SR-BI regulates the synergistic mast cell response by modulating the plasma membrane-associated cholesterol pool.

The high-affinity IgE receptor FcεRI is the mast cell (MC) receptor responsible for the involvement of MCs in IgE-associated allergic disorders. Activation of the FcεRI is achieved via crosslinking by multivalent antigen (Ag) recognized by IgE resulting in degranulation and proinflammatory cytokine production. In comparison to the T- and B-cell receptor complexes, for which several co-receptors orchestrating the initial signaling events have been described, information is scarce about FcεRI-associated proteins. Additionally, it is unclear how FcεRI signaling synergizes with input from other receptors and how regulators affect this synergistic response. We found that the HDL receptor SR-BI (gene name: Scarb1/SCARB1) is expressed in MCs, functionally associates with FcεRI, and regulates the plasma membrane cholesterol content in cholesterol-rich plasma membrane nanodomains. This impacted the activation of MCs upon co-stimulation of the FcεRI with receptors known to synergize with FcεRI signaling. Amongst them, we investigated the co-activation of the FcεRI with the receptor tyrosine kinase KIT, the IL-33 receptor, and GPCRs activated by adenosine or PGE2. Scarb1-deficient bone marrow-derived MCs showed reduced cytokine secretion upon co-stimulation conditions suggesting a role for plasma membrane-associated cholesterol regulating respective MC activation. Mimicking Scarb1 deficiency by cholesterol depletion employing MßCD, we identified PKB and PLCγ1 as cholesterol-sensitive proteins downstream of FcεRI activation in bone marrow-derived MCs. When MCs were co-stimulated with stem cell factor (SCF) and Ag, PLCγ1 activation was boosted, which could be mitigated by cholesterol depletion and SR-BI inhibition. Similarly, SR-BI inhibition attenuated the synergistic response to PGE2 and anti-IgE in the human ROSAKIT WT MC line, suggesting that SR-BI is a crucial regulator of synergistic MC activation.

Serum chemistry profiling and prognostication in systemic mastocytosis: a registry-based study of the ECNM and GREM.

Certain laboratory abnormalities correlate with subvariants of systemic mastocytosis (SM) and are often prognostically relevant. To assess the diagnostic and prognostic value of individual serum chemistry parameters in SM, 2607 patients enrolled within the European Competence Network on Mastocytosis (ECNM) and 575 patients enrolled within the German Registry on Eosinophils and Mast Cells (GREM) were analyzed. For screening and diagnosis of SM, tryptase was identified as the most specific serum parameter. For differentiation between indolent and advanced SM (AdvSM), the following serum parameters were most relevant: tryptase, alkaline phosphatase (AP), ß2-microglobulin, lactate dehydrogenase (LDH), albumin, vitamin B12, and C-reactive protein (P<0.001). With regard to subvariants of AdvSM, an elevated LDH ≥260U/L was associated with multi-lineage expansion (leukocytosis, r=0.37, P<0.001; monocytosis, r=0.26, P<0.001) and the presence of an associated myeloid neoplasm (P<0.001), whereas tryptase levels were highest in mast cell leukemia (MCL vs. non-MCL, 308 µg/L vs. 146 µg/L, P=0.003). Based on multivariable analysis, the hazard-risk weighted assignment of 1 point to lactate dehydrogenase (HR 2.1 [95% CI 1.1-4.0], P=0.018) and 1.5 points each to ß2-microglobulin (HR 2.7 [95% CI 1.4-5.4], P=0.004) and albumin (HR 3.3 [95% CI 1.7-6.5], P=0.001) delineated a highly predictive three-tier risk classification system (0 points, 8.1 years vs. 1 point, 2.5 years, ≥1.5 points, 1.7 years; P<0.001). Moreover, serum chemistry parameters enabled further stratification of IPSM-AdvSM1/2 risk-score classified patients (P=0.027). In conclusion, serum chemistry profiling is a crucial tool in the clinical practice supporting diagnosis and prognostication of SM and its subvariants.

Prevalence of hypersensitivity reactions in various forms of mastocytosis: A pilot study of 2485 adult patients with mastocytosis collected in the ECNM registry.

