KIT D816V Allele Research Articles

The Number of MRGPRX2-Expressing Cells Is Increased in Skin Lesions of Patients With Indolent Systemic Mastocytosis, But Is Not Linked to Symptom Severity.

BackgroundRecently, the expression of the mast cell (MC) receptor Mas-related G protein–coupled receptor X2 (MRGPRX2) has been detected in lesional skin of adult patients with cutaneous mastocytosis. As of yet, little is known about the clinical relevance of MRGPRX2 and its agonists in patients with mastocytosis, including indolent systemic mastocytosis (ISM).MethodsMRGPRX2 and MRGPRX2 agonists, cortistatin (CST), and major basic protein (MBP) were analyzed in lesional and non-lesional skin of patients with ISM and skin of healthy controls by immunohistochemistry. Co-localization of MRGPRX2 and MRGPRX2-mRNA with the MC marker tryptase was assessed by immunofluorescence microscopy and in situ hybridization, respectively. We assessed clinical, demographic, and laboratory data, including mastocytosis activity score (MAS), serum tryptase, and KIT D816V allele burden.ResultsThe number of MRGPRX2-expressing (MRGPRX2+) cells, MRGPRX2-mRNA+ MCs, and CST-expressing (CST+) and MBP-expressing (MBP+) cells was significantly higher in lesional skin as compared to non-lesional skin and/or skin of healthy controls (all p < 0.05). Increased numbers of MRGPRX2+ cells, MRGPRX2-mRNA+ MCs, and CST+ and MBP+ cells were not associated with clinical and laboratory features of ISM, including disease burden, symptom severity, evidence of anaphylaxis, and tryptase levels.ConclusionsSkin lesions of patients with ISM showed high numbers of MRGPRX2+ cells, although they were not linked to symptom severity. Clinical relevance of the MRGPRX2-mediated pathway of MC activation in ISM remains unclear and should be investigated in further studies.

Refined Treatment Response Criteria for Indolent Systemic Mastocytosis Proposed by the ECNM-AIM Consortium

Indolent systemic mastocytosis (ISM) has a favorable prognosis and normal life expectancy. However, many patients suffer from mast cell (MC) mediator-related symptoms, which significantly affect quality of life (QoL). Cutaneous, gastrointestinal, and neurological complaints, musculoskeletal pain, and the presence of skin lesions, anaphylaxis, and osteoporosis are the main symptoms and signs in ISM and must be assessed in all patients before and during treatment. Validated mastocytosis-specific patient-reported outcome measures (PROMs) should be used for this purpose. Serum tryptase and KIT D816V allele burden are recommended as secondary outcome parameters, noting that they do not reflect the severity of signs, symptoms, and related QoL impairment, but indirectly express MC burden. Changes from baseline of 90%, 60%, and 30% indicate complete response >90%, major response 60% to 90%, partial response 30% to 60%, and no response <30% to treatment. To conclude, we recommend the use of PROMs as primary outcome parameters to define treatment response in patients with ISM in clinical trials and in everyday clinical practice.

TET2 regulates immune tolerance in chronically activated mast cells.

Mutation of the TET2 DNA-hydroxymethylase has been associated with a number of immune pathologies. The disparity in phenotype and clinical presentation among these pathologies leads to questions regarding the role of TET2 mutation in promoting disease evolution in different immune cell types. Here we show that, in primary mast cells, Tet2 expression is induced in response to chronic and acute activation signals. In TET2-deficient mast cells, chronic activation via the oncogenic KITD816V allele associated with mastocytosis, selects for a specific epigenetic signature characterized by hypermethylated DNA regions (HMR) at immune response genes. H3K27ac and transcription factor binding is consistent with priming or more open chromatin at both HMR and non-HMR in proximity to immune genes in these cells, and this signature coincides with increased pathological inflammation signals. HMR are also associated with a subset of immune genes that are direct targets of TET2 and repressed in TET2-deficient cells. Repression of these genes results in immune tolerance to acute stimulation that can be rescued with vitamin C treatment or reiterated with a Tet inhibitor. Overall, our data support a model where TET2 plays a direct role in preventing immune tolerance in chronically activated mast cells, supporting TET2 as a viable target to reprogram the innate immune response for innovative therapies.

MPN-395: Efficacy and Safety of ≤200 mg Avapritinib in Patients with Advanced Systemic Mastocytosis: Pooled Results from the Phase 1 EXPLORER and Interim Phase 2 PATHFINDER Studies

<h3>Introduction</h3> Systemic mastocytosis (SM), a clonal neoplasm driven by <i>KIT</i> D816V mutation in ~95% of cases, has limited treatment options. We evaluated pooled efficacy and safety of avapritinib, a selective inhibitor of D816V-mutant <i>KIT,</i> in patients with advanced SM (AdvSM) from the phase I/II EXPLORER (NCT02561988) and from interim analysis of the phase II PATHFINDER (NCT03580655) studies. <h3>Methods</h3> Patients included in this <i>post hoc</i> pooled analysis were ≥18 years of age with centrally confirmed diagnosis of AdvSM and treated with once-daily avapritinib at starting doses up to 200 mg. Overall response rate (ORR), defined as complete remission with full (CR) or partial hematologic recovery (CRh), partial remission (PR), or clinical improvement, was evaluated per modified IWG-MRT-ECNM criteria. Other analyses included overall survival (OS), changes from baseline in mast cell disease burden, and safety. <h3>Results</h3> Efficacy of avapritinib was analyzed in 53 ORR-evaluable patients; median age was 67 years (range, 37–85), 32% had an ECOG PS of 2–3, 66% had prior anti-neoplastic therapy, 47% received prior midostaurin, and 94% had a D816V mutation. Responses occurred in 72% (95% CI, 58–83) of patients, with 57% achieving a PR or better (95% CI, 42–70) and 28% with a CR/CRh. Median duration of response was 38.3 months (95% CI, 21.7–NE), and median time to response was 1.9 months (range, 0.3–26.7), with responses deepening over time. Median estimated 12-month OS rate was 84%. Patients achieved ≥50% reductions from baseline in serum tryptase (49/53, 92.5%), marrow mast cell aggregates (46 of 52 evaluable, 88.5%), and blood KIT D816V allele fraction (37 of 52 evaluable, 71.2%). In all AdvSM patients (safety population) receiving the starting avapritinib dose of 200 mg (n=80), common AEs were edema (79%), diarrhea (28%), nausea (24%), and fatigue/asthenia (23%). Decreased platelets (64%), hemoglobin (55%), and neutrophils (54%) from baseline were the most common laboratory abnormalities. Overall, 4 (5%) patients discontinued treatment due to drug-related AEs and 6 (8%) from disease progression. Of 2 (3%) AE-related deaths, neither were treatment-related. <h3>Conclusions</h3> Pooled study results showed ≤200 mg avapritinib induced rapid and deepening clinical responses and had a manageable safety profile.

