a grant from Fundaçao para a Ciência e Tecnologia (FCT) of Portugal (SFRH/BD/22972/2005).

Systemic mastocytosis (SM) is characterized by accumulation of neoplastic mast cells and is classified into indolent and aggressive forms. The latter include aggressive SM (ASM), mast cell leukemia (MCL), and SM associated with a myeloid neoplasm wherein 1 or both disease compartments exhibit advanced features. These variants, henceforth collectively referred to as advanced SM for the purposes of this report, are typically characterized by organ damage and shortened survival duration. In contrast to indolent SM, in which symptoms are usually managed by noncytotoxic antimediator therapy, cytoreduction is usually necessary for disease control in advanced SM. Unfortunately, current drug treatment of these patients rarely results in complete clinical and histopathologic remissions or improved survival time. Previously defined response criteria were adapted to the heterogeneous presentations of advanced SM and the limited effects of available drugs. However, recent advances in understanding the molecular pathogenesis of SM and the corresponding prospect in targeted therapy make it a priority to modify these criteria. Our current study is the product of an international group of experts and summarizes the challenges in accomplishing this task and forwards a new proposal for response criteria, which builds on prior proposals and should facilitate response evaluation in clinical trials.

Mastocytosis is a term used to denote a heterogeneous group of conditions defined by the expansion and accumulation of clonal (neoplastic) tissue mast cells in various organs. The classification of the World Health Organization (WHO) divides the disease into cutaneous mastocytosis, systemic mastocytosis, and localized mast cell tumors. On the basis of histomorphologic criteria, clinical parameters, and organ involvement, systemic mastocytosis is further divided into indolent systemic mastocytosis and advanced systemic mastocytosis variants, including aggressive systemic mastocytosis and mast cell leukemia. The clinical impact and prognostic value of this classification has been confirmed in numerous studies, and its basic concept remains valid. However, refinements have recently been proposed by the consensus group, the WHO, and the European Competence Network on Mastocytosis. In addition, new treatment options are available for patients with advanced systemic mastocytosis, including allogeneic hematopoietic stem cell transplantation and multikinase inhibitors directed against KIT D816V and other key signaling molecules. Our current article provides an overview of recent advances in the field of mastocytosis, with emphasis on classification, prognostication, and emerging new treatment options in advanced SM., This study was supported by the Austrian Science Funds, SFB grant F47-04 (to P.V.), by the German Research Council, grant HA 2393/6-1 (to K.H.) and by the NIAID Division of Intramural Research (to D.M.).

[Background]: Mastocytosis are rare diseases characterized by an accumulation of clonal mast cells (MCs) in one or multiple organs or tissues. Patients with systemic mastocytosis (SM), whose MCs frequently arbor the activating D816V KIT mutation, may have indolent to aggressive diseases, and they may experience MC mediator related symptoms. Indolent SM with recurrent anaphylaxis or vascular collapse in the absence of skin lesions, ISMs(-), is a specific subtype indolent SM (ISM), and this clonal MC activation disorder represents a significant fraction of all MC activation syndromes. The V560G KIT mutation is extremely rare in patients with SM and its biological and prognostic impact remains unknown. [Case presentation]: A 15-year old boy was referred to our hospital because of repeated episodes of flushing, hypotension and syncope since the age of 3-years, preceded by skin lesions compatible with mastocytosis on histopathology that had disappeared in the late-early childhood. Diagnosis of ISM, more precisely the ISMs(-) variant, was confirmed based on the clinical manifestations together with increased baseline serum tryptase levels and the presence of morphologically atypical, mature appearing (CD117+high, FcεRI+) phenotypically aberrant (CD2+, CD25+) MCs, expressing activation-associated markers (CD63, CD69), in the bone marrow. Molecular genetic studies revealed the presence of the KIT V560G mutation in bone marrow MCs, but not in other bone marrow cells, whereas the screening for mutations in codon 816 of KIT was negative. The patient was treated with oral disodium cromoglycate and the disease had a favorable outcome after an eleven-year follow-up period, during which progressively lower serum tryptase levels together with the fully disappearance of all clinical manifestations was observed. [Conclusions]: To the best of our knowledge this first report of a patient with ISM, whose bone marrow MCs carry the KIT V560G activating mutation, manifesting as recurrent spontaneous episodes of flushing and vascular collapse in the absence of skin lesions at the time of diagnosis, in whom disodium cromoglycate had led to long term clinical remission.

