Detection of c-Kit Mutation in Peripheral Blood vs. Bone Marrow Aspirates in Patients with Systemic Mastocytosis: Comparison Study of Pathological and Clinical Laboratory Findings in Patients with and without Detectable c-Kit Mutation in the Peripheral Blood.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3596-3596 ◽  
Author(s):  
Irina Maric ◽  
Jamie Robyn ◽  
Weiming Fu ◽  
Jennifer Stoddard ◽  
Dean D. Metcalfe ◽  
...  
Keyword(s):  
Mast Cells ◽  
Peripheral Blood ◽  
Mutational Analysis ◽  
Clinical Laboratory ◽  
Systemic Mastocytosis ◽  
Flow Cytometric Analysis ◽  
Serum Tryptase ◽  
Kit Mutation ◽  
Flow Cytometric ◽  
Kit D816v

Abstract The identification of the KIT D816V mutation in patients with systemic mastocytosis (SM) has lately gained a major prognostic significance, largely because of the availability of tyrosine kinase receptor inhibitors such as imatinib. Imatinib was shown to be ineffective in patients carrying KIT D816V mutation, but effective in cases with some other c-kit mutations. Therefore, it is of paramount importance to correctly identify SM patients with KIT D816V mutation. However, the reported frequency of the KIT D816V mutation in SM patients is highly variable in the literature (30%-over 95%). It has been suggested that such variability is due to patient selection, sensitivity of the molecular methods used to detect the mutation or the source of the tested specimen (peripheral blood (PB) vs. bone marrow (BM) aspirate). To date, there has been no systematic study comparing PB and BM mutational findings in SM patients. In this study, we performed mutational analysis of both PB and BM samples in SM patients and compared the results with pathological, clinical laboratory and flow cytometric findings in patients with and without a detectable c-kit mutation in PB. We analyzed in parallel BM aspirates and PB from 55 patients who came to our clinic for evaluation of SM. After diagnostic workup (physical evaluation, measurement of serum tryptase level, study of BM biopsy, flow cytometric analysis of mast cells and mutational analysis by RT-PCR/RFLP), 46 of 55 patients were diagnosed with SM using the WHO diagnostic criteria. Nine patients did not fulfill the WHO diagnostic criteria for SM and all tested negative for c-kit mutation. Out of 46 patients diagnosed with SM, all but two patients (44/46; 95.6%) tested positive for KIT D816V mutation in the BM aspirate, but only 9/46 patients (19.5%) had the mutation detectable in the PB. Two patients who tested negative for KIT D816V mutation in the BM were shown to carry different c-kit mutation by sequencing. No tested patients carried the FIP1L1-PDGFRa fusion gene. 42/46 patients (91%) fulfilled major WHO pathological criteria for diagnosis of SM (dense mast cell aggregates in the BM biopsy). The other 9% had increased atypical spindle-shaped mast cells in the BM biopsy without dense aggregates. Flow cytometric analysis of PB showed no significant increase in circulating mast cells in patients with a detectable KIT D816V mutation in PB (average less than 0.01% mast cells). Comparison of patients with and without a detectable PB mutation showed more extensive BM biopsy involvement by mast cells in PB positive patients (average 45% vs. 15%), higher average serum tryptase levels (266 ng/ml vs. 85 ng/ml) and higher average PB absolute eosinophil counts (710 vs. 234/uL). Flow cytometric analysis of BM mast cells showed that 100% of KIT D816V positive patients had aberrant CD25 expression on mast cells. CD2 expression was more variable, but comparable in both groups of patents (67% vs. 69%). We conclude that the source of the specimen for c-kit analysis is of crucial importance for correct diagnosis, and recommend that all patients with suspected SM should always have BM aspirates tested for the KIT D816V mutation. PB testing yields falsely negative results in over 80% of cases and identifies only SM patients with a markedly increased mast cell burden.