MPN-180: A Novel Mechanism of Action of Midostaurin in Systemic Mastocytosis: Beyond KIT Inhibition

Context: Constitutively active KIT tyrosine kinase harboring the D816V mutation is the major driver of systemic mastocytosis (SM) pathogenesis. We have previously reported that the loss of function of SETD2 histone methyltransferase due to reduced protein stability is a common cooperating event in advanced SM (advSM). Objective: To dissect the mechanisms of action of midostaurin in advSM and to explore the potential of SETD2 as a marker of response to therapy. Materials: The mast cell (MC) leukemia cell line HMC-1 (negative for SETD2) and primary neoplastic MCs from 10 advSM patients, collected at regular time points during midostaurin treatment. Results: This study started from our preliminary observations indicating that aurora kinase A (AKA) is overexpressed and hyperactivated in advSM and is implicated in SETD2 proteasomal degradation. The new gold standard of therapy in advSM is midostaurin, which targets KIT andseveral other kinases, including AKA. We, thus, investigated whether midostaurin treatment may result in efficient AKA inhibition and subsequent SETD2 rescue. To this purpose, the HMC-1 cell line was treated with 5 µM midostaurin for 24 h, and phospho-AKA (T288), SETD2, and H3K36me3 (surrogate marker of SETD2 activity) were evaluated by WB. Midostaurin reduced AKA phosphorylation by 60%, partially restoring SETD2 expression and activity. We next wondered whether the same happens in patients treated with midostaurin. We, thus, collected and performed WB analysis on neoplastic mast cells from advSM patients before and during midostaurin treatment. We found that midostaurin may target AKA and rescue SETD2 expression and function in vivo and that SETD2/H3K36me3 rescue correlates with the clinical response to midostaurin and the reduction of KIT D816V allele burden, as assessed by digital PCR at 3, 6, and 12 months. In 8 patients who responded to midostaurin, SETD2/H3K36me3 rescue was detected by WB as early as 3 months after treatment onset, whereas 2 refractory patients did not show any increase in SETD2/H3K36me3 and maintained an unaltered KIT allele burden. Conclusions: Midostaurin treatment induces AKA partial dephosphorylation and consequent rescue of SETD2 expression and function in vitro and in vivo SETD2/H3K36me3 may serve as a biomarker of response to midostaurin. Supported by AIRC project 23001

Abstract CT023: PATHFINDER: Interim analysis of avapritinib (ava) in patients (pts) with advanced systemic mastocytosis (AdvSM)

Abstract Introduction: Systemic mastocytosis is a clonal, hematologic neoplasm driven by KIT D816V mutation in &amp;gt;90% of cases, with limited treatment options. In a phase I study, ava, a potent inhibitor of KIT D816V, induced durable responses which deepened over time in pts with AdvSM, regardless of prior therapy or AdvSM subtype. PATHFINDER (NCT03580655) is a pivotal open-label, single-arm phase II study of ava in pts with AdvSM. Methods: Pts were aged ≥18 years with centrally confirmed diagnosis of an AdvSM subtype. Primary endpoint was overall response rate (ORR) by modified International Working Group-Myeloproliferative Neoplasms Research and Treatment and European Competence Network on Mastocytosis criteria. This pre-specified interim analysis included 32 response-evaluable pts. Secondary endpoints included mean baseline change in AdvSM-Symptom Assessment Form Total Symptom Score (TSS) (Gotlib J et al. ASH 2018) and safety. Results: At June 23, 2020, 62 pts with AdvSM received ava primarily at 200 mg orally once daily (QD). Median age was 69 years (range 31-88), 31% had ECOG PS 2-3, and 68% had prior systemic treatment (55% with midostaurin). At 10.4 months median follow-up, 52/62 (84%) pts remained on treatment. ORR was 75% (95% CI 57-89; P=1.6×10-9) in 32 response-evaluable pts. Complete remission with full or partial hematologic recovery occurred in 19% of pts. Median time to response was 2 months; responses deepened over time. Median overall survival (OS) was not reached; estimated 12-month OS was 87%. There were ≥50% reductions from baseline serum tryptase (87%; n=54), marrow mast cell aggregates (71%; n=44), and KIT D816V allele fraction (53%; n=33). Mean TSS at baseline (n=56) was 18.3; fatigue, spots, itching, flushing, and abdominal pain were the most severe symptoms. Mean change in TSS was -7.7 (1-sided P&amp;lt;0.001) after 6 months of treatment (n=36). Common (≥25%) adverse events (AEs; all grade, Grade ≥3) were peripheral (50%, 3%) and periorbital (35%, 3%) edema, thrombocytopenia (32%, 8%), and anemia (29%,16%). Overall, 5% of pts discontinued due to a treatment-related AE and 5% due to disease progression. There were 3 (5%) deaths, all unrelated to treatment. Seven (11%) pts had cognitive effect AEs (all Grade 1-2). One pt with pre-treatment severe thrombocytopenia (platelets &amp;lt;50×109/L) had Grade 4 subdural hematoma. Subsequent pts with pre-treatment severe thrombocytopenia were excluded, platelet monitoring was intensified, and dose interruption for severe thrombocytopenia was recommended; no intracranial bleeding events occurred in 57 pts without pre-treatment severe thrombocytopenia. Conclusions: The pivotal PATHFINDER study showed that ava 200 mg QD induced rapid responses, which deepened over the course of treatment, and improved symptoms in pts with AdvSM. Ava was generally well tolerated with a manageable safety profile, including effective thrombocytopenia risk mitigation. Citation Format: Daniel J. DeAngelo, Andreas Reiter, Deepti Radia, Michael W. Deininger, Tracy I. George, Jens Panse, Alessandro M. Vannucchi, Madlen Jentzsch, Iván Alvarez-Twose, Andrzej Mital, Olivier Hermine, Ingunn Dybedal, Elizabeth O. Hexner, Lisa K. Hicks, Lambert Span, Ruben Mesa, Prithviraj Bose, Kristen M. Pettit, Mark L. Heaney, Stephen Oh, Jayita Sen, Hui-Min Lin, Brenton G. Mar, Jason Gotlib. PATHFINDER: Interim analysis of avapritinib (ava) in patients (pts) with advanced systemic mastocytosis (AdvSM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT023.