Key Clinical Message Patients with advanced variants of Systemic Mastocytosis may develop destructive bone lesions when massive mast cell (MC) infiltrates are present. Finding of large osteolyses in indolent systemic mastocytosis, typically characterized by low MC burden, should prompt investigations for an alternative explanation.

Clonal mast cell activation syndromes and indolent systemic mastocytosis without skin involvement are two emerging entities that sometimes might be clinically difficult to distinguish, and they involve a great challenge for the physician from both a diagnostic and a therapeutic point of view. Furthermore, final diagnosis of both entities requires a bone marrow study; it is recommended that this be done in reference centers. In this article, we address the current consensus and guidelines for the suspicion, diagnosis, classification, treatment, and management of these two entities.

Summary Mastocytosis comprises a heterogeneous group of disorders characterized by the presence of clonal mast cells (MC) in organs such as skin, bone marrow (BM), and gastrointestinal tract, among other tissues. The clonal nature of the disease can be established in most adult patients by the demonstration of activating KIT mutations in their BM MC. When highly sensitive techniques capable of identifying cells present at very low frequencies in a sample are applied, BM MC from virtually all systemic mastocytosis patients display unique immunophenotypical features, particularly the aberrant expression of CD25. By contrast, large, multifocal BM MC aggregates (the only World Health Organization major criterion for systemic mastocytosis) are absent in a significant proportion of patients fulfilling at least three minor criteria for systemic mastocytosis, particularly in subjects studied at early stages of the disease with very low MC burden. Moreover, recent molecular and immunophenotypical investigations of BM MC from patients with indolent systemic mastocytosis have revealed a close association of some biological features (e.g., multilineage involvement of hematopoiesis by the KIT mutation and an immature mast cell immunophenotype) with an increased risk for disease progression. These observations support the fact that, although the current consensus diagnostic criteria for systemic mastocytosis have been a major advance for the diagnosis and classification of the disease, rationale usage of the most sensitive diagnostic techniques available nowadays is needed to improve the diagnosis, refine the classification, and reach objective prognostic stratification of adult mastocytosis.

Cutaneous mastocytosis in children is a generally benign disease that can present at birth and is often associated with mast cell mediator-related symptoms including pruritus, flushing, and abdominal pain with diarrhea. The most common form of presentation is urticaria pigmentosa, also referred to as maculopapular mastocytosis. Flares of lesions are induced by triggers such as physical stimuli, changes in temperature, anxiety, medications, and exercise. The skin lesions are typically present on the extremities. Symptoms respond to topical and systemic anti-mediator therapy including antihistamines and cromolyn sodium. Remission at puberty is seen in a majority of cases. Progression to systemic mastocytosis with involvement of extracutaneous organs is not common. The cause of cutaneous mastocytosis is unknown and familial cases are rare. Mutations of c-kit have been observed in the skin of those affected. The diagnosis is established on clinical grounds and the findings on skin biopsy. Bone marrow studies are recommended if there is suspicion of progression of disease to an adult form, if cytoreductive therapy is contemplated, or if skin lesions remain present and/or tryptase levels remain elevated after puberty. The use of chemotherapy, including kinase inhibitors, is strongly discouraged unless severe hematologic disease is present, since malignant evolution is extremely rare.