BACKGROUND: Hypersensitivity reactions (HR) are common in mastocytosis. However, little is known about triggers and risk factors. The registry of the European Competence Network on Mastocytosis (ECNM) enables reliable studies in a larger cohort of mastocytosis patients. We assessed prevalence, triggers and risk factors of HR in adults with mastocytosis in the ECNM registry. METHODS: Data were collected in 27 ECNM centers. We analyzed potential triggers (Hymenoptera venoms, food, drug, inhalant and others) and risk factors at diagnosis and during follow-up. The study group consisted of 2485 adults with mastocytosis, 1379 women (55.5%) and 1106 men (44.5%). Median age was 48.2 years (range 18-91 years). RESULTS: Nine hundred and forty eight patients (38.1%) reported one or more HR`. Most common triggers were Hymenoptera venoms in cutaneous mastocytosis (CM) and indolent systemic mastocytosis (ISM), whereas in advanced SM (advSM), most common elicitors were drugs, including nonsteroidal anti-inflammatory agents and penicillin. In multivariate analyses, tryptase level < 90 ng/mL, <15% infiltration by mast cells in bone marrow biopsy-sections, and diagnosis of ISM were identified as independent risk factors for HR. For drug-induced HR, prominent risk factors were advSM and high tryptase levels. New reactions were observed in 4.8% of all patients during 4 years follow-up. CONCLUSIONS: HR are mainly triggered by Hymenoptera venoms in patients with CM and ISM and by drugs in patients with advSM. Tryptase levels <90 ng/mL, mast cell bone marrow infiltration <15%, and WHO category ISM are predictors of HR. New HR occur in 4.8% of all patients within 4 years.

Efficacy and safety of mammalian target of rapamycin inhibitors in systemic mastocytosis: A nationwide French pilot study.

Systemic mastocytosis (SM) corresponds to a rare and heterogeneous spectrum of diseases characterized by the accumulation of atypical mast cells (MCs). Advanced mastocytosis (Adv-SM) is associated with poor survival; in contrast, patients with non-advanced SM (non-Adv-SM) usually have a normal life expectancy but may experience poor quality of life. Despite recent therapeutic progress including tyrosine kinase inhibitors, new treatment options are needed for refractory and/or intolerant patients with both severely symptomatic and Adv-SM. In vitro, the mTOR pathway is activated in MCs from patients bearing the KIT D816V mutation. Furthermore, rapamycin induces the apoptosis of KIT D816V MCs selectively. In this nationwide study, we report the outcomes of patients diagnosed with SM and treated with a mammalian target of rapamycin inhibitor (imTOR) within the French National Reference Center for mastocytosis (CEREMAST). All patients registered were relapsing, treatment-refractory, or ineligible for other cytoreductive therapy. Non-Adv-SM patients received imTOR as a monotherapy (rapamycin/everolimus), and Adv-SM patients received imTOR as a monotherapy or in combination with cytarabine. The objective response rate (ORR) in non-Adv-SM was 60% (partial response in 40% and major response in 20%), including reductions in skin involvement, mediator release symptoms, and serum tryptase. In the Adv-SM group, the ORR was 20% (including one major response and one partial response, both in patients with a KIT D816V mutation), which enabled a successful bridge to allogeneic stem cell transplantation in one patient. Our results suggest that imTOR treatment has potential benefits in patients with SM harboring a KIT D816V mutation.

Reversible Elevation of Tryptase Over the Individual's Baseline: Why is It the Best Biomarker for Severe Systemic Mast Cell Activation and MCAS?

PURPOSE OF REVIEW: Mast cell (MC) activation syndromes (MCAS) are conditions defined by recurrent episodes of severe systemic anaphylaxis or similar systemic events triggered by MC-derived mediators that can be measured in biological fluids. Since some symptoms of MC activation may occur due to other, non-MC etiologies and lead to confusion over diagnosis, it is of crucial importance to document the involvement of MC and their products in the patients´ symptomatology. RECENT FINDINGS: The most specific and generally accepted marker of severe systemic MC activation is an event-related, transient increase in the serum tryptase level over the individual baseline of the affected individual. However, baseline concentrations of serum tryptase vary among donors, depending on the genetic background, age, kidney function, and underlying disease. As a result, it is of critical importance to provide a flexible equation that defines the diagnostic increase in tryptase qualifying as MCAS criterion in all patients, all situations, and all ranges of baseline serum tryptase. In 2012, the consensus group proposed the 120% + 2 ng/ml formula, which covers the great majority of groups, including cases with low, normal, or elevated basal serum tryptase level. This formula has been validated in subsequent studies and has proven to be a robust and consistent diagnostic criterion of MCAS. The present article is discussing the impact of this formula and possible limitations as well as alternative markers and mediators that may be indicative of MCAS.