Digital Droplet PCR: Next Generation KIT D816V Testing As Advanced Tool for Diagnosis and Prognostication in Mastocytosis

Background: The somatic mutation KIT D816V is a diagnostic criterion for systemic mastocytosis (SM) and can be found in the majority of patients. Therefore, sensitive detection of KIT D816V in blood and bone marrow (BM) is important for diagnosis of SM. Additionally, precise quantification of the KIT D816V allele burden is relevant clinically as it helps to predict multilineage involvement and prognosis in advanced SM and might be useful as a biomarker for treatment response to midostaurin or other interventional drugs. However, despite the high relevance of reliable KIT D816V allele burden measurement, no single assay for the detection and quantification of the KIT mutation has been accepted as a global standard. In addition, differences in calibration material and data normalization hamper the standardization of KIT D816V quantification and complicate the comparison of results obtained in different study groups. Digital droplet PCR (ddPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations.Methods: We performed a validation study of the PrimePCR ddPCR mutation assay for KIT wild-type and the KIT D816V point mutation using the QX-200 droplet-reader on 302 DNA samples from 156 patients with mastocytosis (median age 56 years; 85 females, 71 males) in comparison to melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) (Sotlar et al., 2003, AJP 162, 737-74) and allele-specific quantitative real-time PCR (qPCR) (Kristensen et al., 2011, JMD 13, 180-188). According to WHO criteria, 16 patients were diagnosed with cutaneous mastocytosis, 5 with mastocytosis in the skin, 105 with indolent SM, 7 with smoldering SM, 1 with BM mastocytosis, 8 with aggressive SM, 12 with SM with associated hematologic neoplasm, and 2 with mast cell leukemia. Data were analyzed using the Bland-Altman plot, ordinary least squares regression and Passing-Bablok regression for method comparison, Kaplan-Meier survival plots, and log-rank test.Results: ddPCR was found to be highly sensitive, specific and precise in detecting KIT D816V with a limit of detection of 0.005% variant allele fraction and a coefficient of variation <20%. ddPCR identified the mutation in 90% of all mastocytosis patients compared to 89% with qPCR and 70% with clamp-PCR indicating a superior diagnostic yield of ddPCR over clamp-PCR (p<0.001). In addition, ddPCR was also found to be suitable to detect and quantify KIT D816V in DNA isolated from formalin-fixed paraffin-embedded (FFPE) BM sections. In peripheral blood and BM aspirates, ddPCR for KIT D816V showed a high concordance to qPCR (r=0.99). No systematic deviation for the conversion from qPCR to ddPCR was found (slope of 1.00 of log-transformed data; 95% confidence interval 0.98 to 1.02). In a next step, we confirmed the clinical value of KIT D816V allele burden measurements by ddPCR. In these studies, patients with advanced SM showed a significantly higher KIT D816V allele burden (median 2.43%) compared to patients with indolent SM (median 0.14%; p<0.001). Moreover, ddPCR confirmed the prognostic significance of a high KIT D816V allele burden regarding survival of KIT D816V+ SM patients (n=115) using a cutoff level of 2% mutant alleles. In the group with <2% mutant allele burden the median survival was not reached, whereas in patients with a KIT D816V allele burden of ≥2% the median survival was 7.7 years (p<0.001).Conclusion: We performed a comprehensive validation of ddPCR for KIT D816V in peripheral blood and BM in order to establish a new reliable, quantitative qPCR technique for patients with mastocytosis. ddPCR is highly sensitive and specific for detection and quantification of KIT D816V, gives highly reproducible results even at a very low variant allele fraction level, and confirms the clinical value of KIT D816V allele burden measurement in blood and BM aspirates. In addition, ddPCR for quantification of KIT D816V can be performed in FFPE BM section material. Finally, ddPCR-based quantification of KIT D816V is not limited by the necessity to use generally accepted calibration material for assay standardization. In this regard, we propose to assess the ddPCR for KIT D816V in multicentric studies to increase comparability of allele burden data from different study groups. These studies will be a next crucial step towards the implementation of KIT D816V allele burden measurement as a global standard in clinical practice. [Display omitted] DisclosuresSperr:Teva: Honoraria; Novartis: Other: Register; Meda: Research Funding; Phadia: Research Funding; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Valent:Blueprint: Research Funding; BMS: Honoraria; Incyte: Honoraria; Ariad: Honoraria, Research Funding; Pfizer: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Deciphera: Honoraria, Research Funding; Teva: Honoraria. Hoermann:Gilead: Honoraria, Research Funding; Amgen: Honoraria; Ariad: Honoraria; Novartis: Honoraria.

