Correlation of CD30 Expression on Neoplastic Mast Cells in Systemic Mastocytosis Assessed By Immunohistochemistry Versus Multiparameter Flow Cytometry and Correlation to Clinical Parameters

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1616-1616
Author(s):  
Frauke Bellos ◽  
Karl Sotlar ◽  
Susanne Schnittger ◽  
Claudia Haferlach ◽  
Torsten Haferlach ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mast Cell ◽  
Mast Cells ◽  
Systemic Mastocytosis ◽  
Melting Curve ◽  
Normal Karyotype ◽  
Multiparameter Flow Cytometry ◽  
Employment Equity ◽  
Equity Ownership ◽  
Kit D816v

Abstract Background: Systemic mastocytosis (SM) is rarely diagnosed and presents with highly variable clinical manifestation from taking rather indolent to very aggressive courses, mast cell leukemia being the most aggressive variant. Expression of CD30 (Ki-1 antigen) has been detected on some neoplastic mast cells (MC) by immunohistochemical staining (IHC) in bone marrow (BM) biopsies and presence of CD30 has been reported to be correlated to more aggressive variants of SM. Assessmentof CD30 expression by multiparameter flow cytometry (MFC) might not only contribute to improved diagnostic accuracy but correlation of the hereby detected CD30 expression with clinical SM parameters might also give further insights into disease biology. Aims: Comparison of CD30 expression detected by either MFC or IHC on MC of patients with SM and correlation of results with patient and disease characteristics. Correlation of MFC detected CD30 expression with cytogenetics (CG) determined by chromosome banding analysis and molecular genetics (MG). Methods: For this study, CD30 expression was analyzed in BM samples from 93 patients with SM by MFC using a five color staining assay with monoclonal antibodies against CD30, CD45, CD117, CD2 and CD25. We identified MC based on CD45 positivity and bright expression of CD117. Based on aberrant coexpression of CD2 and/or CD25 neoplastic MC were identified. On those MC, mean and median fluorescence intensities (MFI, medFI) of CD30 were determined and related to CD30 MFI and medFI in lymphocytes to derive CD30 index. Moreover, data on MC infiltration and CD30 expression by IHC was assessed in 22 patients and examination of CG and MG was done in 44 and 80 patients, respectively. KIT D816V mutation was analyzed using melting curve-based DNA mutation analysis applying PNA-mediated PCR clamping according to Sotlar et al. [Am J Pathol. 162: 737-746, 2003]. Results of MFC detected CD30 expression was correlated to those of CD30 expression examined with IHC and to the results of CG and MG. Results: 42 patients were female and 51 male. Median age was 59 years (20-87 years). While we found normal karyotypes in 41 patients, 3 patients showed aberrant karyotypes. KIT D816V mutation was seen in 74/80 patients (93%). Diagnosis of concurrent hematological non-mast cell disease (AHNMD) was made in 14/93 patients (15%). Mean (±SD) MC infiltration was 20%±26% (range, 1.5%-85%) by IHC and 0.4%±1.8% (range, 0.01%-17%) by MFC. Mean (±SD) CD30 index was 19±20 (range, 3-154), mean (±SD) CD30 expression by IHC was 9%±17% (range, 0%-70%). Percentages of MC infiltration detected by IHC and MFC correlated significantly (p=0.002, r=0.819). No correlation of MFC CD30 index (MFI and medFI) with age, sex, concomitant AHNMD, grade of MC infiltration or percentage of CD30 positive MC by IHC was found. Interestingly, a significantly higher medFI CD30 index and a trend to higher MFI CD30 index were seen in patients with normal karyotype (12.2±6.2 vs 6.3±2.7, p=0.037 and 15.1±9.4 vs. 11.6±10.3, n.s., respectively) versus those with an aberrant karyotype. We also detected a trend to higher MFI and medFI CD30 index in patients with KIT D816V mutation (19.8±21.8 vs. 10.2±7.4, n.s. and 24.0±89.0 vs 10.0±5.8, n.s., respectively). Conclusions: Assessment of CD30 expression as a dynamic parameter on neoplastic MC in patients with SM can be reliably performed by MFC. A stronger expression of CD30 expression on neoplastic MC harbouring an aberrant karyotype was found. CD30 expression seems also stronger on MC from patients harbouring KIT D816V mutations compared to those who do not. CD30 expression on neoplastic MC in patients with SM should be further analyzed combining analyses by MFC and IHC to substantiate the present findings. Disclosures Bellos: MLL Munich Leukemia Laboratory: Employment. Sotlar:Ludwig-Maximilians-University: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

A Recombinant Antibody to Siglec-8 Shows Selective ADCC Activity Against Mast Cells from Systemic Mastocytosis Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4092-4092 ◽  
Author(s):  
Rustom Falahati ◽  
Jessica Bright ◽  
Alejandro Dorenbaum ◽  
Christopher Bebbington ◽  
Nenad Tomasevic ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Systemic Mastocytosis ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Adcc Activity ◽  
Employment Equity ◽  
Effector Cells ◽  
Equity Ownership ◽  
Kit D816v

