Immunophenotype Of Mast Cells Research Articles

Multicolor flow cytometric immunophenotyping is highly sensitive and specific in identifying aberrant mast cells in the diagnostic workup of systemic mastocytosis.

Flow cytometric immunophenotyping (FCI) is a fast and sensitive method for characterizing hematolymphoid neoplasms. It is not widely used in the workup of systemic mastocytosis (SM), in part because of the technical challenges and in part because the utility of FCI in assessing mast cells is not well understood. The objectives of this study were to assess the diagnostic utility of FCI in establishing a diagnosis of SM and distinguishing SM from nonneoplastic mast cells and to examine the immunophenotypic findings among SM subtypes. We performed FCI on bone marrow samples suspicious for SM using a panel consisting of CD2, CD25, CD30, CD45, CD117, and HLA-DR. The cohort included 88 SM cases: 67 without an associated hematologic neoplasm (AHN) (PureSM) and 21 with an AHN (SM-AHN). We also assessed 40 normal/reactive controls. Overall, FCI was adequate for interpretation in 87 of 88 (99%) cases and detected at least 1 immunophenotypic aberrancy in 100% of SM cases. CD2, CD25, and CD30 were positive in 78%, 98%, and 90% of SM cases vs 0%, 13%, and 13% of cases with normal/reactive mast cells (P < .0001 for all). Two or 3 abnormalities were observed in 92% of SM cases but not in normal/reactive mast cells. Among SM cases, SM-AHN showed statistically significant less CD2 (38% vs 91%, P < .0001) and less co-expression of all 3 aberrant markers (CD2, CD25, and CD30 positive in 38% vs 86% of cases; P < .0001) than PureSM. Immunohistochemical analysis showed consistently weaker or focal expression of CD2, CD25, and CD30 than FCI, with CD2 and CD30 being falsely negative in 40% and 50% cases, respectively. A KIT D816V mutation was detected in 67% of PureSM cases and 76% of SM-AHN cases. Flow cytometric immunophenotyping is a quick, sensitive, high-yield tool for evaluating the immunophenotype of mast cells. An abnormal FCI finding should prompt careful histologic evaluation and sensitive KIT D816V mutation testing to address the possibility of SM. CD2, CD25, and CD30 are important markers for the detection of immunophenotypic aberrancy of mast cells, and their frequencies of aberrancy differ across SM subtypes.

Immunohistochemical Demonstration of Mast Cells in Bovine Papillomatous Digital Dermatitis of Holstein-Friesian Dairy Cattle

Mast cells (MC)are unique members of immune system; their location and functions are of great importance in health and disease status. This study aims to evaluate MCs distribution and heterogenity in healthy and digital dermatitis lesions of dairy cattle for the first time. A total of 50 skin samples, 25 healthy and 25 with digital dermatitis (DD) lesions were sampled in Holstein-Friesian dairy cattle. All samples were stained with hematoxylin-eosin (H&amp;E) for microscopic examination and also stained with Toluidine blue for MCs demonstration. Epidermal acanthosis and hyperkeratosis with dermal inflammatory cell infiltrations were in consistent with digital dermatitis lesions. Spirochetal agents were successfully demonstrated with Warthin-Starry staining in 22 out of 25 DD samples. Increased number of mast cell were observed in digital dermatitis samples when compared with healthy skin samples. The average number of intact MCs were 4.6 ± 2 and 9.3 ± 1 in healthy and digital dermatitis samples, respectively. Two-fold increase in the number of intact MCs in digital dermatitis samples was observed. Degranulated mast cell numbers in tissue sections were also higher in digital dermatitis samples and 4.25-fold increase was recorded in affected skin samples. Immunophenotype of MCs in skin samples were identified by immmunohistochemical stainings with anti-tryptase and chymase antibodies. Only tryptase positive MCs were observed in healthy and DD samples. In the statistical analysis, differences in the mean intact and degranulated MCs were found to be significant (mean intact: p&lt;0.05, mean degranulated: p&lt;0.01). Our results possibly suggest that MCs may have important roles in the pathogenesis of bovine digital dermatitis.

