Neoplastic Mast Cells Research Articles

Effects of ibrutinib on proliferation and histamine release in canine neoplastic mast cells.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is effective in the treatment of human chronic lymphocytic leukaemia and mantle cell lymphoma. Recent data have shown that ibrutinib also blocks IgE‐dependent activation and histamine release in human basophils (BAs) and mast cells (MCs). The aim of this study was to investigate whether BTK serves as a novel therapeutic target in canine mast cell tumours (MCTs). We evaluated the effects of ibrutinib on two canine MC lines, C2 and NI‐1 and on primary MCs obtained from canine MCTs (n = 3). Using flow cytometry, we found that ibrutinib suppresses phosphorylation of BTK and of downstream STAT5 in both MC lines. In addition, ibrutinib decreased proliferation of neoplastic MCs, with IC50 values ranging between 0.1 and 1 μM in primary MCT cells and between 1 and 3 μM in C2 and NI‐1 cells. In C2 cells, the combination “ibrutinib + midostaurin” produced synergistic growth‐inhibitory effects. At higher concentrations, ibrutinib also induced apoptosis in both MC lines. Finally, ibrutinib was found to suppress IgE‐dependent histamine release in primary MCT cells, with IC50 values ranging from 0.05 to 0.1 μM in NI‐1 cells, and from 0.05 to 1 μM in primary MCT cells. In summary, ibrutinib exerts anti‐proliferative effects in canine neoplastic MCs and counteracts IgE‐dependent histamine release in these cells. Based on our data, ibrutinib may be considered as a novel therapeutic agent for the treatment of canine MCT. The value of BTK inhibition in canine MCT patients remains to be elucidated in clinical trials.

A phase 2 study of brentuximab vedotin in patients with CD30-positive advanced systemic mastocytosis

AbstractThere is an unmet need for effective therapies for advanced systemic mastocytosis (advSM). CD30 is expressed on the surface of neoplastic mast cells (MC) in more than 50% of patients with advSM. Brentuximab vedotin (BV) is a CD30-directed antibody-drug conjugate with preclinical evidence supporting both an antineoplastic effect and an attenuation of immunoglobulin E-associated mediator release. These observations are the basis for this phase 2 trial of BV monotherapy (1.8 mg/kg IV every 3 weeks up to 8 cycles) in patients with CD30-positive advSM. The primary objective was to determine the efficacy of BV according to International Working Group-Myeloproliferative Neoplasms Research and Treatment-European Competence Network on Mastocytosis (IWG-MRT-ECNM) response criteria. Secondary objectives included evaluation of safety, changes in bone marrow (BM) MC burden, serum tryptase level, flow cytometric quantification of MC surface expression of CD30, and self-reported symptom burden. The trial enrolled 10 patients with a diagnosis of CD30+ advSM (aggressive SM, SM with an associated hematologic neoplasm [SM-AHN], or mast cell leukemia [MCL]) with 1 or more signs of SM-related organ damage. According to IWG-MRT-ECNM criteria, none of the patients demonstrated better than stable disease with BV. In addition, there were no significant reductions in BM MC burden, serum tryptase levels, or MC surface expression of CD30. Self-reported symptom scores showed no durable improvement with BV treatment. We conclude that BV is not active as a single agent in CD30+ advSM. This trial was registered at www.clinicaltrials.gov as #NCT01807598.

The upregulation of Pim kinases is essential in coordinating the survival, proliferation, and migration of KIT D816V-mutated neoplastic mast cells

About ˜80% of mast cell neoplasm patients harbor the c-Kit activating mutation D816 V, which is associated with c-Kit inhibitor resistance and poor prognosis. However, the molecular basis for these effects is not fully known. To address this issue, in this study we screened molecules whose expression is altered by KIT D816 V mutation and found that Pim kinases were overexpressed in D816V-mutant neoplastic mast cells. This was accompanied by upregulation of signal transducer and activator of transcription (STAT) and mammalian target of rapamycin (mTOR) and downregulation of Akt and extracellular signal-regulated kinase (ERK1/2). Activated Pim kinases promoted the survival of D816 V cells by maintaining mTOR and p70S6K activation even under nutrient starvation. Conversely, cell proliferation was suppressed by inhibiting Pim kinases. The mRNA level of C-X-C chemokine receptor type 4 (CXCR4) was about 2-fold higher in D816 V cells; this was associated with a 2-fold increase in migratory capacity, which was modulated by Pim kinases. We also confirmed that upregulation of Pim kinases is a feature specific to cells with the D816 V mutation and is not observed in cells with the c-Kit activating N822 K mutation. These data suggest Pim kinases as a promising therapeutic target for the treatment of mast cell neoplasms with KIT D816 V mutation.

PS1437 SETD2 NON‐GENOMIC LOSS OF FUNCTION IN ADVANCED SYSTEMIC MASTOCYTOSIS (SM): PATHOGENETIC AND THERAPEUTIC IMPLICATIONS