Abstract Background: Systemic mastocytosis (SM) is a rare myeloproliferative neoplasm characterized by the accumulation of neoplastic mast cells (MC) in one or more extracutaneous organs. In all forms of SM, anti-mediator drugs are used to control symptoms of MC degranulation. In advanced forms of SM, organ damage is common and patients (pts) exhibit reduced life expectancy. In these individuals, cytoreductive agents such as cladribine and interferon-alpha have been used off-label, and inhibitors of KIT D816V are under investigation. A significant unmet need exists for these patients. Siglec-8 is an inhibitory receptor of the CD33-related family of sialic acid-binding, Ig-like lectins (Siglecs) that is expressed selectively on the surface of mature MC, eosinophils, and basophils. Engagement of Siglec-8 with monoclonal antibodies has been previously shown to inhibit IgE-mediated MC degranulation and to induce apoptosis of cytokine-activated eosinophils. Thus this receptor is a potential target for antibody therapy of SM with or without associated eosinophilia. Anti-Siglec-8 antibodies do not directly affect MC viability but antibodies with effector function can induce antibody cell-mediated cytotoxicity (ADCC). Here we show that a recombinant anti-Siglec-8 IgG1 monoclonal antibody can elicit ADCC activity against MC derived from SM patients ex vivo. Methods: Bone marrow (BM) aspirates from SM patients were evaluated for Siglec-8 cell-surface expression on CD117+ FcεRI+ MC or CD25+ MC by flow cytometry. For ADCC assays, BM MC enriched using CD117-targeting magnetic beads or a Siglec-8 transfected Ramos cell line were used as target cells. Peripheral blood leukocytes (PBL) or NK cells purified from peripheral blood were used as effector cells at an effector:target ratio of 10:1. Recombinant anti-Siglec-8 antibody or an isotype control antibody was added at various concentrations and the percent viable CD117+ FcεRI+ MC remaining after 48 hours of culture was determined by flow cytometry. Results: Samples from 9 pts with SM were included in the analysis (ISM, n=1; SSM, n=1; SM-CMML, n=3; SM-MDS, n=1; SM-CEL, n=1; ASM, n=1; MCL, n=1). Eight pts were KIT D816V positive. At the time of sample collection, treatments included midostaurin (n=3); cladribine (n=1); corticosteroids (n=1); and 4 pts were not receiving any biologic or cytoreductive therapy. All BM samples showed detectable CD117+ MC. Robust and selective cell-surface expression of Siglec-8 was observed in all 6 cases evaluated and 100% of CD117+ FcεRI+ MC were Siglec-8 positive by FACS, including CD25+ MC. Levels of Siglec-8 were comparable to or higher than levels on mature MC isolated from normal skin. In this limited sample size, no difference in Siglec-8 expression was observed between patients receiving different therapies or no therapy. To evaluate the ADCC activity of recombinant anti-Siglec-8 antibody on MC, enriched BM MC were incubated with anti-Siglec-8 antibody or isotype-matched control antibody at 1 μg/ mL in the presence of purified NK effector cells. In two patients evaluated, significant anti-Siglec-8-mediated ADCC activity on MC was observed using non-autologous NK cells (69% reduction, 1 pt) or autologous NK cells (76% reduction, 1 pt) indicating that anti-Siglec-8 has the potential to reduce MC burden in these patients. ADCC activity has been reported to be defective in some cancer patients. To evaluate the ability of effector cells in SM patients to mediate ADCC, an assay was developed using a Siglec-8 transfected target cell line to screen blood samples for ADCC activity induced by anti-Siglec-8 antibody. Using PBL as effector cells, ADCC was observed in all samples tested (5/5). Titration of antibody was performed on 2 samples and potent ADCC activity was observed in both, with an EC50 for target depletion of 49 and 65 ng/mL of anti-Siglec-8 antibody, respectively. Conclusion: These data provide a strong rationale for evaluating the effect of an antibody to Siglec-8 with ADCC activity in patients with SM. Disclosures Falahati: Allakos, Inc.: Employment, Other: Options for Equity Owernship. Bright:Allakos, Inc.: Employment, Other: Options for Equity Owernship. Dorenbaum:Allakos, Inc.: Employment, Equity Ownership. Bebbington:Allakos, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tomasevic:Allakos, Inc.: Employment, Equity Ownership. George:Allakos: Research Funding; Novartis: Consultancy. Gotlib:Allakos, Inc.: Consultancy.

First Selective KIT D816V Inhibitor for Patients with Systemic Mastocytosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3217-3217 ◽  
Author(s):  
Erica K. Evans ◽  
Brian L. Hodous ◽  
Alexandra Gardino ◽  
Julia Zhu ◽  
Adam Shutes ◽  
...  
Keyword(s):  
Mast Cells ◽  
Tumor Growth ◽  
Growth Inhibition ◽  
Systemic Mastocytosis ◽  
Tumor Growth Inhibition ◽  
Employment Equity ◽  
Equity Ownership ◽  
Kit D816v