P1027: RESPONSES TO AVAPRITINIB IN PATIENTS WITH ADVANCED SYSTEMIC MASTOCYTOSIS: HISTOPATHOLOGIC ANALYSES FROM EXPLORER AND PATHFINDER CLINICAL STUDIES

Background: Systemic mastocytosis (SM) is a mast cell (MC) neoplasm driven by the KIT D816V mutation in ~95% of cases. Diagnosis includes evaluation of MC aggregates in extracutaneous organs, atypical MC morphology, and MC immunophenotype. Avapritinib, a potent and selective KIT D816V inhibitor, has been approved in the US for patients with advanced SM (AdvSM) based on the phase 1 EXPLORER and phase 2 PATHFINDER studies. Aims: Here, we describe a comprehensive evaluation of the effect of avapritinib on bone marrow (BM) pathology in patients treated in these studies. Methods: Patients aged ≥18 years with diagnoses of AdvSM per local investigator were treated with once-daily avapritinib <200 mg (n=17), 200 mg (n=126), and ≥300 mg (n=50). Bone marrow biopsies (BMBs), aspirates (BMAs), and peripheral blood (PB) smears were obtained at screening, and after 8 and 24 weeks of treatment with avapritinib. Evaluations were performed using standard Wright-Giemsa and H&E staining, and IHC was performed on formalin-fixed EDTA-decalcified BMBs using standard techniques for CD25 and CD30. Changes in fibrosis were assessed by the European Consensus on grading of BM fibrosis (MF score). Results: As of April 2021, 193 patients enrolled in EXPLORER (n=86) and PATHFINDER (n=107) were included in analyses with centrally confirmed diagnoses of aggressive SM (n=29), SM with an associated hematologic neoplasm (n=119), mast cell leukemia (n=28), indolent SM (n=14), smoldering SM (n = 2) or CMML (n=1). Median (range) age was 67 years (31–88), 56% were male and 88% had a KIT D816V mutation. Following treatment with avapritinib, a decrease in the proportion of patients with multifocal dense aggregates of MCs in BMBs was observed from 93% at screening to 50% by Week 8 (Table). Avapritinib treatment reduced the proportion of aberrant CD25+ and CD30+ MCs in BMBs by Week 8 and Week 24 (Table). In BMAs, avapritinib reduced the mean MC burden from 11% of total nucleated cells at screening to 2% at Week 8 and by Week 24, with reduced proportions of immature and spindle-shaped MCs (Table). Of 9 patients with circulating MCs at screening and post-screening sample measurements (6 with SM with associated hematologic neoplasm diagnoses and 3 with MCL), 8 had no detectable MCs in PB by Week 8. The proportion of patients with fibrosis in BMBs decreased from 96% (178/186) at screening to 90% (142/158) at Week 8 to 84% (103/123) through week 24, and MF scores were reduced during avapritinib treatment in those patients presenting with increased fibrosis at screening (Table). Image:Summary/Conclusion: Patients treated with avapritinib were observed to have rapid (Week 8) and marked (Week 24) reductions in neoplastic BM MCs with a return to a normal morphologic appearance and immunophenotype. This was accompanied by an improvement of fibrosis and a decrease in circulating MCs. These improvements in disease histopathology provide further support that treatment with this highly selective and potent KIT D816V inhibitor is disease modifying in AdvSM.

Utility of flow cytometry in canine primary cutaneous and matched nodal mast cell tumor

Mast cell tumors (MCT) are among the most frequent tumors in dogs, but studies regarding canine mast cell immunophenotype are lacking. The aim of this study was to assess the feasibility of flow cytometric analysis of MCTs, to describe canine MCTs immunophenotype(s), and to evaluate the ability of flow cytometry to detect mast cells in lymph node aspirates. Thirty-four primary canine MCTs and 12 draining lymph nodes were evaluated regarding the expression of CD117, IgE, CD11b, CD18, CD44, CD34, CD25 and CD45.Distinct populations attributable to mast cells and eosinophils were recognized based on light scatters and CD117 positivity. Common antigens (CD18, CD45, CD44) and CD117 were detected in all cases; positivity for IgE and CD11b was found in 28 (82%) and 23 (68%) cases respectively, while CD34 and CD25 were occasionally expressed. A single multicolor tube (IgE/CD117/CD11b/CD21/CD5) allowed the identification of mast cells in lymph nodes, showing a high correlation with cytology in quantifying mast cells infiltration. In conclusion, flow cytometric analysis can be applied to characterize canine MCTs and can be used to detect the presence of mast cells in lymph nodes. The immunophenotype abnormalities observed may be useful to confirm the neoplastic nature of such mast cells but the diagnostic usefulness of atypical antigen expression remains to be clarified.