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

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.

Pioneer Part 2: A Randomized, Double-Blind, Placebo-Controlled, Phase 2 Study to Evaluate Safety and Efficacy of Avapritinib in Indolent Systemic Mastocytosis

Pioneer Part 2: A Randomized, Double-Blind, Placebo-Controlled, Phase 2 Study to Evaluate Safety and Efficacy of Avapritinib in Indolent Systemic Mastocytosis

MDS-227: Digital PCR for Sensitive Detection and Accurate Quantification of KIT D816V Allele Burden in Patients with Suspected Systemic Mastocytosis

Context: Detection of the KIT D816V mutation in the bone marrow (BM) is one of the minor criteria for the diagnosis of systemic mastocytosis (SM). Because in most SM patients, the number of neoplastic mast cells (MCs) in the BM is low, the use of molecular techniques, such as ASO-qPCR, with elevated sensitivity is recommended. Droplet digital PCR (ddPCR) is a promising alternative to ASO-qPCR for D816V testing. Objective: We evaluated a commercial ddPCR assay for the detection of KIT D816V in SM. ddPCR was performed on a BioRad QX200 instrument using the ddPCR™ Mutation Assay: KIT p.D816V, Human (Bio-Rad). Patients: PB samples from 13 healthy donors (HDs) were used as negative controls; the D816V-positive HMC-1.2 cell line was used as positive control. Thirty-five BM samples from patients with suspected SM, either with neoplastic mast cell infiltration Results: In HMC 1.2 cells, ddPCR measured an allele burden (AB) of 50%, consistent with the heterozygous status. No D816V-positive events were detected in the HDs. Comparison between ddPCR and ASO-qPCR revealed high concordance in mutation detection and quantitation, even at low AB levels. ddPCR identified D816V in 70/151 patients prospectively evaluated for diagnosis of SM. According to WHO criteria, 49 were later diagnosed with ISM, 13 with aggressive SM, 4 with smouldering SM, 2 with SM with an associated clonal hematopoietic non-MC disease, and 2 with MC leukemia. The median AB in all positive SM patients was 0.34%. Patients with advanced SM showed a higher D816V AB (median, 15.23%) compared with patients with ISM and smouldering SM (median, 0.27%). All patients positive for D816V in BM also tested positive in the PB. Conclusion: ddPCR proved accurate and precise and was able to detect D816V in PB when the BM was positive. We propose ddPCR as a valid alternative to ASO-qPCR for straightforward and sensitive detection of D816V in SM.

Inhibitory effects of midostaurin and avapritinib on myeloid progenitors derived from patients with KIT D816V positive advanced systemic mastocytosis

Advanced systemic mastocytosis (advSM) is characterized by the presence of an acquired KIT D816V mutation in >90% of patients. In the majority of patients, KIT D816V is not only detected in mast cells but also in other hematopoietic lineages. We sought to investigate the effects of the KIT-inhibitors midostaurin and avapritinib on single-cell-derived myeloid progenitor cells using granulocyte-macrophage colony-forming-units of patients with KIT D816V positive advSM. Colonies obtained prior to treatment were incubated in vitro with midostaurin (n = 10) or avapritinib (n = 11) and showed a marked reduction (≥50%) of KIT D816V positive colonies in 3/10 (30%) and 7/11 (64%) patient samples, respectively. Three of those 7 (43%) avapritinib responders were resistant to midostaurin in both, in vitro and in vivo. Colonies from four patients with high-risk molecular profile and aggressive clinical course were resistant to both drugs. The in vitro activity of midostaurin strongly correlated with clinical and molecular responses, e.g., relative reduction of KIT D816V allele burden and the proportion of KIT D816V positive colonies obtained after six months midostaurin-treatment in vivo. We conclude that the colony inhibition assay provides useful information for prediction of responses on midostaurin and that avapritinib has a superior in vitro activity compared to midostaurin.

Molecular Quantification of Tissue Mast Cell Burden in Systemic Mastocytosis: A New Approach for Diagnostics and Prognostication