Abstract Systemic mastocytosis is a disease characterized by the abnormal proliferation and accumulation of mast cells. In aggressive cases, these mast cells accumulate in organs such as bone marrow, liver and spleen and result in compromised organ function with average patient survival only 3 to 5 years after diagnosis. The mast cells of nearly all systemic mastocytosis patients harbor a heterozygous D816V mutation in the activation loop of KIT conferring constitutive, ligand-independent activation of this receptor tyrosine kinase, suggesting this mutation is a driver of disease. While KIT D816V can be targeted by small molecules such as dasatinib and midostaurin, these agents have activity against many human kinases resulting in dose limiting toxicities in the clinic that prevent complete suppression of KIT D816V activity in vivo. In vitro, their potent activity against multiple kinases leads to uncertainties regarding their mechanism of action. Thus far, selective inhibition of the KIT D816V mutation has not been achieved. However starting with a novel chemical library optimized for kinase selectivity, we have identified BLU-285, a small molecule inhibitor targeting KIT exon 17 mutants including the activated KIT D816V kinase. BLU-285 potently disrupts KIT D816V oncogenic signaling as measured by inhibition of both KIT D816V autophosphorylation and phosphorylation of the downstream substrates Akt and Stat3 in the human mast cell leukemia cell line HMC1.2. In vitro, BLU-285 inhibits proliferation and induces apoptosis in the mouse mastocytoma cell line P815. In vivo, BLU-285 is a well-tolerated, orally bioavailable agent that achieves dose dependent tumor growth inhibition in a P815 mouse xenograft model with tumor regression observed at 30 mg/kg once daily dosing. Tumor growth inhibition correlates with inhibition of KIT autophosphorylation; greater than 80% target suppression throughout the 24-hour dosing period is required for effective tumor growth inhibition. Prolonged target suppression is achievable with BLU-285 but not dasatinib, even when dosed at the MTD in mouse. Furthermore, to more closely mimic the nature of systemic mastocytosis, we have developed a disseminated model of disease whereby the in vivo growth of P815-luciferase expressing cells inoculated intravenously can be measured by whole body bioluminescence. Treatment of mice with systemic disease leads to dose dependent inhibition of disease, with a 3-fold increase in survival time when dosed 30 mg/kg QD. In addition, as anticipated by its selectivity profile, BLU-285 is very well tolerated in vivo with no impact on body weight at efficacious doses. Our data demonstrate that selective inhibition of KIT D816V with BLU-285 achieves complete and prolonged inactivation of the disease-driving kinase and suggests that BLU-285 may provide a compelling new therapy for patients with systemic mastocytosis. Disclosures Evans: Blueprint Medicines: Employment, Equity Ownership. Hodous:Blueprint Medicines: Employment, Equity Ownership. Gardino:Blueprint Medicines: Employment, Equity Ownership. Zhu:Blueprint Medicines: Employment, Equity Ownership. Shutes:Blueprint Medicines: Employment, Equity Ownership. Davis:Blueprint Medicines: Employment, Equity Ownership. Kim:Blueprint Medicines: Employment, Equity Ownership. Wilson:Blueprint Medicines: Employment, Equity Ownership. Wilson:Blueprint Medicines: Employment, Equity Ownership. Zhang:Blueprint Medicines: Employment, Equity Ownership. Kohl:Blueprint Medicines: Employment, Equity Ownership. Guzi:Blueprint Medicines: Employment, Equity Ownership. Lengauer:Blueprint Medicines: Employment, Equity Ownership.

Detection of Activated STAT5 in Neoplastic Mast Cells in Patients with Systemic Mastocytosis: Role of c-kit D816V.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3515-3515 ◽  
Author(s):  
Karoline Sonneck ◽  
Matthias Mayerhofer ◽  
Karoline V. Gleixner ◽  
Marc Kerenyi ◽  
Maria-Theresa Krauth ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Systemic Mastocytosis ◽  
Cell Leukemia ◽  
Oncogenic Signaling ◽  
Kit Mutation ◽  
Knock Out ◽  
Mast Cell Leukemia ◽  
Kit D816v

Abstract Recent data suggest that activated STAT5 contributes to growth and differentiation of mast cells (MC) and that STAT5-knock out mice are MC-deficient. We have recently shown that constitutively activated STAT5 acts as a potent oncogenic signaling molecule in hematopoietic progenitor cells (Cancer Cell2005;7:87–99). In the present study, we examined the expression of activated STAT5 in neoplastic MC in systemic mastocytosis (SM) and asked whether the SM-related oncogene c-kit D816V is involved in STAT5-activation. For the immunohistochemical detection of activated tyrosine phosphorylated STAT5 (P-Y-STAT5), we used the specific monoclonal antibody AX1 (Advantex) which does not react with inactive STAT5. In all patients with SM tested (indolent SM, n=11; smouldering SM, n=2; aggressive SM, n=1; mast cell leukemia, n=1; all exhibiting c-kit D816V), MC were found to display P-Y-STAT5. Expression of activated STAT5 was also demonstrable in the c-kit D816V-positive mast cell leukemia-derived cell line HMC-1. The reactivity of HMC-1 cells with AX1 antibody was abrogated by a STAT5-specific blocking-peptide. To define the role of c-kit D816V in STAT5-activation, Ba/F3 cells with doxycycline-inducible expression of c-kit D816V (Ton.kit) were employed. In these cells, induction of c-kit D816V was followed by a massive increase in phosphorylated STAT5 as determined by a specific DNA-binding assay, whereas the total amounts of STAT5-mRNA and of the STAT5-protein showed only a slight increase or remained unchanged. In summary, these data show that neoplastic MC in SM express activated STAT5 (P-Y-STAT5), and that the transforming c-kit mutation D816V leads to persistent activation of STAT5 in these cells.