Coronavirus Receptor Expression Profiles in Human Mast Cells, Basophils, and Eosinophils.

A major problem in SARS-CoV-2-infected patients is the massive tissue inflammation in certain target organs, including the lungs. Mast cells (MC), basophils (BA), and eosinophils (EO) are key effector cells in inflammatory processes. These cells have recently been implicated in the pathogenesis of SARS-CoV-2 infections. We explored coronavirus receptor (CoV-R) expression profiles in primary human MC, BA, and EO, and in related cell lines (HMC-1, ROSA, MCPV-1, KU812, and EOL-1). As determined using flow cytometry, primary MC, BA, and EO, and their corresponding cell lines, displayed the CoV-R CD13 and CD147. Primary skin MC and BA, as well as EOL-1 cells, also displayed CD26, whereas primary EO and the MC and BA cell lines failed to express CD26. As assessed using qPCR, most cell lines expressed transcripts for CD13, CD147, and ABL2, whereas ACE2 mRNA was not detectable, and CD26 mRNA was only identified in EOL-1 cells. We also screened for drug effects on CoV-R expression. However, dexamethasone, vitamin D, and hydroxychloroquine did not exert substantial effects on the expression of CD13, CD26, or CD147 in the cells. Together, MC, BA, and EO express distinct CoV-R profiles. Whether these receptors mediate virus-cell interactions and thereby virus-induced inflammation remains unknown at present.

Tumor necrosis factor α promotes clonal dominance of KIT D816V+ cells in mastocytosis: role of survivin and impact on prognosis.

ABSTRACT: Systemic mastocytosis (SM) is defined by the expansion and accumulation of neoplastic mast cells (MCs) in the bone marrow (BM) and extracutaneous organs. Most patients harbor a somatic KIT D816V mutation, which leads to growth factor-independent KIT activation and accumulation of MC. Tumor necrosis factor α (TNF) is a proapoptotic and inflammatory cytokine that has been implicated in the clonal selection of neoplastic cells. We found that KIT D816V increases the expression and secretion of TNF. TNF expression in neoplastic MCs is reduced by KIT-targeting drugs. Similarly, knockdown of KIT or targeting the downstream signaling cascade of MAPK and NF-κB signaling reduced TNF expression levels. TNF reduces colony formation in human BM cells, whereas KIT D816V+ cells are less susceptible to the cytokine, potentially contributing to clonal selection. In line, knockout of TNF in neoplastic MC prolonged survival and reduced myelosuppression in a murine xenotransplantation model. Mechanistic studies revealed that the relative resistance of KIT D816V+ cells to TNF is mediated by the apoptosis-regulator BIRC5 (survivin). Expression of BIRC5 in neoplastic MC was confirmed by immunohistochemistry of samples from patients with SM. TNF serum levels are significantly elevated in patients with SM and high TNF levels were identified as a biomarker associated with inferior survival. We here characterized TNF as a KIT D816V-dependent cytokine that promotes clonal dominance. We propose TNF and apoptosis-associated proteins as potential therapeutic targets in SM.

Involvement of an IgE/Mast cell/B cell amplification loop in abdominal aortic aneurysm progression.