Background: The somatic KIT D816V mutation leads to an activation of the receptor tyrosine kinase KIT and represents a diagnostic criterion for systemic mastocytosis (SM). In the majority of patients, only a very few KIT D816V+ mast cells (MC) or MC precursors, if any, are found in peripheral blood (PB) and bone marrow (BM) aspirates. In contrast, the MC count in BM biopsies is typically much higher. Melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) is widely used for detecting KIT mutations in biopsies.1 We have previously proposed dPCR as a new standard method of KIT D816V testing in SM that can also reliably quantify the variant allele fraction (VAF) in formalin-fixed paraffin-embedded (FFPE) material.2 While multilineage involvement of KIT D816V indicated by a high VAF in PB was associated with an aggressive clinical course,3-6 the mutant allele burden in tissue sections has not been studied in a large series of SM patients.Methods: The VAF of KIT D816V in the tissue was assessed by dPCR (PrimePCR ddPCR, Biorad) on DNA isolated from 211 FFPE BM sections from 116 patients with SM (median age 53 years; 58 females, 58 males) and 57 controls (lymphoma patients undergoing staging biopsy). A total of 91 SM patients were diagnosed with indolent SM (ISM) and 25 with advanced SM (aggressive SM, SM with associated hematologic neoplasm, and mast cell leukemia according to WHO criteria). Results of diagnostic samples were compared to qualitative analysis of KIT D816V clamp-PCR. BM MC infiltration was quantified in a representative area of the tryptase stained section and expressed as % of nucleated cells. Total tryptase levels in serum were determined by a fluoroenzyme-immunoassay.Results: The KIT D816V mutation burden in FFPE BM sections of SM patients showed marked differences between patients ranging from 0.027% to 60% VAF (median: 1.9%). The VAF in BM sections was largely independent from that in liquid specimen (PB and BM aspirate). In particular, a number of ISM patients showed substantially higher VAF in FFPE tissue than in liquid specimen (Figure 1A). In line with this, a higher correlation of KIT D816V mutation burden to BM MC infiltration and serum tryptase levels was observed for FFPE tissue (Spearman's coefficient of correlation r = 0.68 and r = 0.68 respectively; Figure 1B-C) than for liquid specimen (r = 0.48 and r = 0.58 respectively). When analyzing subgroups of SM, FFPE BM sections of patients with advanced SM had a significantly higher KIT D816V allele burden (median 23.40%) compared to patients with ISM (median 1.65%; p<0.001 Figure 1D). Finally, we assessed the sensitivity and specificity of dPCR for KIT D816V analysis in FFPE BM section compared to clamp PCR. While a specificity of 100% was observed for both assays, dPCR showed a significantly higher sensitivity to detect the mutation in BM tissue of ISM patients compared to clamp PCR (97% vs. 89%; p<0.05).Conclusion: dPCR is a sensitive method to detect KIT D816V and reliably quantify the mutant allele burden in FFPE BM sections of SM patients. The mutational burden in the tissue represents a new molecular marker of the disease burden in SM since it reflects both the multilineage involvement of KIT D816V and the MC burden while the mutation burden in PB primarily reflects the KIT D816V multilineage involvement. Although the tissue mutational burden in patients with advanced SM is higher compared to ISM, the difference is not as substantial as described for PB. Thus, the prognostic value of KIT D816V allele burden measurement in SM needs to be redefined for BM sections. Potential future applications of KIT D816V quantification in FFPE BM sections include treatment response monitoring and definition of high disease burden in smoldering SM (SSM). We propose to include dPCR-based KIT D816V mutant allele burden measurement in BM tissue as a new biomarker for disease burden in SM in future clinical trials.

Digital PCR: A Sensitive and Precise Method for KIT D816V Quantification in Mastocytosis.

The analytically sensitive detection of KIT D816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the KIT D816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations. We performed a validation study of dPCR for KIT D816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR). dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR (P < 0.001). Moreover, dPCR for KIT D816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of KIT D816V VAF measurements. Thus, patients with advanced SM showed a significantly higher KIT D816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; P < 0.001). Moreover, dPCR confirmed the prognostic significance of a high KIT D816V VAF regarding survival (P < 0.001). dPCR for KIT D816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of KIT D816V allele burden measurement. Thus, dPCR is suitable as a new method for KIT D816V testing in patients with mastocytosis.

Omalizumab prevents anaphylaxis and improves symptoms in systemic mastocytosis: Efficacy and safety observations.

Patients with systemic mastocytosis (SM) may suffer from mast cell (MC) mediator-related symptoms insufficiently controlled by conventional therapy. Omalizumab is an established treatment in other MC-driven diseases, but experiences in SM are limited. To assess the efficacy and safety of omalizumab in SM. In our patient cohort, we evaluated all SM patients treated with omalizumab. A physician global assessment of type and severity of symptoms was performed at baseline, at 3 and 6months and at latest follow-up. Quality of life was assessed by visual analogue scale. S-tryptase and KIT D816V allele burden were monitored. A total of 14 adult SM patients (10 ISM, 2 BMM, 1 SSM, and 1 ASM-AHN) received omalizumab with a median duration of 17months (range: 1-73months). One patient was excluded due to concomitant cytoreductive therapy. In the remaining 13 patients, we observed a significant reduction in symptoms, with complete symptom control in five (38.5%), major response in three (23.1%), and a partial response in three (23.1%) patients, whereas two patients (15.4%) withdrew due to subjective side-effects at first dose. The treatment was most effective for recurrent anaphylaxis and skin symptoms, less for gastrointestinal, musculoskeletal, and neuropsychiatric symptoms. Patient-reported quality of life showed significant improvement. No significant changes in s-tryptase/KIT D816V allele burden were observed. No severe adverse events were recorded. Omalizumab appears to be a promising treatment option in SM, effectively preventing anaphylaxis and improving chronic MC mediator-related symptoms, insufficiently controlled by conventional therapy. Controlled studies are needed to substantiate findings.

Inhibitory Effects of Midostaurin and Blu-285 on Myeloid Progenitor Cells Derived from Patients with Multi-Mutated KIT D816V+ Advanced Systemic Mastocytosis