Characterization of MYD88 mutated Lymphoplasmacytic Lymphoma in Comparison to MYD88 mutated Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 132-132
Author(s):  
Constance Regina Baer ◽  
Frank Dicker ◽  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
Claudia Haferlach
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Lymphocytic Leukemia ◽  
Bone Marrow Infiltration ◽  
Multiparameter Flow Cytometry ◽  
Employment Equity ◽  
Cytogenetic Aberration ◽  
Highly Sensitive ◽  
Equity Ownership

Abstract Introduction: MYD88 (Myeloid Differentiation Primary Response 88) mutations are the most common genetic aberration in Waldenström's macroglobulinemia/lymphoplasmacytic lymphoma (LPL). Since the initial description of MYD88 mutations in LPL, the detection has gained great importance in diagnosing the disease. However, in some patients with other B cell malignancies, including chronic lymphocytic leukemia (CLL), MYD88 mutations are detectable. Aim: We describe the molecular and cytogenetic profile of MYD88 mutated LPL in comparison to CLL, in order to identify aberration patterns potentially useful for diagnostic purposes. Patients and Methods: We analyzed bone marrow samples of 78 LPL patients for MYD88 by highly sensitive allele specific PCR (ASP) for the L265P mutation and by next-generation sequencing (NGS) for MYD88 and CXCR4 (Chemokine (C-X-C Motif) Receptor 4) mutations. For CLL, 784 blood or bone marrow samples were sequenced for MYD88 (by NGS), IGHV, TP53, NOTCH1 and SF3B1 by Sanger or NGS as well as the MYD88 mutated CLL cases for CXCR4. For all samples, cytogenetic and multiparameter flow cytometry data was available. Results: In LPL, 68/78 patients (87%) harbored a MYD88 mutation. In 13 cases with low bone marrow infiltration (median: 3%; range: 1-6%), the MYD88 mutation was detected by ASP only and not by NGS. However, one case was identified by NGS only because of a non-L265P mutation, which cannot be detected by ASP (1/68; 1%). In contrast, in CLL only 17/784 (2%) carried a MYD88 mutation. Interestingly, 5/17 (29%) were non-L265P mutations. Of the MYD88 mutated LPL, 17/68 (25%) carried a genetic lesion in the C-terminal domain of CXCR4. In contrast to MYD88, the mutation spectrum of CXCR4 was much broader including non-sense mutations at amino acid S338 (10/18) but also frame shifts resulting in loss of regulatory serine residues. One patient had two independent CXCR4 mutations (S338* and S341Pfs*25). The mean bone marrow infiltration by flow cytometry was 14% and 9% in the CXCR4 mutated and unmuted subsets, respectively (p=0.17). Besides molecular genetic aberrations, 25% (17/68) of MYD88 mutated LPL cases carried cytogenetic aberration. The most frequent cytogenetic aberration in the MYD88 positive LPL was the deletion of 6q (10/68; 15%). Other recurrent cytogenetic abnormalities were gains of 4q (n=3), 8q (n=2), and 12q (n=4), as well as loss of 11q (n=4), 13q (n=2) and 17p (n=3). In the MYD88 unmutated group, we did neither identify any CXCR4 mutation nor any del(6q), suggesting different genetic driver events in this LPL subcohort. Importantly, in the MYD88 positive CLL cohort, cytogenetic analysis did not reveal any patient with del(6q). Instead, del(13q)(q14) was the most prevalent cytogenetic aberration (12/17; 71%). Neither 11q deletions nor 17p deletions were detected. All MYD88 positive CLL had a mutated IGHV status (MYD88 unmutated CLL: 453/767; 59%; P<0.001). The TP53, NOTCH1 and SF3B1 mutational landscape did not reveal any differences between the MYD88 mutated and unmutated cohort. Finally, CXCR4 mutations were present in none of 15 analyzed MYD88 mutated CLL cases. Conclusion: Besides multiparameter flow cytometry, MYD88 mutations are the most powerful tool in the diagnosis of LPL. MYD88 mutated LPL are characterized by a high frequency of CXCR4 mutations and del(6q), while MYD88 unmutated LPLs are associated with a different pattern of genetic abnormalities. MYD88 mutated CLL is a distinct CLL subset associated with mutated IGHV status, a high frequency of 13q deletions and low frequencies of 11q and 17p deletions. MYD88 mutated CLL differs from MYD88 mutated LPL with respect to the pattern of MYD88 mutations, cytogenetic aberrations and the absence of CXCR4 mutations. Highly sensitive ASP allows the L265P mutation detection even in LPL cases with very low bone marrow infiltration; whereas highly sensitive NGS assay are best applicable for detection of more heterogenic MYD88 mutations in CLL or CXCR mutations in LPL. Thus, an integrated molecular and cytogenetic approach allows the characterization of disease specific genetic patterns and should be analyzed for its clinical impact. Disclosures Baer: MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Mast cell leukemia

Blood ◽  
2013 ◽  
Vol 121 (8) ◽  
pp. 1285-1295 ◽  
Author(s):  
Sophie Georgin-Lavialle ◽  
Ludovic Lhermitte ◽  
Patrice Dubreuil ◽  
Marie-Olivia Chandesris ◽  
Olivier Hermine ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Cell Activation ◽  
De Novo ◽  
Systemic Mastocytosis ◽  
Mast Cell Activation ◽  
Cell Leukemia ◽  
Mast Cell Leukemia ◽  
Kit D816v