Background:We have recently found that advanced SM patients (pts) and the HMC‐1.1 and −1.2 MCL cell lines display histone H3 Lys36 trimethylation (H3K36Me3) deficiency as a result of non‐genomic loss of function of the SETD2 methyltransferase. SETD2 plays a key role in the control of transcription and splicing fidelity and homologous recombination (HR). Inhibition of proteasome‐mediated degradation by bortezomib restored SETD2 protein expression and H3K36Me3.Aims:In this study, we aimed to identify the different players involved in SETD2 loss of function in ASM and MCL and to verify if they may be considered as promising therapeutic targets. Methods:Western blotting (WB) and immunofluorescence (IF) were used to assess proteins expression. Co‐immunoprecipitation assays were performed to investigate proteins interactions. Apoptosis and clonogenic assays were performed to test drugs efficacy.Results:Compared to cells from healthy donors, SETD2‐ and H3K36Me3‐deficient cell lines and pts had significantly higher levels of γH2AX and lower levels of Rad51. RNA‐seq in SETD2‐deficient pts showed evidence of transcription and splicing defects like transcription‐induced chimeras and intron retention not observed in healthy donors. Moreover we found that SETD2 co‐immunoprecipitates (co‐IP) with p53 in HMC‐1.1 and −1.2 cells. Rescuing the interaction between SETD2 and p53 with bortezomib stabilized p53 and upregulated the expression levels of p53 targets including p21, p27, Bax and Gadd45a. This restored inhibition of cell proliferation, block of the transition towards cell cycle checkpoints and consequent activation of apoptosis. The ubiquitin E3 ligase MDM2 was also found to complex with SETD2 after proteasomal inhibition. Treatment with the MDM2 inhibitor SP‐141 rescued SETD2 expression and H3K36Me3, suggesting that MDM2 may play a role in SETD2 degradation in ASM and MCL. Moreover, SP‐141 treatment of HMC‐1 cells at micromolar doses induced cytostatic but not cytotoxic effects. Clonogenic assays supported the cytostatic effects of SP141 in HMC‐1.1 and −1.2 cells. siRNA‐mediated knock‐down of MDM2 also rescued SETD2 expression and activity, supporting the hypothesis that SETD2 hyper‐ubiquitination by MDM2 plays a role in SETD2 reduced stability and proteasomal degradation. Co‐IP also showed that SETD2 interacts with Aurora Kinase A (AKA), that is overexpressed in advanced SM. We found that AKA phosphorylates SETD2 and this phosphorylation may be involved in SETD2 loss of function. In fact, both inhibition by Danusertib and siRNA‐mediated silencing rescued SETD2 expression and activity. Finally, we performed clonogenic assays to evaluate the therapeutic potential of bortezomib, carfilzomib and ixazomib in neoplastic mast cells from 3 patients with advanced SM and we observed in all cases that both first and second generation inhibitors induced a significant reduction of clonogenic activity at nanomolar doses.Summary/Conclusion:AKA triggered MDM2‐mediated ubiquitination contributes to SETD2 non‐genomic loss of function in ASM and MCL. Loss of SETD2 and H3K36Me3 is associated with increased DNA damage and transcription and splicing defects in ASM and MCL pts and is likely to afford an alternative mechanism for the inactivation of the p53‐mediated checkpoint without the need for TP53 gene mutations. Inhibiting AKA or MDM2 activity or proteasome‐mediated degradation are promising therapeutic strategies. Supported by AIRC (project 16996) and AIL.

S888 TNF‐ALPHA PROMOTES CLONAL EXPANSION OF KIT D816V+ CELLS IN SYSTEMIC MASTOCYTOSIS

Background:Systemic mastocytosis (SM) is characterized by clonal mast cell (MC) infiltration in the bone marrow (BM). Most patients harbor the activating KIT mutation D816V. Cytokines are substantially involved in the interactions between neoplastic cells and the BM microenvironment in SM and in classical myeloproliferative neoplasms (MPN) and have been recognized as a major trigger of disease evolution. The pro‐apoptotic/anti‐proliferative and pro‐inflammatory cytokine tumor necrosis factor alpha (TNFa) has been identified as an important driver for the expansion of JAK2 V617F+ clones and the establishment of MPN.Aims:Investigation of the potential role of TNFa in disease evolution of SM patients.Methods:In vitro and in vivo experiments with MC lines and primary material of SM patients.Results:In a first step, lentivirally‐driven expression of the KIT D816V mutant gene in the human Mo7e cells suggested an oncogene‐dependent regulation of the production of TNFa. Correspondingly, the human neoplastic KIT D816V+ MC lines HMC‐1.2 and ROSAKIT D816V expressed and secreted substantial amounts of TNFa and inhibition of KIT D816V either by the recently approved multi‐kinase inhibitor midostaurin or by RNAi‐mediated knockdown of KIT significantly reduced expression of this cytokine. Mechanistically, we studied auto‐/paracrine effects of TNFa on the clonal selection of neoplastic MC using KIT D816V negative (ROSAKIT WT) and positive (ROSAKIT D816V) subclones of ROSA cells. TNFa reduced proliferation of ROSAKIT WT cells (84.9% ± 4.6% of control, p<0.01) whereas ROSAKIT D816V cells were resistant to TNFa (102.7% ± 3.7% of control, n.s.). Preliminary data also indicate a relative resistance of primary neoplastic cells obtained from KIT D816V+ SM patients against TNFa in colony‐forming unit (CFU) assay. To determine the mechanism of TNFa resistance in KIT D816V+ cells, we screened for key regulators of apoptosis and identified the anti‐apoptotic protein survivin to be highly upregulated in KIT D816V+ ROSA cells upon TNFa treatment but not in KIT D816V‐ ROSA cells. We hypothesize that KIT D816V+ MC produce substantial amounts of TNFa that induces apoptosis and suppresses proliferation of normal BM cells while MC themselves are relatively resistant via upregulation of survivin. In a next step, we studied the effect of TNFa on growth of neoplastic MC in vivo in a xenotransplant model using ROSAKIT D816V cells with and without knockout of TNFa in NOD‐SCID IL‐2Rγ‐null (NSG) mice. CRISPR/Cas9‐mediated knockout completely abolished expression and secretion of TNFa in ROSAKIT D816V cells and resulted in a significantly longer survival of mice compared to mice injected with TNFa expressing controls (median 49 vs. 58 days, p < 0.05). Finally, the clinical relevance of TNFa expression was studied in a SM patient's cohort (n = 45). TNFa levels in sera of patients were significantly higher (median 1.95, range 0.72–16.64 pg/ml, p = 0.002) compared to age‐ and sex‐matched controls (median 1.49, range 0.51–5.63 pg/ml). Importantly, when we stratified SM patients into those with clearly elevated TNFa levels and those with TNFa levels within the reference range of controls (≤3.63 pg/ml) a significantly lower overall survival was observed for SM patients with high TNFa serum levels (median 6.3 years vs. not reached, p < 0.005).Summary/Conclusion:In summary, we have identified TNFa as a critical, KIT D816V‐dependent, cytokine‐mediator that promotes clonal expansion of KIT D816V+ MC and may thereby be involved in clonal evolution and disease progression in SM.