The immunophenotype of mast cells and its utility in the diagnostic work-up of systemic mastocytosis.

SM comprises a heterogeneous group of disorders, characterized by an abnormal accumulation of clonal MCs in 1 or more tissues, frequently involving the skin and BM. Despite the fact that most adult patients (>90%) carry the same genetic lesion (D816V KIT mutation), the disease presents with multiple variants with very distinct clinical and biologic features, a diverse prognosis, and different therapeutic requirements. Recent advances in the standardization of the study of BM MC by MFC allowed reproducible identification and characterization of normal/reactive MCs and their precursors, as well as the establishment of the normal MC maturational profiles. Analysis of large groups of patients versus normal/reactive samples has highlighted the existence of aberrant MC phenotypes in SM, which are essential for the diagnosis of the disease. In turn, 3 clearly distinct and altered maturation-associated immunophenotypic profiles have been reported recently in SM, which provide criteria for the distinction between ISM patients with MC-restricted and multilineage KIT mutation; thus, immunphenotyping also contributes to prognostic stratification of ISM, particularly when analysis of the KIT mutation on highly purified BM cells is not routinely available in the diagnostic work-up of the disease.

Anaphylaxis as a clinical manifestation of clonal mast cell disorders.

Clonal mast cell disorders comprise a heterogeneous group of disorders characterized by the presence of gain of function KIT mutations and a constitutively altered activation-associated mast cell immunophenotype frequently associated with clinical manifestations related to the release of mast cells mediators. These disorders do not always fulfil the World Health Organization (WHO)-proposed criteria for mastocytosis, particularly when low-sensitive diagnostic approaches are performed. Anaphylaxis is a frequent presentation of clonal mast cell disorders, particularly in mastocytosis patients without typical skin lesions. The presence of cardiovascular symptoms, e.g., hypotension, occurring after a hymenoptera sting or spontaneously in the absence of cutaneous manifestations such as urticaria is characteristic and differs from the presentation of anaphylaxis in the general population without mastocytosis.

Behavior of the P1.HTR mastocytoma cell line implanted in the chorioallantoic membrane of chick embryos

The P1.HTR cell line includes highly transfectable cells derived from P815 mastocytoma cells originating from mouse breast tissue. Despite its widespread use in immunogenic studies, no data are available about the behavior of P1.HTR cells in the chick embryo chorioallantoic membrane model. The objective of the present investigation was to study the effects of P1.HTR cells implanted on the chorioallantoic membrane of chick embryos. We inoculated P1.HTR cells into the previously prepared chick embryo chorioallantoic membrane and observed the early and late effects of these cells by stereomicroscopy, histochemistry and immunohistochemistry. A highly angiotropic and angiogenic effect occurred early after inoculation and a tumorigenic potential with the development of mastocytoma keeping well mast cells immunophenotype was detected later during the development. The P1.HTR mastocytoma cell line is a good tool for the development of the chick embryo chorioallantoic membrane mastocytoma model and also for other studies concerning the involvement of blood vessels. The chick embryo chorioallantoic membrane model of mastocytoma retains the mast cell immunophenotype under experimental conditions and could be used as an experimental tool for in vivo preliminary testing of antitumor and antivascular drugs.