Abstract Mast cell leukemia (MCL) is a very rare form of aggressive systemic mastocytosis accounting for < 1% of all mastocytosis. It may appear de novo or secondary to previous mastocytosis and shares more clinicopathologic aspects with systemic mastocytosis than with acute myeloid leukemia. Symptoms of mast cell activation—involvement of the liver, spleen, peritoneum, bones, and marrow—are frequent. Diagnosis is based on the presence of ≥ 20% atypical mast cells in the marrow or ≥ 10% in the blood; however, an aleukemic variant is frequently encountered in which the number of circulating mast cells is < 10%. The common phenotypic features of pathologic mast cells encountered in most forms of mastocytosis are unreliable in MCL. Unexpectedly, non-KIT D816V mutations are frequent and therefore, complete gene sequencing is necessary. Therapy usually fails and the median survival time is < 6 months. The role of combination therapies and bone marrow transplantation needs further investigation.

scholarly journals AK002, a Novel Humanized Monoclonal Antibody to Siglec-8, Inhibits Mast Cell Activity and Depletes Eosinophils in Ex Vivo Bone Marrow Tissue from Patients with Systemic Mastocytosis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1104-1104 ◽  
Author(s):  
Bradford A Youngblood ◽  
Emily C Brock ◽  
John Leung ◽  
Alan T Chang ◽  
Christopher Bebbington ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Mast Cell ◽  
Mast Cells ◽  
Ex Vivo ◽  
Systemic Mastocytosis ◽  
Cell Activity ◽  
Receptor Expression ◽  
Long Term Treatment

Abstract INTRODUCTION: Systemic Mastocytosis (SM) is a rare disease characterized by the clonal proliferation and accumulation of mast cells in the bone marrow, respiratory and gastrointestinal tracts, and organs such as the skin, liver, spleen, and brain. Common symptoms include pruritus, flushing, headache, cognitive impairment, fatigue, diarrhea, abdominal pain, hypotension and skin lesions, as well as an increased risk for osteoporosis and anaphylaxis. SM is currently managed with antihistamines, cromolyn sodium, and leukotriene blocking agents, which lack specificity and efficacy. In addition, glucocorticoids can provide temporary relief in some cases; however long-term treatment with steroids is not appropriate due to their many side effects. Siglec-8 is an inhibitory receptor selectively expressed on human mast cells and eosinophils, and therefore represents a novel target for the treatment of SM. Antibodies to Siglec-8 have been shown to inhibit mast cell activity and induce apoptosis of eosinophils. AK002 is a novel, humanized, non-fucosylated IgG1 monoclonal antibody to Siglec-8. This study evaluates the expression of Siglec-8 and ex vivo activity of AK002 on mast cells and eosinophils in bone marrow biopsies from patients with SM. METHODS: Bone marrow aspirates were obtained from patients clinically diagnosed with SM and processed to remove red blood cells. Multi-color flow cytometry was used to quantify eosinophils and mast cells and to evaluate the activation state of mast cells. The ex vivo activity of AK002 against eosinophils and mast cells was evaluated by flow cytometry. The inhibitory activity of AK002 agaist mast cells was also examined by quantifying cytokine levels in cultured bone marrow aspirate supernatants. RESULTS: All mast cells and eosinophils in bone marrow aspirates from SM patients displayed high Siglec-8 receptor expression (Figure 1). These mast cells also expressed the SM specific markers, CD25 (Figure 1) and CD30 and increased levels of cell surface degranulation markers. The expression of CD25 on mast cells significantly decreased following overnight treatment with AK002. AK002 also significantly reduced the level of mast cell-associated cytokines produced in cultured bone marrow supernatants, including IL-6, IL-8, and TNFα (Figure 2A). These changes in mast cell activity after AK002 treatment were not due to a reduction in mast cell numbers. In contrast, overnight incubation of AK002 significantly reduced the number of bone marrow eosinophils compared to an isotype control (Figure 2B). CONCLUSIONS: Bone marrow aspirates from patients with SM had activated mast cells and eosinophils that displayed robust expression of Siglec-8. AK002 demonstrated SM mast cell inhibition in ex vivo bone marrow aspirates. AK002 also had depleting effects on eosinophils, which may be valuable to SM patients with associated eosinophilia. These encouraging results could represent a novel approach for the treatment of SM. Disclosures Youngblood: Allakos, Inc.: Employment. Brock:Allakos, Inc.: Employment. Leung:Allakos, Inc.: Employment. Chang:Allakos, Inc.: Employment. Bebbington:Allakos, Inc.: Employment. Tomasevic:Allakos, Inc.: Employment.

Multiparameter Flow Cytometry in Patients with Suspected Myelodysplastic Syndromes Adds Significant Prognostic Information: A Study on 1,013 Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1722-1722
Author(s):  
Wolfgang Kern ◽  
Claudia Haferlach ◽  
Tamara Alpermann ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Continuous Variable ◽  
The Other ◽  
Multiparameter Flow Cytometry ◽  
Prognostic Information ◽  
Employment Equity ◽  
Aberrant Expression ◽  
Equity Ownership ◽  
Cox Analysis