AIMS: IgE type immunoglobulins and their specific effector cells, mast cells (MCs), are associated with abdominal aortic aneurysm (AAA) progression. In parallel, immunoglobulin-producing B cells, organised in tertiary lymphoid organs (TLOs) within the aortic wall, have also been linked to aneurysmal progression. We aimed at investigating the potential role and mechanism linking local MCs, TLO B cells, and IgE production in aneurysmal progression. METHODS AND RESULTS: Through histological assays conducted on human surgical samples from AAA patients, we uncovered that activated MCs were enriched at sites of unhealed haematomas, due to subclinical aortic wall fissuring, in close proximity to adventitial IgE+ TLO B cells. Remarkably, in vitro the IgEs deriving from these samples enhanced MC production of IL-4, a cytokine which favors IgE class-switching and production by B cells. Finally, the role of MCs in aneurysmal progression was further analysed in vivo in ApoE-/- mice subjected to angiotensin II infusion aneurysm model, through MC-specific depletion after the establishment of dissecting aneurysms. MC-specific depletion improved intramural haematoma healing and reduced aneurysmal progression. CONCLUSIONS: Our data suggest that MC located close to aortic wall fissures are activated by adventitial TLO B cell-produced IgEs and participate to their own activation by providing support for further IgE synthesis through IL-4 production. By preventing prompt repair of aortic subclinical fissures, such a runaway MC activation loop could precipitate aneurysmal progression, suggesting that MC-targeting treatments may represent an interesting adjunctive therapy for reducing AAA progression.

Antibody-Based and Cell Therapies for Advanced Mastocytosis: Established and Novel Concepts.

Advanced systemic mastocytosis (SM) is a heterogeneous group of myeloid neoplasms characterized by an uncontrolled expansion of mast cells (MC) in one or more internal organs, SM-induced tissue damage, and poor prognosis. Advanced SM can be categorized into aggressive SM (ASM), MC leukemia (MCL), and SM with an associated hematologic neoplasm (SM-AHN). In a vast majority of all patients, neoplastic cells display a KIT mutation, mostly D816V and rarely other KIT variants. Additional mutations in other target genes, such as SRSF2, ASXL1, or RUNX1, may also be identified, especially when an AHN is present. During the past 10 years, improved treatment approaches have led to a better quality of life and survival in patients with advanced SM. However, despite the availability of novel potent inhibitors of KIT D816V, not all patients enter remission and others relapse, often with a multi-mutated and sometimes KIT D816V-negative disease exhibiting multi-drug resistance. For these patients, (poly)chemotherapy, antibody-based therapies, and allogeneic hematopoietic stem cell transplantation may be viable treatment alternatives. In this article, we discuss treatment options for patients with drug-resistant advanced SM, including novel KIT-targeting drugs, antibody-based drugs, and stem cell-eradicating therapies.

World Health Organization Classification and Diagnosis of Mastocytosis: Update 2023 and Future Perspectives.

Experts of the European Competence Network on Mastocytosis (ECNM) and the American Initiative on Mast Cell Disorders have discussed and updated diagnostic criteria and the classification of mastocytosis, based on new insights in the field and data collected in recent years, mostly within ECNM registry projects in which studies on several thousand cases have been performed. Based on this proposal, the World Health Organization has updated its classification of mastocytosis. This article discusses the revised classification of mastocytosis in light of a rapidly moving field and the advent of new diagnostic parameters, new prognostication tools, and new therapies.

KIT Mutations and Other Genetic Defects in Mastocytosis: Implications for Disease Pathology and Targeted Therapies.

A KIT activating mutation (usually KIT D816V) is detected in neoplastic cells in greater than 90% of indolent patients with systemic mastocytosis (SM). In more advanced variants of SM, additional genetic defects can be found in several myeloid malignancy-related genes, which can be detected by applying next-generation sequencing. Currently, the techniques recommended to detect the KIT D816V mutation and quantify the mutational burden in peripheral blood, bone marrow, or other organs/tissues are allele specific-quantitative PCR or droplet digital PCR. These techniques are useful for diagnosis, prognostication, follow-up and monitoring of therapeutic efficacy of cytoreductive agents in patients with SM.

The Normal Range of Baseline Tryptase Should Be 1 to 15 ng/mL and Covers Healthy Individuals With HαT.