▪Recent data have highlighted that the molecular pathogenesis of advanced systemic mastocytosis (advSM) is complex. In addition to the phenotypically most important mutations in KIT, e.g. KIT D816V in 80-90% of patients, one or more additional mutations, e.g. in SRSF2, ASXL1, RUNX1, CBL, JAK2 and others, are present in 60-70% of patients (Jawhar et al., Leukemia 30, 2016). In individual patients, a complex mutational profile is detected not only in mature mast cells (MCs) but also in myeloid progenitors derived from granulocyte-macrophage colony-forming progenitor cells (CFU-GM), indicating multi-lineage involvement of all identified mutations in the vast majority of patients (Jawhar et al., Leukemia 29, 2015). Midostaurin, a multi-targeted kinase inhibitor, has demonstrated an overall response rate of 60% in advSM patients (Gotlib et al., NEJM 374, 2016). BLU-285 is a highly selective KIT D816V kinase inhibitor which has demonstrated biochemical activity on the mutated KIT enzyme (KIT D816V IC50 = 0.27 nM). In the current study, we sought to a) investigate the inhibitory effects of midostaurin and BLU-285 on single-cell-derived CFU-GM from bone marrow mononuclear cells derived from multi-mutated KIT D816V+ advSM patients and b) correlate the midostaurin CFU-GM data with clinical and various response parameters in midostaurin-treated advSM patients. The mutational status of CFU-GM colonies (median colonies per patient, n=20; range 10-30) was analyzed for KIT D816V and additional mutations by PCR followed by Sanger Sequencing. In 10 multi-mutated advSM patients (aggressive SM [n=8] or mast cell leukemia [n=2] with an associated hematological neoplasm), CFU-GM colonies were screened prior to midostaurin (month 0, n=10) and, if available, at month 6 on midostaurin (n=8). At month 0, a median of 90% (range, 40-100) CFU-GM colonies were KIT D816V+, while at month 6 a median of 70% (range, 5-100) CFU-GM colonies were KIT D816V+. A significant relative reduction (≥50%) in the proportion of KIT D816V+ colonies at month 6 was observed in 4/8 (50%) patients. Midostaurin-naïve CFU-GM were incubated with midostaurin at concentrations up to 1000 nM and showed a dose-dependent significant reduction (≥50%) of KIT D816V+ colonies in 1/7 (14%) patients. Overall, the in vitro effects correlated with the in vivo effects of midostaurin on CFU-GM and established IWG-MRT-ECNM response criteria (e.g. mast cell infiltration in BM, serum tryptase level) and KIT D816V allele burden in peripheral blood. Midostaurin-naïve CFU-GM from 7/10 (70%) patients were also incubated with different concentrations of BLU-285 ranging from 0 to 75 nM. A dose-dependent, significant relative reduction (≥50%) of KIT D816V+ CFU-GM colonies was observed at concentrations between 45 and 75nM in 5/7 (71%) patients. Of interest, 3/5 (60%) in vitro responders to BLU-285 were resistant to midostaurin (in vivo and in vitro) while CFU-GM colonies from 2 patients resistant to BLU-285 were also resistant to midostaurin. In addition to KIT D816V, recurrent molecular aberrations (median 2/patient, range 1-3) were identified in all patients, most frequently in SRSF2 (n=9), TET2 (n=7) and ASXL1 (n=4). Neither drug had an effect on the relative frequency of additional mutations in CFU-GM colonies. In summary, we conclude that a) the relative reduction of KIT D816V+ CFU-GM colonies between month 0 and month 6 on midostaurin correlates with clinical response, b) the CFU-GM colony assays may provide useful information for prediction of response to midostaurin, c) the highly selective KIT D816V inhibitor BLU-285 has significant activity against KIT D816V, even in cases which are resistant to midostaurin, and d) neither drug had an effect on the prognostically relevant additional mutations. DisclosuresEvans:Blueprint Medicines: Employment, Equity Ownership. Gardino:Blueprint Medicines Corporation: Employment. Lengauer:Blueprint Medicines Corporation: Employment.

Impact of Molecular Markers on Response and Resistance in Midostaurin-Treated Patients with Advanced Systemic Mastocytosis

▪Systemic mastocytosis (SM) arises as a consequence of aberrant activation of KIT signaling, most commonly by acquisition of the KIT D816V mutation (>80-90% of patients). A recently reported phase-II-study exploring the efficacy and safety of midostaurin, a multi-targeted KIT inhibitor, in patients with advanced SM (advSM) has demonstrated major and partial responses in 60% of patients (Gotlib et al., NEJM 374, 2016). However, recent data have highlighted that the molecular pathogenesis of SM is complex. In particular, additional mutations in SRSF2, ASXL1 and/or RUNX1 (S/A/Rpos), seen in 60-70% of patients with advSM, have a significant adverse impact on disease phenotype and prognosis (Jawhar et al., Leukemia 30, 2016). In the current study, we evaluated the impact of molecular markers on response, resistance, progression and overall survival (OS) in midostaurin-treated advSM patients: 1) the S/A/R mutation profile at diagnosis, 2) the reduction of the KIT D816V allele burden (EAB) and 3) the dynamics of the clonal architecture. Between 2009 and 2015, 38 patients (68% male; median age 67 years; range, 48-76) with advSM [SM with an associated hematological neoplasm (SM-AHN), n=22; aggressive SM (ASM), n=3; mast cell leukemia (MCL), n=4; MCL-AHN, n=9] received midostaurin at our institution and were monitored for the KIT D816V EAB by quantitative RT-PCR (RT-qPCR). In addition, serial Next Generation Sequencing (NGS) analyses were performed in 16/25 multi-mutated patients. Median time from diagnosis of advSM to start of midostaurin was 12 months (range, 1-60). Twenty-one of 38 (55%) patients died; median OS was 29 months (range, 0-88) from start of midostaurin and 40 months (range, 5-115) from diagnosis. Three patients (8%) stopped midostaurin early (median 2 months; range, 1-4) because of intolerance. In the remaining 35 (92%) patients, median treatment duration was 13 months (range, 1-88). Progression/death occurred in 6/35 (17%) patients within the first 6 months and 5 (83%) patients were S/A/Rpos. Overall response rate (ORR) according to IWG-MRI-ECNM consensus criteria and OS were significantly different between S/A/Rpos (n=23) vs. S/A/Rneg (n=12) patients [ORR: 35% vs. 75%, P=0.01; OS: P=0.01, HR 4.5 (1.3-16.2), Figure 1A]. The maximal median reduction of KIT D816V EAB in 28 patients treated for >6 months was -29% (range, -100 to 71). Depending on the KIT D816V EAB at month 6, patients were classified as responders (≥25%, n=17) or non-responders (<25%, n=11). Responders were significantly associated with a longer median time on midostaurin (25 vs. 9 months, P=0.01) and achievement of any clinical response according to IWG-MRI-ECNM criteria (13/17 vs. 2/11 patients, P=0.006). All 4 patients who lost their KIT D816V EAB response were S/A/Rpos. In univariate analyses of multiple clinical and molecular response parameters at month 6, three parameters were significantly associated with inferior OS: reduction of KIT D816V EAB <25% (P=0.0004), serum tryptase <50% (P=0.03) and alkaline phosphatase <50% (P=0.04). In multivariate analysis, only KIT D816V EAB reduction <25% remained an independent poor prognostic marker for reduced OS (P=0.004, HR 6.8 [1.8-25.3]) and was superior to the IWG-MRI-ECNM consensus criteria (P=0.005, HR 4.7 [1.5-15.5]) (Figure 1B). Serial NGS analysis of 7 deceased patients revealed acquisition of additional mutations in RUNX1 (n=2), K/NRAS (n=3), IDH2 (n=1) or NPM1 (n=1) and/or increasing mutation allele burdens while KIT D816V EAB was either low (n=2), stable (n=1) or increasing (n=4). In contrast, two patients acquired a JAK2 V617F mutation but remained in remission on midostaurin. In summary, we have found that S/A/Rneg at diagnosis and reduction of the KIT D816V EAB ≥25% at month 6 were the most favorable predictors for survival in midostaurin-treated advSM patients. S/A/Rpos at diagnosis was associated with a more aggressive phenotype, (early) resistance/progression and poor survival. Serial sequencing during treatment also revealed that secondary resistance/transformation may be caused by expansion of sub-clones exhibiting new mutations in critical target genes independent of KIT D816V. In addition to established response parameters, sequential analysis of KIT D816V EAB and other molecular aberrations add relevant information for optimal management and response assessment in midostaurin-treated patients with advSM. [Display omitted] DisclosuresValent:Celegene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria. Meggendorfer:MLL Munich Leukemia Laboratory: Employment.