Abstract Abstract 1722 Backgroundn: Multiparameter flow cytometry (MFC) has been demonstrated capable of identifying aberrant antigen expression related to myelodysplastic syndromes (MDS). The exact role and place of MFC in the diagnostic work-up of patients with suspected MDS, however, remains to be defined. Aim: Evaluation of the diagnostic impact of MFC in relation to cytomorphology (CM) and cytogenetic (CG) by determining the association of MFC results to overall survival (OS). Patients and Methods: In 1,013 patients with suspected MDS bone marrow samples had been analyzed in parallel by MFC, CM, and CG. CM confirmed MDS in 511 patients, excluded MDS in 277 patients, and showed dysplastic features but not sufficient to unequivocally diagnose MDS by CM in 225. The MFC diagnostic result was in agreement with MDS (“MDS by MFC”) in 446 patients including 382/511 patients with MDS proven by CM. CG revealed an aberrant karyotype in 245/1,013 patients. The median follow-up time amounted to 14.8 months, a total of 156 deaths was recorded. Results: The first set of analyses was performed on the cohort of 511 patients with MDS confirmed by CM. The median total number of aberrantly expressed antigens amounted to 3 (range, 0–11) and included expression of mature antigens in myeloid progenitors; abnormal CD13-CD16- and CD11b-CD16-expression patterns, aberrant expression of myeloid markers and reduced side scatter signal (SSC) in granulocytes; reduced expression of myelomonocytic markers in monocytes; aberrant expression of CD71 in erythroid cells; as well as expression of lymphoid markers in all myeloid cell lines. A higher total number of aberrantly expressed antigens as a continuous variable correlated with a shorter OS (Cox analysis, p=0.008). Next, patients were categorized based on the three parameters i) at least 3 aberrantly expressed antigens, ii) significantly reduced SSC in granulocytes, and iii) >5% myeloid progenitor cells in MFC. Patients with at least one of these criteria had a significantly shorter OS than those without (median 48.5 months vs. not reached (n.r.), p<0.001). Overall, the global diagnostic rating of “MDS by MFC” was the strongest MFC parameter: Patients with “MDS by MFC” had a shorter OS as compared to patients without (median 56.8 months vs. n.r., p=0.001). Non-MFC parameters related to OS in univariable Cox analysis included WBC count, thrombocyte count, CG (grouped according to IPSS), % blasts by CM (p<0.001 each), Hb level (p=0.001), and age (p=0.002). In order to determine the clinical relevance of “MDS by MFC” a multivariable analysis for OS was performed on this parameter together with non-MFC parameters (blood counts excluded due to incomplete data sets). It revealed an independent relation between “MDS by MFC” and OS (p=0.045). This was also true for relation of OS to the other parameters (CG, p<0.001; age, p=0.001, % blasts by CM p=0.014). Given this strong prognostic value of “MDS by MFC” in cases with MDS proven by CM a second set of analyses on the relation between MFC findings and OS were performed for the complete cohort of 1,013 patients, i.e. additionally including all cases with a diagnostic result by CM of “no MDS” or “dysplastic features not sufficient to diagnose MDS”. Again, significant relations to OS was found for the total number of aberrantly expressed antigens as a continuous variable (Cox analysis, p<0.001), for at least one of the above mentioned criteria i), ii) or iii) (median 75.6 months vs. n.r., p<0.001), as well as for “MDS by MFC” (median 60.5 months vs. n.r., p<0.001). Again, “MDS by MFC” proved to be the most relevant MFC parameter. Multivariable Cox analysis for OS including “MDS by MFC” and non-MFC parameters revealed a trend only for “MDS by MFC” (p=0.135) and significance for the other parameters (age, p<0.001; CG, p<0.001; blasts by CM, p=0.045). Conclusions: 1) The present data indicates the diagnostic use of MFC for MDS results in independent prognostic information for cases with MDS as proven by CM. 2) Furthermore, the diagnosis of MDS by MFC has a strong prognostic impact even without prove of MDS by CM which strengthens the diagnostic value of MFC even more. 3) This analysis therefore argues in favour of diagnosing MDS not only based on a combination of CM and CG but of adding also MFC for better classification and even prognostication in the future. Disclosures: Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals Response Criteria in Advanced Systemic Mastocytosis: Evolution in the Era of KIT Inhibitors

2021 ◽  
Vol 22 (6) ◽  
pp. 2983
Author(s):  
William Shomali ◽  
Jason Gotlib
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Systemic Mastocytosis ◽  
Myeloproliferative Neoplasms ◽  
Organ Damage ◽  
Response Criteria ◽  
Molecular Response ◽  
Tryptase Level ◽  
Hematologic Neoplasm ◽  
Kit D816v

Systemic mastocytosis (SM) is a rare clonal hematologic neoplasm, driven, in almost all cases, by the activating KIT D816V mutation that leads to the growth and accumulation of neoplastic mast cells. While patients with advanced forms of SM have a poor prognosis, the introduction of KIT inhibitors (e.g., midostaurin, and avapritinib) has changed their outlook. Because of the heterogenous nature of advanced SM (advSM), successive iterations of response criteria have tried to capture different dimensions of the disease, including measures of mast cell burden (percentage of bone marrow mast cells and serum tryptase level), and mast cell-related organ damage (referred to as C findings). Historically, response criteria have been anchored to reversion of one or more organ damage finding(s) as a minimal criterion for response. This is a central principle of the Valent criteria, Mayo criteria, and International Working Group-Myeloproliferative Neoplasms Research and Treatment and European Competence Network on Mastocytosis (IWG-MRT-ECNM) consensus criteria. Irrespective of the response criteria, an ever-present challenge is how to apply response criteria in patients with SM and an associated hematologic neoplasm, where the presence of both diseases complicates assignment of organ damage and adjudication of response. In the context of trials with the selective KIT D816V inhibitor avapritinib, pure pathologic response (PPR) criteria, which rely solely on measures of mast cell burden and exclude consideration of organ damage findings, are being explored as more robust surrogate of overall survival. In addition, the finding that avapritinib can elicit complete molecular responses of KIT D816V allele burden, establishes a new benchmark for advSM and motivates the inclusion of definitions for molecular response in future criteria. Herein, we also outline how the concept of PPR can inform a proposal for new response criteria which use a tiered evaluation of pathologic, molecular, and clinical responses.