The HMGCR Inhibitor Fluvastatin Induces Apoptosis and Autophagy in Primary and Neoplastic Mast Cells

Abstract Mast cell hyperplasia is noted in many inflammatory and allergic diseases. Mast cells can also generate a subtype of acute myelogenous leukemia. Understanding and treating mast cell hyperplasia and leukemia could be beneficial to both inflammatory and malignant diseases. Recently the family of statin drugs, widely employed as HMG CoA Reductase (HMGCR) inhibitors to lower serum cholesterol, have been found to be immunosuppressive. However, statin effects on mast cell survival are not known. We report that fluvastatin induces apoptosis in mouse bone marrow-derived mast cells (BMMC) and mastocytoma cells. Apoptosis was suppressed in p53 KO and Bcl-2 Tg BMMC, consistent with detectable mitochondrial damage and Caspase-9 activation. Because apoptosis induction could be mimicked by suppressing geranylgeranyl transferase (GGT), and fluvastatin effects were reversed by adding the product of the GGT reaction, loss of GGT-mediated protein lipidation is the likely mechanism of cell death. In addition to apoptosis, fluvastatin also induced autophagy. Chemical blockade of autophagy enhanced fluvastatin-mediated apoptosis in primary mast cells, but reduced it in mastocytomas. These data suggest that the FDA-approved statin drugs may be useful in suppressing mast cell hyperplasia and leukemia.

Single-cell analysis reveals the KIT D816V mutation in haematopoietic stem and progenitor cells in systemic mastocytosis.

BackgroundSystemic mastocytosis (SM) is a haematological disease characterised by organ infiltration by neoplastic mast cells. Almost all SM patients have a mutation in the gene encoding the tyrosine kinase receptor KIT causing a D816V substitution and autoactivation of the receptor. Mast cells and CD34+ haematopoietic progenitors can carry the mutation; however, in which progenitor cell subset the mutation arises is unknown. We aimed to investigate the distribution of the D816V mutation in single mast cells and single haematopoietic stem and progenitor cells.MethodsFluorescence-activated single-cell index sorting and KIT D816V mutation assessment were applied to analyse mast cells and >10,000 CD34+ bone marrow progenitors across 10 haematopoietic progenitor subsets. In vitro assays verified cell-forming potential.FindingsWe found that in SM 60–99% of the mast cells harboured the KIT D816V mutation. Despite increased frequencies of mast cells in SM patients compared with control subjects, the haematopoietic progenitor subset frequencies were comparable. Nevertheless, the mutation could be detected throughout the haematopoietic landscape of SM patients, from haematopoietic stem cells to more lineage-primed progenitors. In addition, we demonstrate that FcεRI+ bone marrow progenitors exhibit mast cell-forming potential, and we describe aberrant CD45RA expression on SM mast cells for the first time.InterpretationThe KIT D816V mutation arises in early haematopoietic stem and progenitor cells and the mutation frequency is approaching 100% in mature mast cells, which express the aberrant marker CD45RA.

In vitro and in vivo efficacy of an anti-CD203c conjugated antibody (AGS-16C3F) in mouse models of advanced systemic mastocytosis

AbstractAntibody-drug conjugates (ADCs) are a new class of therapeutics that use antibodies to deliver potent cytotoxic drugs selectively to cancer cells. CD203c, an ecto-nucleotide pyrophosphatase-phosphodiesterase 3, is overexpressed on neoplastic mast cells (MCs) in systemic mastocytosis (SM), thus representing a promising target for antibody-mediated therapy. In this study, we have found that human neoplastic MC lines (ROSAKIT D816V and ROSAKIT D816V-Gluc), which express high levels of CD203c, are highly and specifically sensitive to the antiproliferative effects of an ADC against CD203c (AGS-16C3F). In these cell lines, AGS-16C3F induced cell apoptosis at very low concentrations. To characterize the effects of AGS-16C3F on leukemia progression in vivo, ROSAKIT D816V-Gluc NOD-SCID γ mouse models of advanced SM (AdvSM) were treated with AGS-16C3F or an ADC control for 2 weeks. Whereas AGS-16C3F had no apparent toxicity in xenotransplanted mice, in vivo neoplastic MC burden significantly decreased in both hematopoietic and nonhematopoietic organs. Furthermore, animals treated with AGS-16C3F had prolonged survival compared with the animals treated with control ADC, and AGS-16C3F efficiently prevented disease relapse. In conclusion, these preclinical studies identified CD203c as a novel therapeutic target on neoplastic MCs, and AGS-16C3F as a promising ADC for the treatment of patients with AdvSM.

In-Vitro interactions of neoplastic canine mast cells and stromal fibroblasts

In-Vitro interactions of neoplastic canine mast cells and stromal fibroblasts

The new tool "KIT" in advanced systemic mastocytosis.

Mastocytosis is a rare disease characterized by KIT-driven expansion and accumulation of neoplastic mast cells in various tissues. Although mediator symptoms related to mast cell activation can impose a symptom burden in cutaneous disease and across the spectrum of systemic mastocytosis subtypes, the presence of an associated hematologic neoplasm and/or organ damage denotes advanced disease and the potential for increased morbidity and mortality. In addition to the revised 2016 World Health Organization classification of mastocytosis, a new diagnostic and treatment toolkit, tethered to enhanced molecular characterization and monitoring, is poised to transform the management of patients with advanced systemic mastocytosis (advSM). Although the efficacy of midostaurin and novel selective KIT D816V inhibitors, such as avapritinib (BLU-285), have validated KIT as a therapeutic target, the clinical and biologic heterogeneity of advSM requires that we reimagine the blueprint for tackling these diseases and use tools that move beyond KIT-centric approaches.