PKC412 inhibits in vitro growth of neoplastic human mast cells expressing the D816V-mutated variant of KIT: comparison with AMN107, imatinib, and cladribine (2CdA) and evaluation of cooperative drug effects

Blood ◽  
2006 ◽  
Vol 107 (2) ◽  
pp. 752-759 ◽  
Author(s):  
Karoline V. Gleixner ◽  
Matthias Mayerhofer ◽  
Karl J. Aichberger ◽  
Sophia Derdak ◽  
Karoline Sonneck ◽  
...  
Keyword(s):  
Mast Cells ◽  
Systemic Mastocytosis ◽  
Drug Effects ◽  
Kit Mutation ◽  
Growth Inhibitory ◽  
Ic50 Values ◽  
Tk Inhibitors ◽  
Mast Cell Leukemia ◽  
Kit D816v

AbstractIn most patients with systemic mastocytosis (SM), including aggressive SM and mast cell leukemia (MCL), neoplastic cells express the oncogenic KIT mutation D816V. KIT D816V is associated with constitutive tyrosine kinase (TK) activity and thus represents an attractive drug target. However, imatinib and most other TK inhibitors fail to block the TK activity of KIT D816V. We show that the novel TK-targeting drugs PKC412 and AMN107 counteract TK activity of D816V KIT and inhibit the growth of Ba/F3 cells with doxycycline-inducible expression of KIT D816V as well as the growth of primary neoplastic mast cells and HMC-1 cells harboring this KIT mutation. PKC412 was a superior agent with median inhibitory concentration (IC50) values of 50 to 250 nM without differences seen between HMC-1 cells exhibiting or lacking KIT D816V. By contrast, AMN107 exhibited more potent effects in KIT D816V- HMC-1 cells. Corresponding results were obtained with Ba/F3 cells exhibiting wild-type or D816V-mutated KIT. The growth-inhibitory effects of PKC412 and AMN107 on HMC-1 cells were associated with induction of apoptosis and down-regulation of CD2 and CD63. PKC412 was found to cooperate with AMN107, imatinib, and cladribine (2CdA) in producing growth inhibition in HMC-1, but synergistic drug interactions were observed only in cells lacking KIT D816V. Together, PKC412 and AMN107 represent promising novel agents for targeted therapy of SM. (Blood. 2006;107: 752-759)

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.

Clinical, Biological, and Molecular Characteristics of Well-Differentiated Systemic Mastocytosis: a study in 18 Patients by Spanish Network on Mastocytosis (REMA)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5246-5246
Author(s):  
Iván Alvarez-Twose ◽  
Laura Sánchez-Muñoz ◽  
María Jara-Acevedo ◽  
Cristina Teodosio ◽  
Andrés García-Montero ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Systemic Mastocytosis ◽  
Skin Lesions ◽  
Categorical Variables ◽  
Molecular Characteristics ◽  
Skin Involvement ◽  
Serum Tryptase ◽  
Kit Mutation ◽  
Well Differentiated