Mutational Complexity Is Related To Disease Severity In Systemic Mastocytosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4094-4094
Author(s):  
Juliana Schwaab ◽  
Susanne Schnittger ◽  
Karl Sotlar ◽  
Christoph Walz ◽  
Alice Fabarius ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Conflicts Of Interest ◽  
Systemic Mastocytosis ◽  
Single Gene ◽  
Missense Mutations ◽  
Molecular Feature ◽  
Additional Mutation ◽  
Molecular Aberrations ◽  
Kit D816v

Abstract Systemic mastocytosis (SM) is a rare hematologic neoplasm characterized by abnormal accumulation of mast cells in various tissues, predominantly skin, bone marrow and visceral organs. The morphologic phenotype and extent of organ infiltration/dysfunction are basis for the subclassification of SM into indolent SM (ISM), smoldering SM (SSM), SM with associated hematologic non-mast cell disease (SM-AHNMD), aggressive SM (ASM) and mast cell leukemia (MCL). A somatic point mutation in the kinase domain of the receptor tyrosine kinase (TK) KIT at position 816 (KIT D816V) is present in >95% of patients and plays a central role in the pathogenesis and diagnosis of SM. To further explore mechanisms contributing to the clinical diversity of SM, we analyzed 39 KIT D816V mutated patients with different SM subtypes [ISM, n=10; SSM, n=2; ISM-AHNMD, n=5 (CMML, n=2; MDS/MPNu, n=3); ASM, n=1; ASM-AHNMD, n=14 (CMML, n= 5, MDS/MPNu, n=4, HES/CEL, n=2; AML, n=3); MCL, n=3; MCL-AHNMD n=4 (MDS/MPNu, n=2; HES/CEL, n=1; MDS, n=1)] for the presence of additional mutations. We applied next-generation sequencing to investigate ASXL1, CBL, IDH1/2, JAK2, KRAS, MLL-PTD, NPM1, NRAS, TP53, SRSF2, SF3B1, SETBP1, U2AF1 at mutational hotspot regions, and analyzed the complete coding regions of EZH2, ETV6, RUNX1, and TET2. Additional molecular aberrations were identified in 24/27 (89%) patients with advanced SM (SM-AHNMD, 5/5; ASM/MCL, 19/23) while only 3/12 (25%) ISM/SSM patients carried one additional mutation each (U2AF1, SETBP1, CBL) (p<0.001). In TET2, 9 missense, 4 nonsense, 13 frameshift mutations and one in-frame deletion as well as one splice site mutation were found in 15/39 (39%) patients. Ten of 15 (67%) patients carried more than one TET2 mutation. In SRSF2, missense mutations were identified in 14/39 (36%) patients which were clustered at codon 95 (P95H, n=9; P95L, n=2; P95R, n=2), with one additional mutation identified at codon 18 (V18L, n=1). In RUNX1, 10 mutations (9 missense mutations, 1 frameshift) were identified in 9/39 (23%) patients. Ten CBL mutations were identified in 8 patients (8 missense mutations, 1 duplication, 1 splice mutation) and 8 ASXL1 mutations were identified in 8 patients (7 frameshift and 1 nonsense mutation). In 13 patients, two (TET2, n=8; KRAS, n=1), three (CBL, n=2) or four mutations (TET2, n=2) were found in a single gene. Less frequently affected genes were KRAS (n=4), NRAS, JAK2, U2AF1 (n=2, each), EZH2, SETBP1, and ETV6 (n=1, each). No mutations were identified in MLL, IDH1, IDH2, NPM1, SF3B1 and TP53. In advanced SM, 21/27 patients (78%) carried ³3 mutations and 11/27 patients (41%) exhibited ³5 mutations. The median number of mutations was 4. The concurrent presence of KIT-TET2-SRSF2 (10/39, 26%), KIT-SRSF2-RUNX1 (7/39, 18%), KIT-TET2-CBL (5/39, 13%), KIT-SRSF2-ASXL1 (4/39, 10%) and KIT-TET2-ASXL1 (4/39, 10%) was strongly associated with (A)SM/MCL-AHNMD in 16/16 (100%) of patients (CMML, 7/16, 44%; MDS/MPNu, 6/16, 38%; HES/CEL, 3/16, 19%). Clinical follow-up was available for 38 patients. Six of 38 (16%) patients died, all of whom had at least one additional mutation with 5 patients positive for ³3 and 2 patients for ³5 mutations. The median survival of all 26 patients with at least one additional mutation was 12 months. In contrast, none of the 12 cases with KIT D816V mutation only died. This translated into a significant inferior survival (p=0.019) for patients with additional mutations (figure 1). We therefore conclude that, in addition to the multilineage involvement by KIT D816V, the presence of additional molecular aberrations is a new molecular feature that may contribute essentially to the abnormal phenotype and behaviour of neoplastic mast cells in advanced SM and thus to the clinical diversity and prognosis of advanced mast cell disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Systemic Mastocytosis Patient Experience from Mast Cell Connect, the First Patient-Reported Registry for Mastocytosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4783-4783 ◽  
Author(s):  
Philina Lee ◽  
Tracy I. George ◽  
Hongliang Shi ◽  
Erica K. Evans ◽  
Teghpal Singh ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Mast Cell ◽  
Research Funding ◽  
Disease Burden ◽  
Systemic Mastocytosis ◽  
Employment Equity ◽  
New Therapies ◽  
Patient Reported ◽  
Equity Ownership ◽  
The Impact