Physiological levels of basal serum tryptase vary among healthy individuals, depending on the numbers of mast cells, basal secretion rate, copy numbers of the TPSAB1 gene encoding alpha tryptase, and renal function. Recently, there has been a growing debate about the normal range of tryptase because individuals with the hereditary alpha tryptasemia (HαT) trait may or may not be symptomatic, and if symptomatic, uncertainty exists as to whether this trait directly causes clinical phenotypes or aggravates certain conditions. In fact, most HαT-positive cases are regarded as asymptomatic concerning mast cell activation. To address this point, experts of the European Competence Network on Mastocytosis (ECNM) and the American Initiative in Mast Cell Diseases met at the 2022 Annual ECNM meeting and discussed the physiological tryptase range. Based on this discussion, our faculty concluded that the normal serum tryptase range should be defined in asymptomatic controls, inclusive of individuals with HαT, and based on 2 SDs covering the 95% confidence interval. By applying this definition in a literature screen, the normal basal tryptase in asymptomatic controls (HαT-positive persons included) ranges between 1 and 15 ng/mL. This definition should avoid overinterpretation, unnecessary referrals, and unnecessary anxiety or anticipatory fear of illness in healthy individuals.

SETD2 non genomic loss of function in advanced systemic mastocytosis is mediated by an Aurora kinase A/MDM2 axis and can be therapeutically targeted.

BACKGROUND: The SETD2 tumor suppressor gene encodes a histone methyltransferase that safeguards transcription fidelity and genomic integrity via trimethylation of histone H3 lysine 36 (H3K36Me3). SETD2 loss of function has been observed in solid and hematologic malignancies. We have recently reported that most patients with advanced systemic mastocytosis (AdvSM) and some with indolent or smoldering SM display H3K36Me3 deficiency as a result of a reversible loss of SETD2 due to reduced protein stability. METHODS: Experiments were conducted in SETD2-proficient (ROSAKIT D816V) and -deficient (HMC-1.2) cell lines and in primary cells from patients with various SM subtypes. A short interfering RNA approach was used to silence SETD2 (in ROSAKIT D816V cells), MDM2 and AURKA (in HMC-1.2 cells). Protein expression and post-translational modifications were assessed by WB and immunoblotting. Protein interactions were tested by using co-immunoprecipitation. Apoptotic cell death was evaluated by flow cytometry after annexin V and propidium iodide staining, respectively. Drug cytotoxicity in in vitro experiments was evaluated by clonogenic assays. RESULTS: Here, we show that the proteasome inhibitors suppress cell growth and induce apoptosis in neoplastic mast cells by promoting SETD2/H3K36Me3 re-expression. Moreover, we found that Aurora kinase A and MDM2 are implicated in SETD2 loss of function in AdvSM. In line with this observation, direct or indirect targeting of Aurora kinase A with alisertib or volasertib induced reduction of clonogenic potential and apoptosis in human mast cell lines and primary neoplastic cells from patients with AdvSM. Efficacy of Aurora A or proteasome inhibitors was comparable to that of the KIT inhibitor avapritinib. Moreover, combination of alisertib (Aurora A inhibitor) or bortezomib (proteasome inhibitor) with avapritinib allowed to use lower doses of each drug to achieve comparable cytotoxic effects. CONCLUSIONS: Our mechanistic insights into SETD2 non-genomic loss of function in AdvSM highlight the potential value of novel therapeutic targets and agents for the treatment of patients who fail or do not tolerate midostaurin or avapritinib.

Antineoplastic efficacy profiles of avapritinib and nintedanib in KIT D816V+ systemic mastocytosis: a preclinical study.