The CDK4/6 Inhibitor Palbociclib Exerts Growth-Inhibitory Effects on Neoplastic Mast Cells and Synergizes with Midostaurin in Producing Growth Arrest

Aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL) are rare, malignant diseases with an unfavorable prognosis. In a majority of patients, the transforming KIT mutation D816V is detectable. Currently, several drugs are available for the treatment of ASM/MCL, including midostaurin, a KIT D816V-targeting drug that has recently been approved for the treatment of advanced SM in the US and in Europe. However, when applied as single drug, midostaurin usually fails to induce durable remissions in patients with ASM/MCL, and the same holds true for all other drugs tested in the ASM/MCL context so far. Therefore, drug combinations, including established drugs and novel targeted drugs are currently being examined for their anti-neoplastic effects in ASM/MCL. CDK4 and CDK6 are kinases that play an essential role in cell cycle-initiation in normal and neoplastic cells. However, the role of CDK4/6 as potential therapeutic targets in neoplastic mast cells (MC) has not been analyzed so far. Recently, three CDK4/6 inhibitors, palbociclib, ribociclib and abemaciclib, have been translated into clinical application. The aim of the current study was to evaluate the effects of these CDK4/6 inhibitors on cell cycle progression, proliferation and survival of neoplastic MC. In initial experiments, we employed the MCL-related cell lines HMC-1.1 (lacking KIT D816V), HMC-1.2 (KIT D816V+), ROSAKIT WT, ROSAKIT D816V and MCPV-1 (expressing RAS G12V, Large T and hTert). In 3H-thymidine incorporation experiments, all three CDK4/6-inhibitors were found to block proliferation in both HMC-1 sub-clones and both ROSA sub-clones, with comparable IC50 values (<0.5 µM). In MCPV-1 cells, similar results were obtained, but higher concentrations of palbociclib, ribociclib and abemaciclib were required to block proliferation (IC50 1-5 µM). These data suggest that CDK4/6-inhibitors exert anti-proliferative effects in neoplastic MC independent of the presence of KIT D816V. In a next step, we examined drug effects on primary bone marrow cells obtained from patients with KIT D816V+ indolent SM (n=3), ASM (n=1), SM with an associated hematologic neoplasm, ASM-AHN (n=5) and MCL (n=2). As determined by 3H-thymidine uptake, palbociclib was found to inhibit cell proliferation at pharmacologically meaningful concentrations in all donors tested, with IC50 values ranging between 5 nM and 250 nM. Similar effects were obtained when applying ribociclib (25-500 nM) and abemaciclib (5-500 nM). To learn more about the mechanisms underlying the effects of the CDK4/6 inhibitors on neoplastic MC, cell cycle progression and apoptosis were examined in HMC-1.1 and HMC-1.2 cells after drug exposure. In both cell lines, the palbociclib-induced growth inhibition was found to be accompanied by cell cycle arrest in the G1-phase. Moreover, all three CDK4/6 inhibitors were found to produce time- and dose-dependent apoptosis in HMC-1.1 and HMC-1.2 cells during 72 hours of incubation. In a next step, Western blot experiments were performed using antibodies against the main downstream target of CDK6, retinoblastoma protein-1 (Rb-1). The Rb-1 antigen was found to be expressed in phosphorylated form (p-Rb-1) in HMC-1.1 and HMC-1.2 cells. As expected, all 3 CDK4/6 inhibitors were found to suppress p-Rb-1 expression in both HMC-1 cell lines, suggesting specific drug effects. In a final step, we examined potential cooperative drug effects using palbociclib and the KIT D816V-targeting drug midostaurin. In these experiments, palbociclib was found to synergize with midostaurin in inducing growth inhibition in HMC-1 cells. In conclusion our data suggest that inhibition of CDK4/6 may be a new promising approach for the treatment of patients with advanced SM. In addition, our data suggest that CDK4/6 inhibitors may represent promising combination partners for midostaurin in the treatment of ASM/MCL. Whether treatment with CDK4/6 inhibitors alone or in combination with KIT inhibition, is indeed sufficient to control proliferation of neoplastic MC in vivo in patients with advanced SM remains to be determined in forthcoming studies. DisclosuresHoermann:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria. Sperr:Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria. Reiter:Incyte: Consultancy, Honoraria. Valent:Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria.

MDM2 and Aurora Kinase a Contribute to SETD2 Loss of Function in Advanced Systemic Mastocytosis: Implications for Pathogenesis and Treatment