Abstract Background: Systemic mastocytosis (SM) is a rare mast cell disorder associated with a range of debilitating symptoms and a shortage of effective treatment options. Little is known about the impact of the disease from the patients' perspective. Systematically characterizing the natural history of SM and its impact on patients will facilitate the development of new therapies. Registries that engage patients have proven valuable in other rare diseases by expanding knowledge of current treatment approaches, evaluating disease burden and accelerating clinical trials. Methods: Mast Cell Connect (NCT02620254, www.mastcellconnect.org) is an observational database that captures demographic, socioeconomic and disease information from patients with mastocytosis. It is the first patient-reported registry for mastocytosis to our knowledge. Key objectives are to improve collective understanding of the disease burden on patients and to facilitate development of new therapies by increasing participation in clinical trials. Enrollment is "site-agnostic" as participants register and take surveys on a secure online portal. Since many US patients are seen outside large clinical centers, this enables broader participation and collection of real-world data. Inclusion criteria are a diagnosis of SM or cutaneous mastocytosis (CM). Diagnoses are self-reported and participants are asked to provide medical reports that will be used to confirm diagnoses in the future. Informed consent is required to join this IRB-approved study. Results: Mast Cell Connect opened on December 1, 2015. As of May 31, 2016, 166 participants had registered, of whom 157 had responded to the online survey. Of the 153 participants who reported their diagnosis, 107 had SM and 38 had CM. Those who reported a diagnosis of SM were categorized as SM participants if they also reported other diagnoses. The current analyses are based on data provided by the 107 SM participants. Of the 107 SM participants, 60 (56%) had Indolent SM (ISM), 8 (7%) had Smoldering SM (SSM), 10 (9%) had Advanced SM (AdvSM, defined as aggressive SM, SM with an associated hematological neoplasm, and Mast Cell Leukemia) and 29 (27%) did not know their subtype. Median age was 50 years (range 4-77). More females participated (74%) than males (26%). Median time from symptom-onset to diagnosis was 7 years. Participants saw a median of 3 specialists prior to diagnosis, most frequently dermatology (67%), allergy/immunology (67%), hematology/oncology (63%) and general practitioner/internal medicine (63%). Specialists diagnosing SM most often included dermatology (36%), allergy/immunology (25%) and hematology/oncology (23%). AdvSM participants were typically diagnosed by hematology/oncology (70%). The medications taken most often at the time of survey were anti-histamines (85% H1 blockers, 72% H2 blockers), leukotriene inhibitors (38%) and cromolyn sodium (33%). Agents to reduce mast cell burden were taken less often (imatinib: 7%, interferon-α: 2%, hydroxyurea: 1% and investigational agents: 1%). Symptoms reported as moderately to severely bothersome included: fatigue (69%), difficulty concentrating (63%), abdominal pain (55%), pain in other locations (53%), difficulty sleeping (53%), itching (46%), diarrhea (45%), bloating (43%), nausea (42%), headache (40%), skin changes (40%), anxiety (39%) and flushing (37%). Participants reported being limited in work or other daily activities "quite a bit" to "very much" in 47% of ISM, 43% of SSM, and 70% of AdvSM cases. Participants' physical condition or medical treatment interfered with their family life "quite a bit" or "very much" in 59% of ISM, 43% of SSM, and 80% of AdvSM cases. Conclusions: Over 160 participants joined Mast Cell Connect in the first 6 months, indicating the mastocytosis community, including The Mastocytosis Society, is highly motivated to participate in research. SM participants reported a diagnostic odyssey, seeing multiple specialists over a median of 7 years prior to diagnosis. Despite frequent use of symptom-directed medications, symptom burden remains substantial and considerably impacts quality of life. Novel therapies targeting the underlying disease are needed. Establishing US centers of excellence may hasten the time to diagnosis. Continued collaboration between researchers, patient advocates and industry is needed to advance the care of patients with SM. Disclosures Lee: Blueprint Medicines: Employment, Equity Ownership. George:Allakos: Research Funding; Novartis: Consultancy; Blueprint Medicines: Consultancy; Allakos: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Consultancy, Research Funding; Incyte: Consultancy; GLG: Consultancy; Wiley Blackwell: Consultancy; American Registry of Pathology: Patents & Royalties; Wolters Kluwer: Patents & Royalties; UpToDate: Patents & Royalties. Shi:Blueprint Medicines: Employment, Equity Ownership. Evans:Blueprint Medicines: Employment, Equity Ownership. Singh:Blueprint Medicines: Employment, Equity Ownership. Boral:Blueprint Medicines: Employment, Equity Ownership. Rangel Miller:PatientCrossroads: Employment, Equity Ownership.

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