[Background]: The impact of pregnancy on mast cell (MC)-related symptoms and newborn outcome in women with mastocytosis is not well described. We report a series of 30 women who had 45 pregnancies. [Methods]: Patients completed a specific questionnaire concerning MC mediator release symptoms graded according to their frequency to detect clinical changes occurring during pregestation and pregnancy as well as postpartum. Information about the medications received during pregnancy and labor and about newborn medical complications was also recorded. [Results]: Worsening of MC-related symptoms during pregnancy was observed in 10 cases (22%); additionally, 1 woman developed skin lesions as a manifestation of indolent systemic mastocytosis (ISM) within the third trimester of pregnancy. Conversely, 15 cases (33%) experienced clinical improvement during pregnancy, with a complete resolution of pregestational symptoms in 7 cases. MC mediator release symptoms intrapartum were observed in 5 cases (11%) without any fatal outcome. Newborn medical complications (e.g. prematurity, low birth weight, and respiratory distress) were detected in 7 infants (16%) who were all successfully managed with conservative measures. One infant developed cutaneous mastocytosis several years after birth. [Conclusions]: Mastocytosis has a heterogeneous clinical behavior during pregnancy: the profile of MC-related symptoms remained unchanged in half of the cases, while in the other half pregnant women experienced either an improvement or an exacerbation of the symptoms, with the manifestation of ISM during pregnancy in 1 case. To prevent potential life-threatening MC-related symptoms, adequate prophylactic antimediator therapy intrapartum should be systematically administered. The absence of both maternal and infant severe complications suggests that patients with nonaggressive categories of mastocytosis should not be advised against pregnancy. Copyright © 2011 S. Karger AG, Basel.

[Background]: Indolent systemic mastocytosis is a group of rare diseases for which reliable predictors of progression and outcome are still lacking. [Objective]: Here we investigate the prognostic impact of the clinical, biological, phenotypic, histopathological, and molecular disease characteristics in adults with indolent systemic mastocytosis, who were followed using conservative therapy. [Methods]: A total of 145 consecutive patients were prospectively followed between January 1983 and July 2008; in addition, from 1967 to 1983, 20 patients were retrospectively studied. [Results]: Multivariate analysis showed that serum β2-microglobulin (P = .003) together with the presence of mast/stem cell growth factor receptor gene (KIT) mutation in mast cells plus myeloid and lymphoid hematopoietic lineages (P = .02) was the best combination of independent parameters for predicting disease progression (cumulative probability of disease progression of 1.7% ± 1.2% at 5-10 years and of 8.4% ± 5.0% at 20-25 years). Regarding overall survival, the best predictive model included age >60 years (P = .005) and development of an associated clonal hematological non-mast cell disorder (P = .03) with a cumulative probability of death of 2.2% ± 1.3% at 5 years and of 11% ± 5.9% at 25 years. [Conclusions]: Indolent systemic mastocytosis in adults has a low disease progression rate, and the great majority of patients have a normal life expectancy, with the presence of KIT mutation in all hematopoietic lineages and increased serum β2-microglobulin the most powerful independent parameters for predicting transformation into a more aggressive form of the disease., Supported by grants from the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (REMA G03/007, FIS050769, FIS060529, FIS061377, and RETICS RD06/0020/0035-FEDER); Comunidad Autónoma de Madrid (GR/SAL/0133/2004), Junta de Castilla y León (Grant SAN196/SA10/07); Junta de Comunidades de Castilla La Mancha (FISCAM 2007/36); and Fundación MMA. A.G.-M. is supported by a grant from FIS/FEDER (CP03/00035) and C.T. from the FCT of Portugal (SFRH/BD/17545/2004).