Additional Mutations in SRSF2, ASXL1 and/or RUNX1 Identify a High Risk Group of Patients with KIT D816V+ Advanced Systemic Mastocytosis

The presence of multiple additional mutations was recently reported in >90% of patients with KIT D816V+ advanced systemic mastocytosis (SM) (Schwaab et al., Blood 2013). In the vast majority of cases, additional mutations occur prior to KIT D816V indicating a multi-mutated stem cell disease with phenotype modification through KIT D816V (Jawhar et al., Leukemia 2015). We now sought to clarify the prognostic impact of individual mutations and the number of mutated genes in 70 multi-mutated patients with ISM (n=26), aggressive SM (ASM, n=34) and mast cell leukemia (MCL, n=10). All patients had an associated clonal hematologic non-mast-cell disease (AHNMD), such as a myelodysplastic/myeloproliferative syndrome unclassified (MDS/MPNu, n=28), chronic myelomonocytic leukemia (CMML, n=21), chronic eosinophilia leukemia (CEL, n=9), acute myeloid leukemia (AML, n=8) or primary mylofibrosis (PMF, n=4). Mutational analysis was performed by next-generation sequencing in the following genes: ASXL1, CBL, ETV6, EZH2, IDH2, JAK2, KRAS, NRAS, RUNX1, SF3B1, SRSF2, TET2 and U2AF1. The most frequently identified mutated genes were TET2 (n=33, 47% of cases), SRSF2 (n=30, 43%), ASXL1 (n=20, 29%), RUNX1 (n=16, 23%) and JAK2 (n=11, 16%). The molecular data were correlated with overall survival (OS), clinical characteristics (e.g. B- and C-findings), bone marrow (BM) findings, serum tryptase levels and KIT D816V allele burden in peripheral blood. With a median observation time of 3.7 years, 25 of 70 patients (36%) have died. Three-year OS probability was 71%. In univariate analysis, OS was adversely influenced by mutations in SRSF2 ([hazard ratio (HR) 5.9, 95% confidence interval (CI)] [2.2-15.4], p<0.0001), ASXL1 (HR 3.4 [1.5-7.7], p=0.002) and RUNX1 (HR 2.4 [1.1-5.4], p=0.03) but was not influenced by mutations in TET2 (p=0.3) or JAK2 (p=0.1). In multivariate analysis of the most frequently mutated genes, SRSF2 remained as the best independent poor risk marker for OS. Furthermore, inferior OS was significantly associated with the number of mutated genes in SRSF2/ASXL1/RUNX1 (S/A/R), with median OS not reached for 0 mutation (n=25), 3.9 years for 1 mutation (n=27) and 2.7 years for >=2 mutations (n=18). The 3-year OS was 90% in patients with 0 mutation, 73% in patients with 1 mutation and 42% in patients with >=2 mutations in the S/A/R gene panel. Pairwise significantly different OS probabilities were observed for the comparisons 0 vs. 1 mutation (HR 9.2 [2.0-42.3], p=0.001), 0 vs. >=2 mutations (HR 4.1 [1.9-8.8], p<0.0001) and 1 and >=2 mutations in the S/A/R gene panel (HR 2.5 [1.1-6.0], p=0.04) (Figure 1). Regarding clinical characteristics, the S/A/R mutation profile is significantly related to C-findings including hepatosplenomegaly, albumin <35g/L, alkaline phosphatase >150 IU/L, ascites and weight loss, and other important disease-related parameters including bone marrow mast cell infiltration, serum tryptase levels and KIT D816V+ allele burden. We therefore conclude that a) presence and number of mutations in the S/A/R gene panel are adversely associated with advanced disease and poor survival in KIT D816V+ SM patients and b) the inclusion of molecular markers should be considered in upcoming prognostic scoring systems for SM patients. [Display omitted] DisclosuresSchnittger:MLL: Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Sotlar:Ludwig-Maximilians-University: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Valent:Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria; Ariad: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Cross:Qiagen: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

KIT mutation analysis in mast cell neoplasms: recommendations of the European Competence Network on Mastocytosis.

