Proliferation Of Neoplastic Mast Cells Research Articles

Advanced systemic mastocytosis-Revised classification, new drugs and how we treat.

Mastocytosis constitutes the neoplastic proliferation of mast cells and is broadly classified into systemic mastocytosis (SM), cutaneous mastocytosis and mast cell sarcoma. SM is further partitioned into advanced (AdvSM) and non-advanced (SM-non-Adv) subcategories. AdvSM includes aggressive SM (ASM), SM with an associated haematological neoplasm (SM-AHN) and mast cell leukaemia (MCL). In 2022, two separate expert committees representing the 5th edition of the World Health Organization (WHO5) and the International Consensus (ICC) classification systems submitted revised classification criteria for SM, highlighted by the ICC-proposed incorporation of mast cell cytomorphology in the diagnostic criteria for MCL and myeloid-lineage restriction for the AHN component in SM-AHN. Recent developments in SM also include the introduction of KIT-targeting tyrosine kinase inhibitors (KITi), including midostaurin and avapritinib, both drugs have shown potent activity in reducing mast cell and mutant KIT burden and alleviating mast cell-associated organopathy and mediator symptoms; however, their overall impact on survival or superiority over pre-KITi era treatment options (e.g. cladribine) has not been studied in a controlled setting. In the current review, we provide a summary of recent changes in disease classification and an analysis of recent clinical trials and their impact on our current treatment approach in AdvSM.

Systemic mastocytosis mimicking blastic plasmacytoid dendritic cell neoplasm: a case report

BackgroundSystemic mastocytosis (SM), a rare myeloid neoplasm, is defined as a clonal and neoplastic proliferation of mast cells in at least one extracutaneous organ(s). The pathologic diagnosis and treatment of SM are challenging.Case presentationWe presented a 44-year-old male patient who had endured abdomen discomfort for 4 years and diarrhea for 5 months. Colonoscopy and PET/CT found a protuberant lesion in the cecum with adjacent lymphadenopathy. Histopathology of the cecum biopsy showed diffuse infiltration of medium-sized round/oval cells in lamina propria with immunohistochemical expressions of CD45, CD117, CD25, CD68, CD123, CD56, CD4, and CD35, mimicking blastic plasmacytoid dendritic cell neoplasm. Sanger sequencing revealed missense mutation (D816V) in the exon 17 of KIT gene. Serum tryptase level was 38.56 ng/ml. No abnormality was found in skin examination and bone marrow biopsy. No primitive cells were observed in bone marrow smear and peripheral blood smear. The diagnosis of aggressive SM with intestinal tract involvement was established. The patient received avapritinib treatment at an initial dosage of 200 mg once daily and exhibited dramatic clinical improvement but memory impairment within 1 month. No recurrence was observed in 1-year follow-up at the adjusted avapritinib dose (75 mg once daily).ConclusionsSM is very rare and should be considered in patients with long-term diarrhea symptoms and hematopoietic/lymphoid-appearing tumors. KIT D816V mutation contributes to the differentiation of CD123, CD4, and CD56 immunoreactive SM from blastic plasmacytoid dendritic cell neoplasm. The rare side-effect of memory impairment in this case helps to accumulate the experience of avapritinib in treating KIT D816V-mutant SM.

Systemic Lymphadenopathic Mastocytosis with Eosinophilia.

Systemic mastocytosis is a neoplastic proliferation of mast cells that most frequently involves cutaneous sites. Mastocytosis involves various extracutaneous sites, but the lymph node is rare. We present an interesting image of systemic mastocytosis in the lymph node with marked eosinophilia. It is a rare subtype of systemic mastocytosis requiring high suspicion levels for the correct diagnosis.

Mastocytosis in the skin accompanied by pseudo-Kaposi's sarcoma.