The SETD2 gene encodes the only methyltransferase responsible for histone H3 lysine 36 trimethylation (H3K36Me3) in humans. H3K36me3 play a key role in preserving the fidelity of transcription elongation and splicing. In addition, SETD2/H3K36me3 have more recently been implicated in the maintenance of genomic integrity by regulating homologous recombination (HR) repair, Mismatch Repair (MMR) mitotic spindle assembly and chromosome segregation. SETD2 deletions and/or inactivating mutations occur in many solid tumors and have recently been found also in acute leukemias. We have reported that the HMC-1.1 and -1.2 mast cell leukemia (MCL) cell lines and many advanced systemic mastocytosis (SM) patients (pts) display H3K36Me3 deficiency as a result of non-genomic loss of function of SETD2. Proteasome inhibition restored SETD2 protein expression and H3K36me3, suggesting that a functional protein is produced but rapidly degraded. In an attempt to uncover the mechanisms underlying this phenomenon, we used an in silico approach to identify candidate SETD2-interacting proteins, followed by experimental confirmation by co-immunoprecipitation (co-IP). We found that, after proteasomal inhibition, SETD2 co-immunoprecipitates with the ubiquitin E3 ligase MDM2. Treatment with the MDM2 inhibitor SP-141 rescued SETD2 expression and H3K36Me3, suggesting that MDM2 may play a role in SETD2 degradation in ASM and MCL. Moreover, SP-141 treatment of HMC-1 cells at micromolar doses induced cytostatic but not cytotoxic effects as shown by cell growth curves. Clonogenic assays supported the cytostatic effects of SP141 in HMC-1.1 and -1.2 cells. siRNA-mediated knock-down of MDM2 also rescued SETD2 expression and activity, further supporting the hypothesis that SETD2 hyper-ubiquitination by MDM2 plays a role in SETD2 reduced stability and proteasomal degradation. Co-IP also showed that SETD2 interacts with Aurora Kinase A, as it was suggested in silico. We found that Aurora A is overexpressed in advanced SM and may target SETD2 for phosphorylation. Both pharmacological inhibition by Danusertib and siRNA-mediated silencing of Aurora A rescued SETD2 expression and activity, raising the hypothesis that phosphorylation by Aurora A might be the trigger for MDM-2 mediated degradation of SETD2.To evaluate whether increased DNA damage and reduced HR proficiency can be observed in SETD2/H3K36Me3-deficient SM, we used western blotting (WB) and immunofluorescence (IF) to assess phosphorylated histone 2A.X (γH2AX) and Rad51 foci. Compared to cells from healthy controls, SETD2- and H3K36Me3-deficient cell lines and pts had significantly higher levels of γH2AX and lower levels of Rad51. RNA-seq in SETD2-deficient pts showed evidence of transcription and splicing defects like transcription-induced chimeras, intron retention and non-canonical splicing patterns not observed in healthy donors.Next, the ROSAD816V cell line, which displays SETD2 and H3K36me3 levels superimposable to healthy donors, was studied by WB and IF to assess γH2AX and Rad51 in steady state and after sub-lethal DNA damage by UV exposure. The same experiments were carried out after SETD2 silencing for 2 months. Cells with silenced SETD2 had significantly higher levels of γH2AX and were unable to activate the HR repair. Interestingly, clonogenic assays in ROSAD816V cells before and after SETD2 silencing showed that reduction of clonogenic potential after proteasomal or MDM2 inhibition is indeed SETD2-dependent (Figure 1A).Finally, we performed clonogenic assays to evaluate the therapeutic potential of bortezomib, carfilzomib and ixazomib in neoplastic mast cells from 3 patients with advanced SM and we observed in all cases that both first and second generation inhibitors induced a significant reduction of clonogenic activity at nanomolar doses (Figure 1B).Taken together, our results suggest that AKA and MDM2-mediated post-translational modifications contribute to SETD2 non-genomic loss of function in advanced SM. Loss of SETD2 and H3K36me3 is associated with increased DNA damage and transcription and splicing defects in patients. Inhibiting AKA or MDM2 activity or proteasome-mediated degradation are promising therapeutic strategies in patients with low SETD2 expression levels.Acknowledgments: study supported by AIRC (project code 16996) and AIL (Associazione Italiana contro le Leucemia, Linfomi e Mieloma). [Display omitted] DisclosuresBonifacio:Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Pagano:Janssen: Speakers Bureau; Merck: Speakers Bureau; Gilead: Speakers Bureau; Basilea: Speakers Bureau; Pfizer: Speakers Bureau. Valent:Novartis: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Soverini:Novartis: Consultancy; Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy.

Comprehensive Cytokine Profiling of Patients with Advanced Systemic Mastocytosis Treated with Midostaurin

Background: Advanced systemic mastocytosis (AdvSM) comprises a group of hematologic neoplasms primarily driven by the oncogenic KIT D816V mutation. Morbidity and mortality relate to organ damage caused by neoplastic mast cell infiltration and mast cell mediator symptoms. Midostaurin, an oral multikinase/KIT inhibitor, produced reversion of organ damage in 60-70% of AdvSM patients (Gotlib et al, NEJM, 2016; DeAngelo et al, Leukemia, 2018), which was associated with decreases in bone marrow mast cell burden, serum tryptase levels, and splenomegaly in the majority of patients. Similar to myelofibrosis (MF) patients treated with JAK inhibitors, midostaurin improves disease symptoms and quality of life (Gotlib et al, NEJM, 2016). We conducted a cytokine profiling study to better understand the biologic effects of midostaurin in this patient population.Methods: Plasma cytokine levels were analyzed in 19 patients with AdvSM (2 aggressive SM, 10 SM with an associated hematologic neoplasm, and 7 with mast cell leukemia) treated with midostaurin 100 mg twice daily. Seventeen patients were treated on the phase II investigator-initiated trial (DeAngelo et al,Leukemia, 2018), and two were treated on a compassionate-use basis. Cytokine levels were compared to 10 patients with MF (primary MF [n=4]; post PV/ET MF [n=6]), treated with JAK inhibitors (ruxolitinib, fedratinib, or momelotinib) and 10 healthy patients whose age range matched the SM group. Plasma samples were obtained pretreatment, and after 4-8 weeks of therapy. Each patient sample was evaluated for 62 cytokines using a Luminex assay (eBioscience) performed at the Stanford Human Immune Monitoring Center. Sparse partial least squares discriminant analysis (SPLS-DA) was used to identify the most predictive cytokines from the expression data that help classify patient samples based on disease states, and SPLS regression was used to identify cytokines correlated with survival (Rohart et al, mixOmics. R package version 6.3.2). Overall survival was calculated from the time of initiation of midostaurin to time of death.Results: Plasma levels of several cytokines were higher at baseline in AdvSM and MF patients compared to healthy controls (Fig 1A-B). In patients with AdvSM and MF, pretreatment levels of leptin (false discovery rate (FDR) < 0.002), epidermal growth factor (EGF; FDR < 2e-5), and brain-derived neurotrophic factor (BDNF; FDR < 3.5e-7) were lower than healthy controls. Using SPLS-DA, we identified a distinct baseline cytokine expression profile that could classify patient samples as either AdvSM, MF, or healthy with an error rate of less than 0.10 (Fig 1C). The six most predictive cytokines were nerve growth factor, EGF, BDNF, interferon alpha, intercellular adhesion molecule-1 (ICAM1), and leukemia inhibitory factor. Corroborating prior data (Verstovsek et al, NEJM 2010), we found that several plasma cytokine levels significantly decreased in MF patients after 1-2 cycles of JAK inhibition (Fig 1B). Although we observed similar decreases in plasma cytokine levels for AdvSM patients on midostaurin (Fig 1A), the results were more variable and did not reach statistical significance. AdvSM patients that responded to midostaurin (major or partial response per Valent criteria) expressed a different baseline cytokine expression profile when compared to non-responders. SPLS-DA was able to distinguish these two classes of patients with an error rate of 0.35 (Fig 1D). SPLS regression identified baseline levels of interleukin-7 (IL7), ICAM1, interleukin 12 subunit p40 (IL12p40), EGF, and platelet-derived growth factor BB (PDGFBB) as potential predictors of survival for patients with AdvSM treated with midostaurin. High pretreatment levels of IL7 (109 months (m) vs. 33 m; p=0.01), EGF (92 m vs. 34 m; p=0.04), and PDGFBB (80 m vs. 37 m; p=0.09) and low pretreatment levels of ICAM1 (91 m vs. 19 m; p=0.02) and IL12p40 (91 m vs. 19 m; p=0.03) were associated with increased survival.Conclusions: Cytokine expression profiling can distinguish patients with AdvSM or MF from healthy controls. Compared to the use of JAK inhibitors in MF, midostaurin's activity in AdvSM appears less related to reversion of pro-inflammatory cytokines. Investigation of a larger cohort of patients is needed to determine whether such analyses of plasma cytokines will provide added value to new scoring prognostic systems of AdvSM that incorporate clinical and molecular data. [Display omitted] DisclosuresDeAngelo:Glycomimetics: Research Funding; Amgen: Consultancy; ARIAD: Consultancy, Research Funding; Takeda: Honoraria; BMS: Consultancy; Incyte: Consultancy, Honoraria; BMS: Consultancy; ARIAD: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria; Shire: Honoraria; Amgen: Consultancy; Pfizer Inc: Consultancy, Honoraria; Blueprint Medicines: Honoraria, Research Funding. George:Blueprint Medicines: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Gotlib:Deciphera: Consultancy, Honoraria, Research Funding; Kartos: Consultancy; Promedior: Research Funding; Gilead: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Blueprint Medicines: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