Systemic mastocytosis (SM) is a hematopoietic neoplasm with a complex pathology and a variable clinical course. Clinical symptoms result from organ infiltration by mast cells (MC) and the effects of pro-inflammatory mediators released during MC activation. In SM, growth and survival of MC are triggered by various oncogenic mutant-forms of the tyrosine kinase KIT. The most prevalent variant, D816V, confers resistance against various KIT-targeting drugs, including imatinib. We examined the effects of two novel promising KIT D816V-targeting drugs, avapritinib and nintedanib, on growth, survival, and activation of neoplastic MC and compared their activity profiles with that of midostaurin. Avapritinib was found to suppress growth of HMC-1.1 cells (KIT V560G) and HMC-1.2 cells (KIT V560G + KIT D816V) with comparable IC50 values (0.1-0.25 µM). In addition, avapritinib was found to inhibit the proliferation of ROSAKIT WT cells, (IC50: 0.1-0.25 µM), ROSAKIT D816V cells (IC50: 1-5 µM), and ROSAKIT K509I cells (IC50: 0.1-0.25 µM). Nintedanib exerted even stronger growth-inhibitory effects in these cells (IC50 in HMC-1.1: 0.001-0.01 µM; HMC-1.2: 0.25-0.5 µM; ROSAKIT WT: 0.01-0.1 µM; ROSAKIT D816V: 0.5-1 µM; ROSAKIT K509I: 0.01-0.1 µM). Avapritinib and nintedanib also suppressed the growth of primary neoplastic cells in most patients with SM examined (avapritinib IC50: 0.5-5 µM; nintedanib IC50: 0.1-5 µM). Growth-inhibitory effects of avapritinib and nintedanib were accompanied by signs of apoptosis and decreased surface expression of the transferrin receptor CD71 in neoplastic MC. Finally, we were able to show that avapritinib counteracts IgE-dependent histamine secretion in basophils and MC in patients with SM. These effects of avapritinib may explain the rapid clinical improvement seen during treatment with this KIT inhibitor in patients with SM. In conclusion, avapritinib and nintedanib are new potent inhibitors of growth and survival of neoplastic MC expressing various KIT mutant forms, including D816V, V560G, and K509I, which favors the clinical development and application of these new drugs in advanced SM. Avapritinib is of particular interest as it also blocks mediator secretion in neoplastic MC.

European Competence Network on Mastocytosis (ECNM): 20-Year Jubilee, Updates, and Future Perspectives.

In 2002, the European Competence Network on Mastocytosis (ECNM) was launched as a multidisciplinary collaborative initiative to increase the awareness and to improve diagnosis and management of patients with mast cell (MC) disorders. The ECNM consists of a net of specialized centers, expert physicians, and scientists who dedicate their work to MC diseases. One essential aim of the ECNM is to timely distribute all available information about the disease to patients, doctors, and scientists. In the past 20 years, the ECNM has expanded substantially and contributed successfully to the development of new diagnostic concepts, and to the classification, prognostication, and treatments of patients with mastocytosis and MC activation disorders. The ECNM also organized annual meetings and several working conferences, thereby supporting the development of the World Health Organization classification between 2002 and 2022. In addition, the ECNM established a robust and rapidly expanding patient registry and supported the development of new prognostic scoring systems and new treatment approaches. In all projects, ECNM representatives collaborated closely with their U.S. colleagues, various patient organizations, and other scientific networks. Finally, ECNM members have started several collaborations with industrial partners, leading to the preclinical development and clinical testing of KIT-targeting drugs in systemic mastocytosis, and some of these drugs received licensing approval in recent years. All these networking activities and collaborations have strengthened the ECNM and supported our efforts to increase awareness of MC disorders and to improve diagnosis, prognostication, and therapy in patients.

Human C1q Tumor Necrosis Factor 8 (CTRP8) defines a novel tryptase+ mast cell subpopulation in the prostate cancer microenvironment.

The adipokine C1q Tumor Necrosis Factor 8 (CTRP8) is the least known member of the 15 CTRP proteins and a ligand of the relaxin receptor RXFP1. We previously demonstrated the ability of the CTRP8-RXFP1 interaction to promote motility, matrix invasion, and drug resistance. The lack of specific tools to detect CTRP8 protein severely limits our knowledge on CTRP8 biological functions in normal and tumor tissues. Here, we have generated and characterized the first specific antiserum to human CTRP8 which identified CTRP8 as a novel marker of tryptase+ mast cells (MCT) in normal human tissues and in the prostate cancer (PC) microenvironment. Using human PC tissue microarrays composed of neoplastic and corresponding tumor-adjacent prostate tissues, we have identified a significantly higher number of CTRP8+ MCT in the peritumor versus intratumor compartment of PC tissues of Gleason scores 6 and 7. Higher numbers of CTRP8+ MCT correlated with the clinical parameter of biochemical recurrence. We showed that the human MC line ROSAKIT WT expressed RXFP1 transcripts and responded to CTRP8 treatment with a small but significant increase in cell proliferation. Like the cognate RXFP1 ligand RLN-2 and the small molecule RXFP1 agonist ML-290, CTRP8 reduced degranulation of ROSAKIT WT MC stimulated by the Ca2+-ionophore A14187. In conclusion, this is the first report to identify the RXFP1 agonist CTRP8 as a novel marker of MCT and autocrine/paracrine oncogenic factor within the PC microenvironment.