Transgenic mice expressing mutant human amyloid precursor protein (APP) develop an age-dependent amyloid pathology and memory deficits, but no overt neuronal loss. Here, in mice overexpressing wild-type human APP (hAPP(wt)) we found an early memory impairment, particularly in the water maze and to a lesser extent in the object recognition task, but beta-amyloid peptide (Abeta(42)) was barely detectable in the hippocampus. In these mice, hAPP processing was basically non-amyloidogenic, with high levels of APP carboxy-terminal fragments, C83 and APP intracellular domain. A tau pathology with an early increase in the levels of phosphorylated tau in the hippocampus, a likely consequence of enhanced ERK1/2 activation, was also observed. Furthermore, these mice presented a loss of synapse-associated proteins: PSD95, AMPA and NMDA receptor subunits and phosphorylated CaMKII. Importantly, signs of neurodegeneration were found in the hippocampal CA1 subfield and in the entorhinal cortex that were associated to a marked loss of MAP2 immunoreactivity. Conversely, in mice expressing mutant hAPP, high levels of Abeta(42) were found in the hippocampus, but no signs of neurodegeneration were apparent. The results support the notion of Abeta-independent pathogenic pathways in Alzheimer's disease.

Clinical trials with rosiglitazone, a potent agonist at peroxisome proliferator-activated receptor gamma (PPARgamma) suggest an improvement of cognitive function in Alzheimer's disease (AD) patients. The mechanisms mediating this potential beneficial effect remain to be fully elucidated. In mice overexpressing mutant human amyloid precursor protein (hAPP), a model of AD, we found that memory impairment in the object recognition test was prevented and also reversed by chronic rosiglitazone treatment. Given the possible involvement of glucocorticoid receptors (GR) in the actions of PPARgamma-ligands, we studied the effect of chronic rosiglitazone treatment on GR levels in the hippocampus of hAPP mice. An early down-regulation of GR, not related to elevated plasma corticosterone levels, was found in different hippocampal subfields of the transgenic mice and this decrease was prevented by rosiglitazone. In parallel with behavioural studies, rosiglitazone also normalized GR levels in older animals. This effect may contribute to explain the attenuation of memory decline by PPARgamma activation in an AD mouse model.

Cutaneous lesions in patients with mastocytosis are highly heterogeneous and encompass localized and disseminated forms. Although a classification and criteria for cutaneous mastocytosis (CM) have been proposed, there remains a need to better define subforms of cutaneous manifestations in patients with mastocytosis. To address this unmet need, an international task force involving experts from different organizations (including the European Competence Network on Mastocytosis; the American Academy of Allergy, Asthma & Immunology; and the European Academy of Allergology and Clinical Immunology) met several times between 2010 and 2014 to discuss the classification and criteria for diagnosis of cutaneous manifestations in patients with mastocytosis. This article provides the major outcomes of these meetings and a proposal for a revised definition and criteria. In particular, we recommend that the typical maculopapular cutaneous lesions (urticaria pigmentosa) should be subdivided into 2 variants, namely a monomorphic variant with small maculopapular lesions, which is typically seen in adult patients, and a polymorphic variant with larger lesions of variable size and shape, which is typically seen in pediatric patients. Clinical observations suggest that the monomorphic variant, if it develops in children, often persists into adulthood, whereas the polymorphic variant may resolve around puberty. This delineation might have important prognostic implications, and its implementation in diagnostic algorithms and future mastocytosis classifications is recommended. Refinements are also suggested for the diagnostic criteria of CM, removal of telangiectasia macularis eruptiva perstans from the current classification of CM, and removal of the adjunct solitary from the term solitary mastocytoma., Supported by research grants from the German Research Council (DFG; HA 2393/6-1; CRC/SFB832, project A14; to K.H.); from the Division of Intramural Research, National Institute of Allergy and Infectious Diseases (to D.D.M.); and from the Austrian Science Funds (FWF; projects SFB F4611 and SFB F4707-B20; to P.V.). The Consensus Conference on Mastocytosis in Boston, Massachusetts (October 2012), was supported by the American Academy of Allergy, Asthma & Immunolog. A task force/consensus panel on mastocytosis between 2010 and 2012 was supported by the European Academy of Allergy and Clinical Immunology.