Mastocytosis is a heterogeneous group of diseases characterized by abnormal proliferation of neoplastic mast cells in the skin and/or other extracutaneous tissues. Most patients with skin involvement can be subclassified into one of the three subtypes of cutaneous mastocytosis currently recognized by the World Health Organization (i.e., mastocytoma, maculopapular cutaneous mastocytosis and diffuse cutaneous mastocytosis); however, some patients may occasionally present with atypical skin lesions that cannot be ascribed to any of these disease subtypes. Here, we report three patients diagnosed with mastocytosis and an unusual cutaneous involvement mimicking Kaposi's sarcoma. Skin biopsies showed neoplastic mast cell infiltrates together with features commonly seen in acroangiodermatitis, and immunohistochemistry for human herpesvirus 8 was negative. One patient fulfilled the criteria for aggressive systemic mastocytosis, showed no response to cytoreductive therapy, and died because of disease progression. The remaining two patients had indolent and smoldering systemic mastocytosis, respectively, but they showed several features associated with an unfavorable prognosis such as extensive involvement of the hematopoiesis by the KIT D816V mutation, increased serum β2-microglobulin, and decreased serum lactate dehydrogenase. The presence of pseudo-Kaposi's sarcoma skin lesions is an uncommon finding in mastocytosis which may alert physicians to the possible existence of underlying features indicative of a poor prognosis.

Hypertryptasemia and Mast Cell-Related Disorders in Severe Osteoporotic Patients.

Purpose Systemic mastocytosis (SM) is characterized by a clonal proliferation of neoplastic mast cells (MCs) in one or more extracutaneous organs including the bone marrow (BM). SM is often associated with osteoporosis (OP) and fractures. Hypertryptasemia usually occurs in SM. We investigated the prevalence of hypertryptasemia in a series of severe osteoporotic patients, the performance of the tryptase test in diagnosing SM in these patients, and their bone features. Methods The medical records of 232 patients (168 females and 64 males) with a diagnosis of OP (50.4% with fractures) and a serum tryptase assessment were reviewed. BM assessment was performed in a subset of hypertryptasemic patients; clinical, biochemical, and radiographic data were collected. Results Hypertryptasemia was detected in 33 patients. BM assessment (n = 16) was normal in 8 hypertryptasemic patients, while BM criteria for the diagnosis of SM were met in 3 patients, MC alterations were detected in 4 patients, and one patient presented a polycythemia vera. Serum tryptase levels were higher than 11.4 ng/ml in all patients with BM alterations. The best cut-off of tryptase level related to BM alterations was 17.9 ng/ml, with a sensibility and sensitivity of 75% (AUC = 0.797 and P = 0.015 by ROC analysis). All osteoporotic patients with hypertryptasemia experienced at least one vertebral fracture associated with a severe reduction of the lumbar bone mineral density. Conclusions The prevalence of MC-related disorders in severe OP was 3.0%, accounting for the 7.4% of the secondary causes of OP. MC-related disorders may be involved in bone fragility and assessment of serum tryptase is useful to detect MC-related disorders.

In vitro effects of histamine receptor 1 antagonists on proliferation and histamine release in canine neoplastic mast cells.