A Retrospective Case Series Describing the Efficacy of Ketotifen in Systemic Mastocytosis

IntroductionSystemic Mastocytosis (SM) represents a heterogeneous group of disorders characterized by the accumulation of neoplastic mast cells (MCs) in one or more organ systems. Patients with SM present with a broad range of symptoms resulting from excessive mast cell mediator release, especially histamine that frequently overlap with those of allergic disease. Episodes of life-threatening anaphylaxis are a recognized feature of SM. Here at St. Michael's Hospital, we currently follow more than 50 SM patients, the largest patient cohort in Canada. Ketotifen is a dual action mast cell stabilizer used to treat asthma, anaphylaxis and other mast cell, allergic-type disorders. Along with cromolyn, another mast cell stabilizer, ketotifen is known to be the most effective at controlling mast cell activation. At St. Michael's Hospital, ketotifen is used as an add-on, off-label therapy in SM patients at risk for recurrent anaphylaxis. The literature has yet to confirm the efficacy of using ketotifen to treat SM. Few centres besides St. Michael's use ketotifen as a potential therapy.ObjectivesOur primary objective was to describe the response to treatment by ketotifen in patients with SM in a tertiary care centre.MethodsThis is an observational, retrospective study (n=10) of SM patients treated with ketotifen at St. Michael's Hospital from January 2015 to June 2018. Electronic medical records were reviewed for mastocytosis treatment and symptom progression. All patients included in the study were diagnosed with SM according to the 2016 WHO criteria by undergoing a bone marrow biopsy. Symptoms were assessed at baseline (2-5 months pre-ketotifen initiation) and again at follow-up (4-8 months post initiation). An independent statistician produced binomial proportion confidence intervals for each reported symptom.ResultsOur study consisted of 7 females and 3 males, with an average age of 49 years old [IQR 33-67]. 5 of the 10 patients were diagnosed with indolent SM, the more moderate of the six SM subtypes. Table 1 demonstrates the 5 most frequently reported symptoms at baseline.(Refer to Table 1) For nausea/vomiting, presyncope/syncope and urticaria, 100% of patients who reported these symptoms responded to treatment. For diarrhea and flushing, 86% and 67% of patients who reported these symptoms responded to treatment.ConclusionsKetotifen appears to be an effective therapy for patients with SM with anaphylaxis. It should be readily considered in the management of this population. Further investigation is warranted given our small sample size and single centre recruitment. Next steps include following these patients prospectively to better capture the efficacy of ketotifen within this population.DisclosuresNo conflicts of interest to declare DisclosuresNo relevant conflicts of interest to declare.

Systemic Mastocytosis: Management and Outcome. Data Analysis from the Greek Registry