[Background]: Well-differentiated systemic mastocytosis (WDSM) is a rare variant of systemic mastocytosis (SM) characterized by bone marrow (BM) infiltration by mature-appearing mast cells (MCs) often lacking exon 17 KIT mutations. Because of its rarity, the clinical and biological features of WDSM remain poorly defined. [Objective]: We sought to determine the clinical, biological, and molecular features of a cohort of 33 patients with mastocytosis in the skin in association with BM infiltration by well-differentiated MCs and to establish potential diagnostic criteria for WDSM. Methods Thirty-three patients with mastocytosis in the skin plus BM aggregates of round, fully granulated MCs lacking strong CD25 and CD2 expression in association with clonal MC features were studied. [Results]: Our cohort of patients showed female predominance (female/male ratio, 4:1) and childhood onset of the disease (91%) with frequent familial aggregation (39%). Skin involvement was heterogeneous, including maculopapular (82%), nodular (6%), and diffuse cutaneous (12%) mastocytosis. KIT mutations were detected in only 10 (30%) of 33 patients, including the KIT D816V (n = 5), K509I (n = 3), N819Y (n = 1), and I817V (n = 1) mutations. BM MCs displayed a unique immunophenotypic pattern consisting of increased light scatter features, overexpression of cytoplasmic carboxypeptidase, and aberrant expression of CD30, together with absent (79%) or low (21%) positivity for CD25, CD2, or both. Despite only 9 (27%) of 33 patients fulfilling the World Health Organization criteria for SM, our findings allowed us to establish the systemic nature of the disease, which fit with the definition of WDSM. [Conclusions]: WDSM represents a rare clinically and molecularly heterogeneous variant of SM that requires unique diagnostic criteria to avoid a misdiagnosis of cutaneous mastocytosis per current World Health Organization criteria., Supported by grants from Asociación Española de Mastocitosis, Madrid, Spain (grant AEDM 2014); Instituto de Salud Carlos III, FEDER, Ministry of Economy and Competitivity, Madrid, Spain (grant PI11/02399); Fundación Ramón Areces, Madrid, Spain (grant CIVP16A1806); and Novartis Farmacéutica, S.A., Spain. I. Alvarez-Twose has received research support from Novartis Farmacéutica, S.A., Spain. A. García-Montero has received research support from Fundacion Ramon Areces (grant no. CIVP16A1806) and ISCIII Ministerio de Economia y Competitividad (grant no. PI11/02399). A. Orfao has received research support from Fundacion Ramon Areces (grant no. CIVP16A1806).

Mast cell (MC) leukemia (MCL) is a subtype of systemic mastocytosis (SM) defined by the World Health Organization as ⩾ 20% of MCs in the bone marrow (BM) aspirate, with (leukemic variant) or without (aleukemic variant) ⩾ 10% of MCs in peripheral blood (PB). The European/American Consensus Group on Mastocytosis has recently proposed a new subclassification of MCL that distinguishes acute vs chronic MCL based on the presence vs absence of organ damage, respectively.

Systemic mastocytosis (SM) is a heterogeneous disease with altered interleukin (IL)-6 and IL13 plasma levels. However, no study has simultaneously investigated the plasma levels of IL1ß, IL6, IL13, CCL23 and clusterin in SM at diagnosis and correlated them with disease outcome. Here we investigated IL1ß, IL6, IL13, CCL23 and clusterin plasma levels in 75 SM patients--66 indolent SM (ISM) and 9 aggressive SM¿and analyzed their prognostic impact among ISM cases grouped according to the extent of hematopoietic involvement of the bone marrow cells by the KIT D816V mutation. Although increased IL1ß, IL6 and CCL23 levels were detected in SM patients versus healthy controls, only IL6 and CCL23 levels gradually increased with disease severity. Moreover, increased IL6 plasma levels were associated with ISM progression to more aggressive disease, in particular among ISM patients with multilineal KIT mutation (ISM-ML), these patients also showing a higher frequency of organomegalies, versus other ISM-ML patients. Of note, all ISM patients who progressed had increased IL6 plasma levels already at diagnosis. Our results indicate that SM patients display an altered plasma cytokine profile already at diagnosis, increased IL6 plasma levels emerging as an early marker for disease progression among ISM cases, in particular among high-risk ISM patients who carry multilineage KIT mutation.