Abstract BACKGROUND: Well-differentiated systemic mastocytosis (WDSM) has recently been described as a novel form of mast cell disease. WDSM is characterized by a marked increase of bone marrow (BM) mast cells, usually with compact aggregates, with normal phenotype and morphology as well as the absence of the typical D816V somatic KIT mutation. OBJECTIVE: To describe and compare clinical, morphological, biological and molecular characteristics in a group of 18 patients who fullfilled criteria for WDSM with a group of 32 patients with indolent systemic mastocytosis (ISM). PATIENTS AND METHODS: All the patients were diagnosed on the basis of BM aspirate and biopsy findings after they were thought to have a systemic mastocytosis. A rigorous skin examination together with a clinical work-up and a complete laboratory analysis including peripheral blood count, routine biochemistry and serum tryptase levels were performed. The Mann-Whitney U and the chi-square tests were used to assess the statistical differences of continuous and categorical variables, respectively. RESULTS: WDSM patients were 4 males and 14 females with a median (range) age of 24 years (2–72) at diagnosis. Median (range) age at the time of the first observation of skin lesions was 2 years (0–41). In 16 of the 18 patients (89%), skin lesions appeared under the age of 14, five of them being younger than 1 year old. All the WDSM patients had skin involvement but the typical maculo-papular lesions were found only in 18% of patients while in the remaining 82% of cases, cutaneous lesions were plaques or nodules. Interestingly, 78% of WDSM patients had cutaneous neck involvement in contrast with only 13% in the ISM group (p<0.001). There were no significant differences in mast cell mediators-related symptoms such as pruritus, flushing, abdominal pain or diahrrea between the two groups while anaphylactic reactions were significantly more frequent in the WDSM group than in the ISM group (59% vs 19%, p=0.004). Overall, bone loss was found in 38% and 37% of WDSM and ISM patients, respectively. Among all the biochemical parameters analyzed, only median serum cholesterol (mg/dL), ferritin (ng/mL) and tryptase levels (ng/mL) were significantly lower in patients with WDSM when compared to ISM patients (150 vs 168.5, p=0.043; 31.3 vs 56.6, p=0.004; 11 vs 31.6, p<0.001; respectively). There were no significant differences in the median percentage of mast cells in the BM study as assesed by flow cytometry between both groups (0.055% in WDSM group and 0.08% in ISM group). In contrast, the presence of both fibrosis and lymphoid aggregates in the BM biopsy were more frequent in patients with ISM than in patients with WDSM (52% vs 0%, p<0.001; 55% vs 14%, p=0.01; respectively). Somatic KIT mutation at codon 816 was found in 31 of the 32 ISM patients (97%) while only in one (6%) WDSM patient (p<0.001). Additionally, 2 WDSM patients were found to carry variant mutations at codons other than at codon 816 (N819Y and I817V, respectively). From the remaining 15 WDSM patients, clonality of mast cell population was demonstrated by human androgen receptor (HUMARA) assay in the 5 female patients in whom the assay was made. CONCLUSIONS: WDSM is a variant of systemic mastocytosis with several characteristics that are distinguishable from ISM, such as: early onset of skin lesions, atypical skin involvement (plaques or nodules and cutaneous neck involvement), frequent anaphylactic episodes, lower serum tryptase levels than ISM despite no differences in BM mast cell infiltration, and infrequent detection of typical activating D816V KIT mutation.

Aggressive Systemic Mastocytosis Is Associated with a High Kit D816V Allele Burden in Peripheral Blood.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2899-2899
Author(s):  
Philipp Erben ◽  
Georg Bolz ◽  
Juliana Popa ◽  
Georgia Metzgeroth ◽  
Martin C Müller ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Detection Limit ◽  
Mast Cells ◽  
Tyrosine Kinase ◽  
Peripheral Blood ◽  
Kinase Inhibitors ◽  
Systemic Mastocytosis ◽  
Allele Burden ◽  
Minor Criteria ◽  
Kit D816v