Behavior of the P1.HTR mastocytoma cell line implanted in the chorioallantoic membrane of chick embryos

The P1.HTR cell line includes highly transfectable cells derived from P815 mastocytoma cells originating from mouse breast tissue. Despite its widespread use in immunogenic studies, no data are available about the behavior of P1.HTR cells in the chick embryo chorioallantoic membrane model. The objective of the present investigation was to study the effects of P1.HTR cells implanted on the chorioallantoic membrane of chick embryos. We inoculated P1.HTR cells into the previously prepared chick embryo chorioallantoic membrane and observed the early and late effects of these cells by stereomicroscopy, histochemistry and immunohistochemistry. A highly angiotropic and angiogenic effect occurred early after inoculation and a tumorigenic potential with the development of mastocytoma keeping well mast cells immunophenotype was detected later during the development. The P1.HTR mastocytoma cell line is a good tool for the development of the chick embryo chorioallantoic membrane mastocytoma model and also for other studies concerning the involvement of blood vessels. The chick embryo chorioallantoic membrane model of mastocytoma retains the mast cell immunophenotype under experimental conditions and could be used as an experimental tool for in vivo preliminary testing of antitumor and antivascular drugs.

Current state of biology and diagnosis of clonal mast cell diseases in adults

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.

An immature immunophenotype of bone marrow mast cells predicts for multilineage D816V KIT mutation in systemic mastocytosis

D816V KIT mutation of bone marrow (BM) mast cells (MC) is a common feature to systemic mastocytosis (SM) patients. Nevertheless, occurrence of the KIT mutation in BM cell compartments other than MC is associated with progression to more aggressive forms of the disease and poor outcome in indolent SM (ISM). Here, we assessed the potential association between the immunophenotype of MC and multilineage KIT mutation in the BM of SM patients through the investigation of the flow cytometric protein expression profile (PEP) of bone marrow mast cells (BMMC) from 70 control individuals and 206 SM patients, classified according to the WHO (World Health Organization), and the degree of involvement of BM hematopoiesis by the D816V KIT mutation; additionally, we developed a score-based class prediction algorithm for the detection of SM cases with multilineage mutation. Our results show that aberrant expression of CD25 with a FcɛRI(lo), FSC(lo), SSC(lo) and CD45(lo) immature phenotype of BMMC, in the absence of coexisting normal MC in the BM, was associated with multilineage involvement by the D816V KIT mutation, regardless of the diagnostic subtype of the disease (for example, indolent vs aggressive SM), which supports the utility of the immunophenotype of BMMC as a surrogate marker to screen for multilineage KIT mutation in ISM.

Flow cytometry for systemic mastocytosis

Mastocytosis is a heterogeneous group of disorders characterised by abnormal proliferation of mast cells. In most cases of adult mastocytosis it is possible to demonstrate evidence of clonal proliferation. As is the case with other clonal disorders of haematopoietic lineage, flow cytometry has become a major diagnostic modality. Flow cytometry is particularly useful in the diagnosis of systemic mastocytosis, where the mast cells may still occur at a very low frequency in the bone marrow, despite neoplastic growth. Numerous studies have compared immunophenotype of mast cells in mastocytosis and those in normal and diseased controls. Some aberrantly expressed antigens on mast cells are of such high specificity for systemic mastocytosis that their expression on mast cells has become a minor World Health Organisation (WHO) criterion for diagnosis (CD2, CD25). This presentation will discuss the phenotype of normal mast cell and compare with abnormal immunophenotype – both in the context of the WHO diagnostic criteria as well as other diagnostically useful antigens that are not part of the criteria.

Imatinib Is Not a Potential Alternative Treatment for Uterine Leiomyosarcoma

To the Editor: We read with great interest the article written by Raspollini et al. ([1][1]). The authors studied a series including 32 cases of uterine leiomyosarcomas, which showed a 50% rate of positive immunostaining for KIT. Based on these results,the authors suggested the possibility of

Aleukemic Mast Cell Leukemia with Abnormal Immunophenotype and C-kit Mutation D816V