Background and Methods: Systemic Mastocytosis (SM) is a rare haematologic malignancy characterized by the abnormal growth and accumulation of neoplastic mast cells in one or more organs. The cKIT D816V mutation is a common genetic finding in most cases. A subset of patients appears to be at increased risk for mediator release related symptoms as well as organ dysfunction, including skeletal problems such as osteoporosis, osteolytic lesions and fractures. The diversity of clinical manifestations results in both delayed diagnosis and therapeutic dilemmas. SM cases diagnosed in Greece, between 1987 and 2018, are presented. This project is included in the current activities of Myeloproliferative Neoplasm Working Party of Hellenic Society of Haematology for registry and research development. The medical files of the patients were retrospectively evaluated for disease characteristics, treatment and outcome.Results: Overall 59 patients, median age 52.0 years, with SM were included in the study. Median time of symptoms onset to diagnosis was two years. Twenty-one patients were categorized as indolent SM (ISM), 19 as SM with an associated haematological neoplasm (SM-AHN), 18 as aggressive SM (ASM) and one with mast cell leukaemia (MCL). The main characteristics of the disease are shown in Table 1. Several haematologic neoplasms were associated with SM. Although myeloid malignancies were the most common, including MDS or MDS/MPN (n=9), CMML (n=2), AML (n=1), CML (n=1) and ET (n=1), lymphoid malignancies were also reported and included NHL (n=3), HL (n=1) and B-ALL (n=1). SM and the AHN were diagnosed simultaneously in 12 cases. AHN diagnosis preceded the diagnosis of SM in 4 cases (median time: 22 months), while the opposite occurred in three cases (median time: 7.0 months). Most of the patients with ISM (16/21) did not receive any specific treatment. As far as the remaining five are concerned, one was treated with imatinib, one with hydroxyurea and the rest three received corticosteroids to control the mediators' related symptoms. One of the latter had diarrhoea without infiltration of the gastrointestinal tract and received consecutively Interferon alpha (IFNα), imatinib and corticosteroids without resolution of the syndrome. Eight patients with SM-AHN received treatment for both the SM and the co-existing haematological neoplasm, six only for the AHN, three only for SM, while three did not require treatment yet. The most common treatment for SM was IFNα (n=6), followed by imatinib (n=4), cladribine (2-CdA) (n=3) and dasatinib (n=1). Patients with ASM received either IFNα (n=8) or 2-CdA (n=5) as first line treatment. Second line treatment included imatinib (n=2), 2-CdA due to IFNα intolerance (n=1) and corticosteroids (n=2). Two patients with vertebrae fractures required surgical intervention. All patients with skeletal involvement received additionally biphosphonates. The patient with MCL received consecutively 2-CdA, chemotherapy (FLAG-Ida) and dasatinib achieving partial response and proceeded to allogeneic stem cell transplantation. He died 6 months later due to complications related to graft versus host disease. With a median follow-up of 31 months the median overall survival in the entire cohort is not reached. The median follow-up for patients with ISM and ASM is 33.5 and 18.5 months respectively, and all of them are alive with adequate disease control. Seven deaths were reported only in the group of patients with SM-AHN. Six patients died due to acute leukaemia and one due to infection, indicating that the aggressiveness of the underlying haematological malignancy is the strongest factor that affects survival in this group.Conclusion: Systemic Mastocytosis is a rare disease with variable manifestations and outcome. Nowadays, several therapeutic modalities are available for effective disease management over time. Novel targeted therapies seem to be promising to further improve the outcome, but still early and accurate diagnosis, in accordance to WHO classification, remains important. DisclosuresGavriilaki:European Hematology Association: Research Funding. Terpos:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Grant, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Grant, Patents & Royalties; Genesis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Grant, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Grant, Patents & Royalties; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria.

A Retrospective Case Series Describing the Efficacy of Omalizumab in Systemic Mastocytosis

IntroductionSystemic mastocytosis (SM) represents a heterogeneous group of disorders characterized by accumulation of neoplastic mast cells (MCs) in one or more organ systems. Patients with SM present with a broad range of symptoms resulting from excessive mast cell mediator release, especially histamine, that frequently overlap with those of allergic disease. Episodes of life-threatening anaphylaxis are a recognized feature of SM. Here at St. Michael's Hospital we currently follow more than 50 SM patients, the largest patient cohort in Canada. Omalizumab is a subcutaneously administered monoclonal antibody which acts on circulating IgE, reducing binding to the high-affinity IgE receptor (FCεR1) on mast cells, thereby reducing the potential reactivity of these cells. At St. Michael's Hospital, omalizumab is used as an add-on, off-label therapy in SM patients at risk for recurrent anaphylaxis. The efficacy of omalizumab treatment for SM patients remains unclear. Typically, highly symptomatic patients who are refractory to all other medication are candidates for omalizumab therapy.ObjectivesOur primary objective was to describe the response to treatment by omalizumab in patients with SM in a tertiary care centre. Our secondary objective was to compare the markers of disease in SM patients between those who were non-responsive versus responsive to omalizumab. The clinical and biological markers to be studied are symptoms and tryptase levels.MethodsThis is an observational, retrospective study (n=6) of SM patients treated with omalizumab at St. Michael's Hospital between January, 2014 and June, 2018. Electronic medical records were reviewed for mastocytosis treatment, symptom progression and tryptase levels, if available. All patients included in the study were diagnosed with SM according to the 2016 WHO criteria by undergoing a bone marrow biopsy. A baseline was established 2-5 months pre omalizumab exposure, as well as two follow-ups, each ranging from 2-8 months post omalizumab exposure (av. 4.7 months). The Brown Anaphylaxis score was used to capture severity of anaphylaxis. Mild (1), moderate (2), and severe (3) scores were associated with cutaneous manifestations, systemic (GI, respiratory, cardiovascular) involvement and systemic (hypoxia, hypotension, neurological compromise) collapse, respectively.ResultsOur study consisted of 4 females and 2 males, with an average age of 49 years old [IQR 36-74]. All 6 patients were diagnosed with indolent SM, the more moderate of the six SM subtypes.In every system, except for respiratory, it appears that symptoms decreased once therapy began. From baseline to first follow-up: all three patients who were experiencing systemic symptoms, three of the six manifesting cutaneous symptoms, and two of the three with cardiovascular involvement, responded fully to treatment. At second follow-up, patient 1 presented to clinic asymptomatically. Overall, 100% of patients responded to treatment with responses ranging from 17% to 100% improvement of mastocytosis-related symptoms.The grading of anaphylaxis severity reported three of the six patients improving from scores of 3 (severe) to 1 (mild). The other three patients remained at scores of 2.Patients 1 and 2 (only patients with available tryptase levels at both baseline and follow-up) saw a decrease in tryptase level from 134 to 84.1 and 11.4 to 8.3, respectively.ConclusionsOmalizumab appears to be an effective therapy for patients with SM with anaphylaxis and reduces tryptase levels. It should be readily considered in the management of this population. Next steps include following these patients prospectively to better capture the efficacy of omalizumab within this population.DisclosuresNo conflicts of interest to declare DisclosuresNo relevant conflicts of interest to declare.