Mast cells are multipotent effector cells of the immune system. They are able to induce and enhance angiogenesis via multiple pathways. (-)-Epigallocatechin-3-gallate (EGCG), a major component of green tea and a putative chemopreventive agent, was reported to inhibit tumor invasion and angiogenesis, processes that are essential for tumor growth and metastasis. Using the human mast cell line HMC-1 and commercial cDNA macroarrays, we evaluated the effect of EGCG on the expression of angiogenesis-related genes. Our data show that among other effects, EGCG treatment reduces expression of two integrins (α5 and β3) and a chemokine (MCP1), resulting in a lower adhesion of mast cells associated with a decreased potential to produce signals eliciting monocyte recruitment. These effects on gene expression levels are functionally validated by showing inhibitory effects in adhesion, aggregation, migration and recruitment assays. © 2007 Birkhäuser Verlag., This work was supported by PI05–0327, the Spanish Network of Mastocytosis and CIBERER (MSC, Spain), Ramón-Areces Foundation, grants SAF2005–01812 (MEC, Spain), grants CVI-657 and CVI-267 (Andalusian Research Program, PAI, Andalusia, Spain) to, F.S.J. and Fondo de Investigaciones Sanitarias (FIS, MSC, Spain) (06/0529) and Fundación MMA, Spain to, L.E.

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.

The diagnosis of 'rare diseases', such as mastocytosis, remains a challenge. Despite this, the precise benefits of referral of mastocytosis patients to highly specialized reference centres are poorly defined and whether patients should be managed at non-specialized versus reference centres remains a matter of debate. To evaluate the quality and efficiency of diagnostic procedures performed at the reference centres for mastocytosis in Spain (REMA) versus other non-reference centres, we retrospectively analysed a series of 122 patients, for the overall degree of agreement obtained for the World Health Organization (WHO) diagnostic and classification criteria betwen the referring and REMA centres. Our results showed that not all WHO diagnostic criteria were frequently investigated at the referring centres. Among the five WHO diagnostic criteria, the highest degree of agreement was obtained for serum tryptase levels [median 90% (95% confidence interval 84-96%)]; in turn, the overall agreement was significantly lower for the major histopathological criterion [80% (72-89%)], and the other three minor criteria: cytomorphology [68% (56-80%)] immunophenotyping of BM mast cells [75% (62-87%)] and detection of the KIT mutation [34% (8-60%)]. Referral of patients with diagnostic suspicion of mastocytosis to a multidisciplinary reference centre improves diagnostic efficiency and quality., This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS) and RTICC of the Instituto de Salud Carlos III, Ministry of Economy and Competitivity, Madrid, Spain (grants PI11/02399 and RD12/0036/0048, FEDER); Ayudas a Proyectos de Investigacion en Salud de la Fundacion Mutua Madrileña 2014; Ayuda de la Asociacion Española de Enfermos de Mastocitosis (AEDM 2014); and Fundacion Ramon Areces, Madrid, Spain (grant CIVP16A1806). AM was supported by RTICC.

Human mast cells (MCs) are directly derived from human pluripotent CD34+ stem and progenitor hematopoietic cells with stem cell factor being a critical growth factor supporting human MC proliferation, differentiation, and survival. Because of the advantages that flow cytometry offers (it allows rapid, objective, and sensitive multiparameter analysis of high numbers of cells from a sample, with information being provided on the basis of a single cell), it has become the method of choice in the past decade for immunophenotypic identification, enumeration, and characterization of human MCs in bone marrow and other tissue specimens.