Mastocytosis comprises a heterogeneous group of disorders characterized by proliferation and accumulation of mast cells in 1 or more organ systems. Mast cell leukemia (MCL) is an extremely rare subtype of mastocytosis in which a leukemic spread of mast cells and a rapid progression of disease is seen. In typical cases, mast cells are found in the peripheral blood. However, an aleukemic variant of MCL (formerly termed malignant mastocytosis) has also been described. We here report a case of aleukemic MCL with abnormal immunophenotype of mast cells and the classical c-kit point mutation Asp-816-Val ( = D816V). The 75-year-old male patient had a short history of weight loss and lymphadenopathy. There were no urticaria pigmentosa-like skin lesions. The bone marrow was diffusely infiltrated with atypical mast cells that comprised more than 80% of all nucleated cells on a bone marrow smears. As assessed by immunohistochemistry, neoplastic mast cells expressed tryptase, chymase, CD2, CD25, CD68, and the KIT protein (CD117). Mutation analysis revealed the c-kit mutation D816V. Since circulating mast cells could not be detected in the peripheral blood, the diagnosis of aleukemic MCL was established in accordance to the updated WHO consensus classification. This case further supports the notion that the pathogenesis (c-kit mutation D816V) in MCL is closely related to that found in indolent mast cell disorders. However, additional (but yet unknown) molecular (genetic) defects have to be considered to explain the extremely heterogenous clinical course in these patients.

Bone marrow mast cell immunophenotyping in adults with mast cell disease: a prospective study of 33 patients

The aberrant co-expression of CD2 and CD25 antigens is the immunophenotypic hallmark of neoplastic mast cells, and has been consistently identified on bone marrow mast cells from patients with indolent mast cell disease (MCD). We prospectively analyzed the bone marrow mast cell immunophenotype by multiparametric flow cytometry (FC) for 33 MCD cases, to examine the role of CD2 and CD25 expression in establishing diagnosis, detecting histologically occult bone marrow mast cell infiltration, and assessing treatment response. While CD25 was almost uniformly expressed, only 6 of 13 patients with indolent MCD, 1 of 8 with aggressive MCD, 2 of 7 with MCD and an associated hematological disorder, and none of the 2 patients with either mast cell leukemia or smoldering systemic mastocytosis, expressed CD2. One of three patients with cutaneous mastocytosis had an aberrant CD2+/CD25+ mast cell population suggesting histologically occult bone marrow involvement. CD25 expression was lost in one patient who achieved complete histologic remission with therapy, but not in two patients who achieved a partial remission. In conclusion, CD25, but not CD2, is a reliable marker for neoplastic mast cells, and CD25 expression indicates histologically occult bone marrow infiltration and residual disease after therapy.

Evolution of Urticaria Pigmentosa into Indolent Systemic Mastocytosis: Abnormal Immunophenotype of Mast Cells without Evidence of c-kit Mutation ASP-816-VAL

Mastocytosis comprises a heterogeneous group of hematological disorders which are morphologically defined by proliferation and accumulation of tissue mast cells in one or more organs. Clinical manifestations of mastocytosis range from disseminated maculopapular skin lesions (=urticaria pigmentosa [UP]) that may spontaneously regress to highly aggressive neoplasms like mast cell leukemia or mast cell sarcoma. Recently, it could be shown that systemic mastocytosis (SM) is a clonal disorder often exhibiting mutations of c-kit, a protooncogene encoding the tyrosine kinase receptor for stem cell factor (SCF). Mutations of c-kit are considered to play a key role in the pathogenesis of mastocytosis. Therefore, we investigated the unique case of a 36 year-old male patient with indolent systemic mastocytosis (ISM) evolving from UP (cutaneous mastocytosis) by means of histology, immunophenotyping and molecular biology. At the time of initial diagnosis the bone marrow showed only a mild diffuse increase in mast cells but compact infiltrates were missing. The serum tryptase levels were normal. Five years later, however, the bone marrow histology displayed patchy compact mast cell infiltrates, which now allowed to establish the diagnosis of an ISM. The serum tryptase levels at this time were markedly elevated. At both time points, mast cells were analyzed by immunohistochemistry using anti-tryptase antibody AA1, by flow cytometry using antibodies against CD2 and CD25, and nested polymerase chain reaction (PCR) on laser-microdissected, single pooled mast cells. Immunohistochemistry revealed strong tryptase-positivity of mast cells in both cutaneous and bone marrow infiltrates. Flow cytometry yielded an aberrant expression of CD2 and CD25 on bone marrow mast cells. However, repeated thorough PCR analysis failed to unveil c-kit mutation in atypical mast cells of skin and bone marrow samples of both dates. These findings clearly show that ISM can evolve from UP. Moreover, our study provides further evidence that the c-kit mutation Asp-816-Val is not invariably present in ISM.