Prognostic Impact of Organomegaly in Mastocytosis: An Analysis of the European Competence Network on Mastocytosis.

BACKGROUND: Organomegaly, including splenomegaly, hepatomegaly, and/or lymphadenopathy, are important diagnostic and prognostic features in patients with cutaneous mastocytosis (CM) or systemic mastocytosis (SM). OBJECTIVES: To investigate the prevalence and prognostic impact of 1 or more organomegalies on clinical course and survival in patients with CM/SM. METHODS: Therefore, 3155 patients with CM (n = 1002 [32%]) or SM (n = 2153 [68%]) enrolled within the registry of the European Competence Network on Mastocytosis were analyzed. RESULTS: Overall survival (OS) was adversely affected by the number of organomegalies (OS: #0 vs #1 hazard ratio [HR], 4.9; 95% CI, 3.4-7.1, P < .001; #1 vs #2 HR, 2.1, 95% CI, 1.4-3.1, P < .001; #2 vs #3 HR, 1.7, 95% CI, 1.2-2.5, P = .004). Lymphadenopathy was frequently detected in patients with smoldering SM (SSM, 18 of 60 [30%]) or advanced SM (AdvSM, 137 of 344 [40%]). Its presence confered an inferior outcome in patients with AdvSM compared with patients with AdvSM without lymphadenopathy (median OS, 3.8 vs 2.6 years; HR, 1.6; 95% CI, 1.2-2.2; P = .003). OS was not different between patients having organomegaly with either ISM or SSM (median, 25.5 years vs not reached; P = .435). At time of disease progression, a new occurrence of any organomegaly was observed in 17 of 40 (43%) patients with ISM, 4 of 10 (40%) patients with SSM, and 33 of 86 (38%) patients with AdvSM, respectively. CONCLUSIONS: Organomegalies including lymphadenopathy are often found in SSM and AdvSM. ISM with organomegaly has a similar course and prognosis compared with SSM. The number of organomegalies is adversely associated with OS. A new occurrence of organomegaly in all variants of SM may indicate disease progression.

Capitalizing on paradoxical activation of the mitogen-activated protein kinase pathway for treatment of Imatinib-resistant mast cell leukemia.

Prevention of fatal side effects during cancer therapy of cancer patients with high-dosed pharmacological inhibitors is to date a major challenge. Moreover, the development of drug resistance poses severe problems for the treatment of patients with leukemia or solid tumors. Particularly drug-mediated dimerization of RAF kinases can be the cause of acquired resistance, also called "paradoxical activation." In the present work we re-analyzed the effects of different tyrosine kinase inhibitors (TKIs) on the proliferation, metabolic activity, and survival of the Imatinib-resistant, KIT V560G, D816V-expressing human mast cell (MC) leukemia (MCL) cell line HMC-1.2. We observed that low concentrations of the TKIs Nilotinib and Ponatinib resulted in enhanced proliferation, suggesting paradoxical activation of the MAPK pathway. Indeed, these TKIs caused BRAF-CRAF dimerization, resulting in ERK1/2 activation. The combination of Ponatinib with the MEK inhibitor Trametinib, at nanomolar concentrations, effectively suppressed HMC-1.2 proliferation, metabolic activity, and induced apoptotic cell death. Effectiveness of this drug combination was recapitulated in the human KIT D816V MC line ROSAKIT D816V and in KIT D816V hematopoietic progenitors obtained from patient-derived induced pluripotent stem cells (iPS cells) and systemic mastocytosis patient samples. In conclusion, mutated KIT-driven Imatinib resistance and possible TKI-induced paradoxical activation can be efficiently overcome by a low concentration Ponatinib and Trametinib co-treatment, potentially reducing the negative side effects associated with MCL therapy.