Although acquired mutations in KIT are commonly detected in various categories of mastocytosis, the methodologies applied to detect and quantify the mutant type and allele burden in various cells and tissues are poorly defined. We here propose a consensus on methodologies used to detect KIT mutations in patients with mastocytosis at diagnosis and during follow-up with sufficient precision and sensitivity in daily practice. In addition, we provide recommendations for sampling and storage of diagnostic material as well as a robust diagnostic algorithm. Using highly sensitive assays, KIT D816V can be detected in peripheral blood leukocytes from most patients with systemic mastocytosis (SM) that is a major step forward in screening and SM diagnosis. In addition, the KIT D816V allele burden can be followed quantitatively during the natural course or during therapy. Our recommendations should greatly facilitate diagnostic and follow-up investigations in SM in daily practice as well as in clinical trials. In addition, the new tools and algorithms proposed should lead to a more effective screen, early diagnosis of SM and help to avoid unnecessary referrals.

Molecular Profiling of Myeloid Progenitor Cells in Multi-Mutated Advanced Systemic Mastocytosis Identifies KIT D816V As a Distinct and Late Event

Systemic mastocytosis (SM) is characterized by abnormal proliferation and accumulation of mast cells (MC) in various tissues and organs, predominantly skin, bone marrow (BM) and visceral organs. The extent of organ infiltration and subsequent organ damage is the basis for the classification of SM into indolent SM (ISM), smoldering SM (SSM), SM with associated clonal hematologic non-MC lineage disease (SM-AHNMD), aggressive SM (ASM) and MC leukemia (MCL). Depending on the subtype of SM, cell source (BM or peripheral blood) and assay sensitivity, an acquired mutation in the receptor tyrosine kinase KIT, usually KIT D816V, is detectable in 80-90% of patients. Next-Generation Deep Amplicon Sequencing (NGS) was performed to investigate 18 candidate genes at known mutational hotspot regions as previously described. Additional mutations in genes encoding for signaling molecules (JAK2, CBL, KRAS, NRAS), transcription factors (RUNX1), epigenetic regulators (ASXL1, DNMT3A, EZH2, TET2) or splicing factors (SRSF2, SF3B1, U2AF1) are detectable in the vast majority of patients KIT D816V+ advanced SM. In order to gain more insight into clonal evolution of myeloid progenitors in indolent and advanced SM with or without AHNMD, we explored the mutation profile of single-cell-derived CFU-GM colonies - by using Sanger sequencing - in 19 KIT D816V+ SM patients (investigated colonies, n=285; median per patient, n=15; range 10-30). The study included 7 patients with ISM/SSM/ASM (0 additional mutations), 4 patients with SM-AHNMD (median 1 additional mutation, range 1-4) and 8 patients with ASM-AHNMD (median 3 additional mutations, range 1-4). KIT D816V+ CFU-GM could be identified in all 8 patients with ASM ± AHNMD but in only 20% (1/5) of patients with SM-AHNMD, while all CFU-GM colonies derived from ISM patients were completely KIT D816V negative. On the other hand, CFU-GM colonies from individual ASM ± AHNMD patients were never entirely KIT D816V+ (median 60%, range 0-95). In contrast to KIT D816V, additional mutations were identified in CFU-GM colonies from all 12 multi-mutated (A)SM-AHNMD patients and many of these mutations were present in 100% of the CFU-GM-derived colonies analyzed. In 8 patients, different subclones with variable proportions of the number of mutated genes were identified and allowed to generate putative evolutionary trees. These mutations included TET2 and SRSF2 mutations in 6/6 and 4/4 patients with (A)SM-AHNMD. In contrast, ASXL1 mutations were not identified in all TET2/SRSF2 positive CFU-GM colonies suggesting that they are likely to occur later than TET2 and SRSF2. When additional mutations and KIT D816V were detected concomitantly in individual single-cell-derived CFU-GM colonies, the overall frequency of CFU-GM colonies positive for additional mutations was always higher than those that were KIT D816V positive, indicating that the KIT D816V mutation was a secondary event. In contrast to advanced SM, all ISM patients were negative for additional mutations. CFU-GM colonies and also microdissected CD15+ cells derived from BM biopsies were entirely KIT D816V negative in these patients, highlighting that the KIT D816V mutation may be restricted to other (probably later) stages of stem cell development and possibly only to the MC lineage. In general, the relative proportion of KIT D816V+ progenitors correlated well with established parameters for quantification of disease burden, e.g. BM MC infiltration, serum tryptase levels and KIT D816V allele burden in individual patients. In conclusion, the presence of multi-mutated myeloid non-MC lineage progenitors of the CFU-GM-type suggests an initial clonal expansion at an early stage of hematopoietic development due to mutations other than KIT D816V with a subsequent phenotype modification towards SM due to a later acquisition of KIT D816V. In contrast, ISM/SSM is not affected by mutations at the CFU-GM level which may at least in part explain its excellent prognosis. DisclosuresSchnittger:MLL Munich Leukemia Laboratory: Other. Horny:Novartis: Consultancy, Honoraria. Haferlach:MLL: Equity Ownership. Valent:Novartis: Consultancy. Reiter:Novartis: Consultancy, Honoraria.

The KIT D816V allele burden predicts survival in patients with mastocytosis and correlates with the WHO type of the disease.

KIT D816V is present in a majority of patients with systemic mastocytosis (SM). We determined the KIT D816V allele burden by quantitative real-time PCR in bone marrow and peripheral blood of 105 patients with mastocytosis. KIT D816V was detected in 92/105 patients (88%). Significant differences in the median allele burden were observed between disease subgroups: cutaneous mastocytosis (0.042%), indolent SM (0.285%), smoldering SM (5.991%), aggressive SM (9.346%), and SM with associated hematologic non-mast cell lineage disease (3.761%) (P < 0.001). The KIT D816V burden also correlated with serum tryptase (R = 0.5, P < 0.005) but not with mast cell infiltration in bone marrow or mediator symptoms. Moreover, the allele burden was of prognostic significance regarding survival (P < 0.01). Patients responding to cytoreductive therapy showed a significant decrease in KIT D816V (P < 0.05). To conclude, the KIT D816V burden correlates with the variant of mastocytosis, predicts survival, and is a valuable follow-up parameter in SM.