Abstract Abstract 2899 Poster Board II-875 According to 2008 WHO definitions, diagnosis of systemic mastocytosis (SM) is based on the presence of one major criteria (multifocal dense infiltrates of mast cells in bone marrow or organ biopsies) and at least one of the following minor criteria (i) >25% are atypical cells on bone marrow smears or are spindle-shaped in mast cell infiltrates of visceral organs, ii) KIT point mutation at codon 816 in the bone marrow or other extracutaneous organs, iii) mast cells express CD2 and/or CD25, iv) baseline serum tryptase concentration >20 ng/mL) or the presence of at least three minor criteria. Patients with aggressive SM (ASM) have to present with one or more C-findings (i) neutrophils <1,000/μL, Hb <10g/dL, or platelets <100,000/μL, ii) hepatomegaly with impaired liver function – elevated transaminases and/or bilirubin levels and/or hypoalbuminemia, iii) palpable splenomegaly with signs of hypersplenism, iv) malabsorption with significant hypoalbuminemia and/or significant loss. ASM and MCL are clearly associated with an inferior survival. The activating KIT D816V point mutation is thought to be pivotal for pathogenesis and potential targeted therapy with novel tyrosine kinase inhibitors. However, conventional sequencing (CS) of RNA/DNA extracted from bone marrow (BM) samples of SM patients reveals the mutation in less than 50-60% of patients while peripheral blood (PB) samples are frequently negative as consequence of low numbers of malignant cells and poor assay sensitivity. For better qualitative and additional quantitative assessment of the KIT D816V allele burden (expressed as ratio KIT D816V vs. KIT wildtype), we sought to establish (i) a D-HPLC (denaturing-high performance liquid chromatography) assay combined with direct sequencing in case of a positive D-HPLC signal and (ii) a LightCyclerTM based quantitative PCR (RQ-PCR). The assay sensitivities were calculated through serial cell (KIT D816V positive HMC-1 cells in NB4 cells) and RNA dilutions. The detection limit was estimated between 0.1 and 0.5% for both assays which was thus significantly improved in comparison to the detection limit of CS which was at 10–15%. Patient material was available from BM (n=134) and PB (n=93) from 173 patients (88 m, 85 f, median age 54 years) with diagnosis of SM including 10 patients with confirmed ASM (n=9) or aleukemic variant of MCL (n=1) according to WHO definitions. At diagnosis, D-HPLC, RQ-PCR and CS were positive in 78% (104/134), 84% (112/134) and 60% (80/134) of BM samples, respectively. Two patients had a KIT D816H and one patient had a KIT D816L mutation. In 54 cases with contemporaneously collected BM and PB samples, the KIT D816V mutation was found in PB of BM positive patients by D-HPLC, RQ-PCR and CS in 47% (22/46), 55% (27/49) and 38% (14/37) of cases, respectively. This information allowed the detection of the KIT D816V mutation in an additional 27 of 39 (69%) patients without available bone marrow biopsies. All ASM patients were KIT D816V positive in PB with a median KIT D816V allele burden of 36% (range 26–98%) vs. 7.8% (range 0.1–61%) in other stages of SM (p=0.0021). Eleven of 124 (8.9%) SM patients with a KIT D816V allele burden >20% are currently explored for the potential diagnosis of ASM or MCL. In conclusion, D-HPLC is a reliable and sensitive method for the screening of variable KIT mutations in BM and PB of SM patients and is clearly superior to CS. Application of RQ-PCR assays for the most common D816V mutation may overlook rare mutations but allows quantification of the KIT D816V allele burden which may be useful for diagnosis of ASM and monitoring of residual disease during treatment with novel tyrosine kinase inhibitors, e.g. midostaurin or dasatinib. Disclosures: No relevant conflicts of interest to declare.

Identification of MCL1 as a novel target in neoplastic mast cells in systemic mastocytosis: inhibition of mast cell survival by MCL1 antisense oligonucleotides and synergism with PKC412

Blood ◽  
2006 ◽  
Vol 109 (7) ◽  
pp. 3031-3041 ◽  
Author(s):  
Karl J. Aichberger ◽  
Matthias Mayerhofer ◽  
Karoline V. Gleixner ◽  
Maria-Theresa Krauth ◽  
Alexander Gruze ◽  
...  
Keyword(s):  
Mast Cells ◽  
Antisense Oligonucleotides ◽  
Kinase Inhibitors ◽  
Systemic Mastocytosis ◽  
Synergistic Effects ◽  
Kit Mutation ◽  
Myeloid Neoplasm ◽  
Novel Target ◽  
Kit D816v

Abstract MCL-1 is a Bcl-2 family member that has been described as antiapoptotic in various myeloid neoplasms. Therefore, MCL-1 has been suggested as a potential new therapeutic target. Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In the present study, we examined the expression and functional role of MCL-1 in neoplastic MCs and sought to determine whether MCL-1 could serve as a target in SM. As assessed by RT-PCR and immunohistochemical examination, primary neoplastic MCs expressed MCL-1 mRNA and the MCL-1 protein in all SM patients examined. Moreover, MCL-1 was detectable in both subclones of the MC line HMC-1—HMC-1.1 cells, which lack the SM-related KIT mutation D816V, and HMC-1.2 cells, which carry KIT D816V. Exposure of HMC-1.1 cells or HMC-1.2 cells to MCL-1–specific antisense oligonucleotides (ASOs) or MCL-1–specific siRNA resulted in reduced survival and increased apoptosis compared with untreated cells. Moreover, MCL-1 ASOs were found to cooperate with various tyrosine kinase inhibitors in producing growth inhibition in neoplastic MCs, with synergistic effects observed with PKC412, AMN107, and imatinib in HMC-1.1 cells and with PKC412 in HMC-1.2 cells. Together, these data show that MCL-1 is a novel survival factor and an attractive target in neoplastic MCs.