CD44 is a RAS/STAT5-regulated invasion receptor that triggers disease expansion in advanced mastocytosis

AbstractThe Hermes receptor CD44 is a multifunctional adhesion molecule that plays an essential role in the homing and invasion of neoplastic stem cells in various myeloid malignancies. Although mast cells (MCs) reportedly express CD44, little is known about the regulation and function of this receptor in neoplastic cells in systemic mastocytosis (SM). We found that clonal CD34+/CD38− stem cells, CD34+/CD38+ progenitor cells, and CD117++/CD34− MCs invariably express CD44 in patients with indolent SM (ISM), SM with an associated hematologic neoplasm, aggressive SM, and MC leukemia (MCL). In addition, all human MCL-like cell lines examined (HMC-1, ROSA, and MCPV-1) displayed cytoplasmic and cell-surface CD44. We also found that expression of CD44 in neoplastic MCs depends on RAS-MEK and STAT5 signaling and increases with the aggressiveness of SM. Correspondingly, higher levels of soluble CD44 were measured in the sera of patients with advanced SM compared with ISM or cutaneous mastocytosis and were found to correlate with overall and progression-free survival. To investigate the functional role of CD44, a xenotransplantation model was employed using severe combined immunodeficient (SCID) mice, HMC-1.2 cells, and a short hairpin RNA (shRNA) against CD44. In this model, the shRNA-mediated knockdown of CD44 resulted in reduced MC expansion and tumor formation and prolonged survival in SCID mice compared with HMC-1.2 cells transduced with control shRNA. Together, our data show that CD44 is a RAS-MEK/STAT5-driven MC invasion receptor that correlates with the aggressiveness of SM. Whether CD44 can serve as therapeutic target in advanced SM remains to be determined in forthcoming studies.

A Population-Based Study of Neoplastic Mast Cell Disease

Neoplastic mast cell disease (NMCD) is rare and exhibits a spectrum of severity from indolent cutaneous lesions to leukemia. To date few population-based studies have examined demographic and clinical correlates of overall survival (OS) in patients with NMCD. We aimed to investigate this using the Surveillance, Epidemiology, and End Results (SEER) database. We included 601 patients from the SEER 18 registries diagnosed with malignant NMCD from 1975-2014. These patients were defined by the following ICD-O-3 codes: 9740/3, mast cell sarcoma (MCS); 9741/3, systemic mastocytosis (SM); and 9742/3, mast cell leukemia (MCL). Kaplan-Meier analyses, log-rank tests, and Cox proportional hazards models were used to assess the impact of demographic and clinical factors on OS. The cohort was predominantly white (n=553, 92%). Roughly half of the patients were female (n=303, 50.4%). Patient age at diagnosis ranged from 0-91 years (median 55 years). The majority of patients were coded as having SM (n=558, 92.8%). Median OS for those with SM, MCS, and MCL were 186, 133, and 14 months respectively (p<0.001 log-rank test). With Cox regression accounting for age at diagnosis, year of diagnosis, race, sex, histology, site of disease, surgery for NMCD, radiation for NMCD, and chemotherapy for NMCD, female sex was associated with an improved OS (HR=0.60, 95% CI 0.44-0.80, p<0.001). Black race was associated with poorer OS (HR=2.63, 95% CI 1.46-4.73, p<0.01). OS was comparable across different sites of disease, with the exception of worse OS with disease coded as in the lymph nodes (HR 5.41, 95% CI 1.58-18.51, p<0.01). History of surgery for NMCD (HR=3.13, 95% CI 1.52-6.42, p<0.01) and chemotherapy for NMCD (HR=3.07, 95% CI 2.33-4.22, p<0.001) were associated with poorer OS relative to those that did not receive these interventions. Radiotherapy for NMCD did not significantly affect OS (HR=0.63, 95% CI 0.24-1.47, p=0.29). This hypothesis-generating population-based study suggests that NMCD has a variable prognosis—those coded as having MCS and MCL had poorer OS compared to those with SM. Prognosis is affected by sex and race. The poorer OS associated with surgery for NMCD, and chemotherapy for NMCD likely represents selection bias. Further study is needed to overcome the biases inherent in population-based retrospective analyses and better characterize NMCD prognostic factors. Prospective study is needed to develop paradigms to treat patients with NMCD optimally.

Comparative oncology: The paradigmatic example of canine and human mast cell neoplasms.

In humans, advanced mast cell (MC) neoplasms are rare malignancies with a poor prognosis. Only a few preclinical models are available, and current treatment options are limited. In dogs, MC neoplasms are the most frequent malignant skin tumours. Unlike low‐grade MC neoplasms, high‐grade MC disorders usually have a poor prognosis with short survival. In both species, neoplastic MCs display activating KIT mutations, which are considered to contribute to disease evolution. Therefore, tyrosine kinase inhibitors against KIT have been developed. Unfortunately, clinical responses are unpredictable and often transient, which remains a clinical challenge in both species. Therefore, current efforts focus on the development of new improved treatment strategies. The field of comparative oncology may assist in these efforts and accelerate human and canine research regarding diagnosis, prognostication, and novel therapies. In this article, we review the current status of comparative oncology approaches and perspectives in the field of MC neoplasms.

Clinical Validation of KIT Inhibition in Advanced Systemic Mastocytosis.

We discuss recent developments in the treatment of advanced systemic mastocytosis (advSM) with inhibitors of the KIT receptor tyrosine kinase. advSM is a heterogeneous group of neoplasms of poor prognosis characterized by the accumulation of neoplastic mast cells. The canonical KIT D816V mutation is present in approximately 90% of SM patients, and its detection is critical for both diagnosis and therapeutic decision-making. The multikinase/KIT inhibitor midostaurin was recently approved for advSM. This agent can reverse SM-related organ damage and disease symptoms, and decrease the bone marrow mast cell burden and splenomegaly. However, complete remissions are rare and durability of responses is variable. Potent and selective KIT D816V inhibitors including avapritinib (BLU-285) and DCC-2618 have entered clinical trials, and rational combination strategies are under development. The clinical efficacy of KIT inhibitors validate KIT as a key oncogenic driver in mast cell neoplasms. An improved understanding of the genetic heterogeneity beyond KIT will help inform the dynamics of response and relapse.