The human mast cell line (HMC-1) has been used to study the relationship between intracellular pH and cytosolic calcium (Ca 2+ ) in mast cells. Thapsigargin (TG) caused store-operated Ca 2+ entry, that is enhanced by the PKC activator PMA. NH 4 Cl-induced alkalinization showed an inhibitory effect on TG-sensitive stores depletion (not on TG-insensitive stores), and also on final cytosolic Ca 2+ levels reached in response to both TG and the ionophore ionomycin. Loperamide, a positive modulator of store-operated channels, induced a slight Ca 2+ entry by itself, and also increased TG-induced Ca 2+ entry. This enhancement was not enough to reverse the inhibitory effect of NH 4 Cl-induced alkalinization. When comparing the effect of NH 4 Cl-induced alkalinization on Ca 2+ levels, with those observed using Ca 2+ channel blockers (namely Ni 2+ and SKF-96365), cytosolic profiles for this ion are different, either in modified saline solution or in HCO 3 - -free medium. Thus, it seems unlikely that the inhibitory effect of NH 4 Cl-induced alkalinization on Ca 2+ is taking place by blockage of Ca 2+ entry. Furthermore, inhibition of the plasma membrane Ca 2+ -ATPase (an important mechanism for Ca 2+ efflux) with sodium orthovanadate (SO) matches with the inhibition of the negative effect on Ca 2+ levels elicited by NH 4 Cl. Data indicate that NH 4 Cl-induced alkalinization might be activating Ca 2+ efflux from the cell, by stimulation of the plasma membrane Ca 2+ -ATPase, and also confirm our previous finding that Ca 2+ is a secondary signal to activate HMC-1 cells. © 2006 Wiley-Liss, Inc.

We read with great interest the article by Pozdnyakova et al,1 particularly in dealing with the proposal for a new flow cytometric criterion for bone marrow (BM) involvement in systemic mastocytosis (SM), as defined by the observation of a discrete population of CD117 bright mast cells (MCs). The authors claim that this new criterion could increase the sensitivity of the immunophenotypic criterion proposed by the World Health Organization for the analysis of their BM samples from SM patients, without harming specificity. However, we noted some apparent inconsistencies in the article with regard to the exact numbers of BM samples and SM cases analyzed, while at the same time few negative control samples were studied to definitively prove the specificity of the new proposed criterion. In addition, Pozdnyakova et al1 also include in their article a high percentage (11/39; 28%) of SM cases, which showed a rather uncommon CD2–, CD25– immunophenotype. These 3 points are discussed below in more detail. First, a careful analysis of …

Background Mastocytosis is a term used for a heterogeneous group of disorders characterized by an abnormal proliferation and accumulation of mast cells (MCs) in one or multiple tissues including skin, bone marrow, liver, spleen, and lymph nodes, among others. Methods In recent years, multiparameter flow cytometric studies have shown that pathologic MCs from patients with mastocytosis display unique aberrant immunophenotypic characteristics as compared with normal MCs. Results Among other features, pathologic MCs show aberrant expression of CD25 and CD2 antigens and abnormally high levels of the CD11c and CD35 complement receptors, the CD59 complement regulatory molecule, the CD63 lysosomal membrane antigen, and the CD69 early-activation antigen. In addition, MCs from mastocytosis express abnormally low levels of CD117 and unexpectedly high light scatter and autofluorescence characteristics. Conclusions These aberrant immunophenotypic features are of great relevance for the assessment of tissue involvement in mastocytosis with consequences in the diagnosis, classification, and follow-up of the disease and in its differential diagnosis with other entities. In this paper we provide the reader with information for the objective and reproducible identification of pathologic MCs by using quantitative multiparametric flow cytometry, information for their phenotypic characterization, and the criteria currently used for a correct interpretation of the immunophenotypic results obtained. © 2004 Wiley-Liss, Inc.