Evidence for the involvement of a hematopoietic progenitor cell in systemic mastocytosis from single-cell analysis of mutations in the c-kit gene

Blood ◽  
2002 ◽  
Vol 100 (2) ◽  
pp. 661-665 ◽  
Author(s):  
A. Selim Yavuz ◽  
Peter E. Lipsky ◽  
Sule Yavuz ◽  
Dean D. Metcalfe ◽  
Cem Akin
Keyword(s):  
B Cells ◽  
Mast Cells ◽  
Peripheral Blood ◽  
Genomic Dna ◽  
Progenitor Cell ◽  
Systemic Mastocytosis ◽  
Point Mutations ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Kit Mutation

Abstract Mast cells are derived from multipotential hematopoietic progenitors and are clonally increased in systemic mastocytosis, a disease associated with point mutations of codon 816 (most commonly Asp816Val) of c-kit. To study the lineage relationship and the extent of expansion of cells derived from the mutated clone, we examined the occurrence of the Asp816Val c-kit mutation in genomic DNA of individual sorted peripheral blood T cells, B cells, and monocytes in patients with indolent systemic mastocytosis. The mutation was detected in varying frequencies in the genomic DNA of individual B cells and monocytes and bone marrow mast cells in patients with extensive disease. In B cells, the immunoglobulin repertoire was polyclonal, indicating that the mutation occurred before VH/(D)/JH recombination. These results show that mastocytosis is a disorder of a pluripotential hematopoietic progenitor cell that gives rise to B cells and monocytes in addition to mast cells and that the affected clone shows variable expansion in these lineages in the peripheral blood of patients with systemic mastocytosis.

c-kit D816V Provides a Strong Signal for Myelomastocytic Differentiation and Cluster Formation in Murine Ba/F3 Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 485-485 ◽  
Author(s):  
Matthias Mayerhofer ◽  
Karl J. Aichberger ◽  
Stefan Florian ◽  
Maria-Theresa Krauth ◽  
Martin Bilban ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Histidine Decarboxylase ◽  
Systemic Mastocytosis ◽  
Cluster Formation ◽  
Kit Mutation ◽  
Strong Signal ◽  
Mouse Mast Cell ◽  
Kit D816v

Abstract The pathologic hallmark of systemic mastocytosis (SM) is differentiation and cluster formation of mast cells (MC) in hematopoietic tissues. The somatic c-kit mutation D816V is detectable in a majority of all SM patients independent of the proliferation-status of MC or subtype (indolent or aggressive) of disease. To investigate the role of c-kit D816V in the pathogenesis of SM, we established a Ba/F3 cell line with doxycycline-inducible expression of c-kit D816V. We found that c-kit D816V provides a strong signal for mast cell differentiation and cluster formation in Ba/F3 hematopoietic progenitor cells without enhancing their growth thereby resembling the clinical presentation of indolent SM (ISM). As assessed by gene chip analysis, induction of c-kit D816V resulted in expression of various differentiation antigens including mouse mast cell protease 5, mi transcription factor, histidine decarboxylase (HDC), secretory granule proteoglycan, IL-4 receptor, ICAM-1, and CD63 consistent with an early phase of mastopoiesis. By contrast, c-kit D816V did neither induce expression of granulo-monocytic antigens such as myeloperoxidase, IL-3 receptor, or GM-CSF receptor, nor expression of ‘late stage’ mast cell antigens such as FcεRI. The c-kit D816V-induced synthesis of histamine in Ba/F3 cells was confirmed by RIA. To examine the role of c-kit D816V in the pathogenesis of mastocytosis, we extended our analysis to bone marrow biopsy sections obtained from patients with ISM. In these experiments, the D816V-mutated form of c-kit was detected more frequently in micro-dissected tryptase-positive MC obtained from dense compact infiltrates (44.2%) than in diffusely spread MC in these patients (22.6%) (p&lt;0.05). In summary, our data establish a role for c-kit D816V in differentiation and cluster formation of neoplastic (mast) cells. Additional genetic hits, apart from c-kit D816V, may be responsible for aggressive growth of MC in advanced MC neoplasms.

Increased Detection of KIT D816V Mutation in Peripheral Blood Samples from Patients with Indolent Systemic Mastocytosis (ISM) in the Phase 2 Pioneer Study Using a High Sensitivity Droplet Digital (dd) PCR Assay Compared with Next Generation Sequencing (NGS)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-8
Author(s):  
Tracy I. George ◽  
Gerard Hoehn ◽  
Hui-Min Lin ◽  
Stephen Miller ◽  
Cem Akin
Keyword(s):  
Peripheral Blood ◽  
Systemic Mastocytosis ◽  
High Sensitivity ◽  
Diagnostic Sensitivity ◽  
Assay Method ◽  
Phase 2 ◽  
Serum Tryptase ◽  
Publicly Traded ◽  
Kit Receptor ◽  
Kit D816v

Introduction: Systemic mastocytosis (SM) is a rare, clonal, hematopoietic neoplasm characterized by excessive proliferation and hyperactivation of abnormal mast cells (MCs). Approximately 95% of patients with SM harbor the KIT D816V mutation within the KIT receptor tyrosine kinase, which results in constitutive activation of the KIT receptor. Abnormal activation of KIT leads to increased proliferation, survival, and hyperactivation of MCs, which can cause debilitating constitutional symptoms such as pruritus, flushing, headaches, bone pain, nausea, vomiting, diarrhea, and life-threatening anaphylaxis, across all SM subtypes.Polymerase chain reaction (PCR)-based detectionof the KIT D816V mutation in bone marrow (BM) aspirates or other extracutaneous tissues is an important tool to confirm clinical and pathological diagnosis. In addition, genomic determination of the KIT D816V variant allelic fraction (VAF) in peripheral blood (PB) or BM can serve as a potential prognostic tool and may also be used to monitor treatment efficacy over time. However, low numbers of neoplastic MCs in BM or PB samples, especially from patients with non-advanced forms of the disease (ISM and smoldering SM) can result in misdiagnosis and underestimation of disease burden. Here, we report on the performance of an adapted ddPCR assay compared with NGS for measuring the KIT D816V VAF in PB samples from patients with moderate to severe ISM and symptoms inadequately controlled with best supportive care enrolled in part 1 (randomized dose finding part to determine recommended dose) of the ongoing, randomized, double-blind, placebo-controlled phase 2 PIONEER clinical study (NCT03731260) of avapritinib, a potent and selective inhibitor of KIT D816V mutant kinases. Methods : Part 1 of PIONEER included 39 patients (3 cohorts with 10 patients at 25, 50 and 100 mg of avapritinib, and a placebo cohort of 9 patients) with a confirmed diagnosis of ISM based on World Health Organization criteria and moderate-to-severe symptoms based on Total Symptom Score as measured on the ISM-System Assessment Form (ISM-SAF). Samples of PB were collected at screening for all enrolled patients in part 1 and were evaluated by local quantitative and qualitative KIT testing, central ddPCR assay for detection of KIT D816V, and central NGS (TruSight™ Myeloid Panel) for KIT D816V and other co-mutations (central assays performed by MolecularMD, Portland, OR). Results were expressed as the percentage of patient PB samples testing positive for KIT D816V mutation (all genomic assays) and the log percent of VAF as measured by both central assay methods. Correlations of KIT D816V VAF to objective measure of MC burden, including serum tryptase levels and BM MC numbers, were performed. KIT D816V VAF was also evaluated serially in each patient over the treatment period. Results: The central ddPCR assay method detected the KIT D816V mutation in 37/39 (95%) of PB samples compared with 11/39 (28%) assayed by NGS and 30/39 (80%) of PB samples assayed locally (Table). ddPCR also demonstrated ~30-fold greater sensitivity over NGS for measuring VAF: median percent VAF (range) by ddPCR was 0.36 (0.02-30) and by NGS was 11 (1.9-31) (Figure). The lower limit of detection of VAF by ddPCR on PB samples was 95-fold lower than that of NGS (0.02% and 1.9%, respectively). In addition, ddPCR provided greater diagnostic sensitivity for ISM compared with serum tryptase &gt;20 ng/mL (30/39, 77%) and presence of BM MC aggregates (35/39, 90%). Conclusions: The high-sensitivity ddPCR assay method detected the KIT D816V mutation in 95% of PB samples from patients with previously confirmed ISM, which provided greater sensitivity for detection of KIT D816V mutation than either NGS or local assessments, greater sensitivity over NGS for KIT D816V VAF in PB samples, and higher diagnostic sensitivity than clinical assessments of serum tryptase and BM MC aggregates. These results have implications for assay sensitivity in the diagnosis of SM and suggest that higher sensitivity testing for KIT D816V mutation using a minimally invasive PB ddPCR assay could be used as a screening tool to facilitate identification of ISM patients, including those with variable disease characteristics or low MC burden. Disclosures George: Blueprint Medicines Corporation: Consultancy, Other: I have received no funding for this research. ARUP Laboratories, owned by the University of Utah, has received funding; Allakos: Consultancy; Celgene: Consultancy; Deciphera: Other: consultancy, but has received no financial compensation for the past 12 months. Hoehn:Blueprint Medicines Corporation: Current Employment, Current equity holder in publicly-traded company. Lin:Blueprint Medicines Corporation: Current Employment, Current equity holder in publicly-traded company. Miller:Blueprint Medicines Corporation: Current Employment, Current equity holder in publicly-traded company. Akin:Novartis: Consultancy; Blueprint Medicines Corporation: Consultancy, Research Funding.

Elevated serum tryptase levels identify a subset of patients with a myeloproliferative variant of idiopathic hypereosinophilic syndrome associated with tissue fibrosis, poor prognosis, and imatinib responsiveness

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4660-4666 ◽  
Author(s):  
Amy D. Klion ◽  
Pierre Noel ◽  
Cem Akin ◽  
Melissa A. Law ◽  
D. Gary Gilliland ◽  
...  
Keyword(s):  
Mast Cells ◽  
Poor Prognosis ◽  
Systemic Mastocytosis ◽  
Hypereosinophilic Syndrome ◽  
Elevated Serum ◽  
Clinical Manifestations ◽  
Serum Tryptase ◽  
Bone Marrow Biopsies ◽  
Kit Mutation ◽  
Tissue Fibrosis

Abstract Since serum tryptase levels are elevated in some patients with myeloproliferative disorders, we examined their utility in identifying a subset of patients with hypereosinophilic syndrome (HES) and an underlying myeloproliferative disorder. Elevated serum tryptase levels (&gt; 11.5 ng/mL) were present in 9 of 15 patients with HES and were associated with other markers of myeloproliferation, including elevated B12 levels and splenomegaly. Although bone marrow biopsies in these patients showed increased numbers of CD25+ mast cells and atypical spindle-shaped mast cells, patients with HES and elevated serum tryptase could be distinguished from patients with systemic mastocytosis and eosinophilia by their clinical manifestations, the absence of mast cell aggregates, the lack of a somatic KIT mutation, and the presence of the recently described fusion of the Fip1–like 1 (FIP1L1) gene to the platelet-derived growth factor receptor α gene (PDGFRA). Patients with HES and elevated serum tryptase were more likely to develop fibroproliferative end organ damage, and 3 of 9 died within 5 years of diagnosis in contrast to 0 of 6 patients with normal serum tryptase levels. All 6 patients with HES and elevated tryptase treated with imatinib demonstrated a clinical and hematologic response. In summary, elevated serum tryptase appears to be a sensitive marker of a myeloproliferative variant of HES that is characterized by tissue fibrosis, poor prognosis, and imatinib responsiveness.

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