Canine mastocytomas (MCTs) are characterized by rapid proliferation of neoplastic mast cells (MCs) and clinical signs caused by MC‐derived mediators. In dogs suffering from MCT, histamine receptor 1 (HR1) antagonists are frequently used to control mediator‐related clinical symptoms. Previous studies have shown that the HR1 antagonists loratadine and terfenadine exert some growth‐inhibitory effects on neoplastic MCs. We examined whether other HR1 antagonists used in clinical practice (desloratadine, rupatadine, cyproheptadine, dimetindene, diphenhydramine) affect proliferation and survival of neoplastic MCs. Furthermore, we analysed whether these HR1 antagonists counteract IgE‐dependent histamine release from a MC line harbouring a functional IgE‐receptor. HR1 antagonists were applied on two canine MC lines, C2 and NI‐1, and on primary MCs obtained from three MCT samples. The HR1 antagonists desloratadine, rupatadine and cyproheptadine were found to be more potent in decreasing proliferation of C2 and NI‐1 cells when compared with dimetindene and diphenhydramine. Similar effects were seen in primary neoplastic MCs, except for diphenhydramine, which exerted more potent growth‐inhibitory effects than the other HR1 antagonists. Drug‐induced growth‐inhibition in C2 and NI‐1 cells was accompanied by apoptosis. Loratadine, desloratadine and rupatadine also suppressed IgE‐dependent histamine release in NI‐1 cells. However, drug concentrations required to elicit substantial effects on growth or histamine release were relatively high (>10 µM). Therefore, it remains unknown whether these drugs or similar, more potent, HR1‐targeting drugs can suppress growth or activation of canine neoplastic MCs in vivo.

Prevalence of CD30 immunostaining in neoplastic mast cells: A retrospective immunohistochemical study.

Mastocytosis is a rare disease characterized by clonal neoplastic proliferation of mast cells (MCs). It ranges from skin lesions as cutaneous mastocytosis (CM) which may spontaneously regress to highly aggressive neoplasms with multiorgan involvement corresponding to some aggressive systemic mastocytosis (ASM), mast cell leukemia (MCL), and/or mast cell sarcoma (MCS).There is increasing evidence of CD30 expression in neoplastic MCs of the bone marrow. This expression has been described almost exclusively in aggressive forms of systemic mastocytosis (SM).The aim of the present study is to evaluate CD30 expression both in cutaneous and systemic forms of mastocytosis. Forty-two mastocytosis cases were reviewed, including cutaneous (n = 29) and systemic (n = 13) forms to assess the prevalence of CD30 expression. Thirty-nine out of 42 (92.8%) cases were CD30 positive. In cases of CM, 28/29 (96.5%) cases were CD30 positive, 11/13 cases of SM (84.6%) were positive for CD30. MCs in normal skin biopsies and in urticaria lesions were CD30-negative. This study found that CD30 is also frequently expressed in CM as well as in systemic forms. This finding is a major departure from the prevailing concept that CD30 expression is often related to aggressive systemic forms of mastocytosis.

Sphingosine Kinase Inhibitors Effectively Inhibit Growth of Mast Cells with D816V-KIT Mutations

Abstract Sphingosine-1-phosphate (S1P) is a lipid mediator that promotes cell proliferation and prevents apoptosis in a variety of cells and its increased synthesis by Sphingosine Kinase (SphK) has been associated with oncogenesis and cancer prognosis. Mastocytosis is a disorder characterized by excessive mast cell (MC) proliferation and accumulation in tissues. Treatment therapies for aggressive mastocytosis have to date been partially effective, and thus the search of new pharmacological targets is warranted. We thus investigated the involvement of SphK isoforms (SphK1 and SphK2) and generation of S1P in the regulation of abnormal MC growth. The specific SphK1 and SphK2 inhibitors, SKI-178 and ABC294640, respectively, were used in this study. The effect of SphK1 and SphK2 inhibition on human (LAD2, HMC1.1 and HMC1.2) and murine (P815) neoplastic MC proliferation and survival was evaluated. SphK2 inhibition retarded entry into the cell cycle in all MC lines with no significant effect on cell survival. However, SphK1 inhibition led to cell cycle arrest in G2/M and apoptosis predominantly in MCs carrying D816V-KIT mutation. The effect of SKI-178 was associated with an alteration in the damage response cascade. This included activation of checkpoint kinase 2 (chk2) combined with a depletion of chk1, leading respectively to p53- and caspase2-induced apoptosis and cell cycle arrest mediated by cdc25c depletion. Both SphK inhibitors reduced MC accumulation in an in vivo mouse model of aggressive mastocytosis and the number of cultured bone marrow MCs and MC progenitors from a patient with systemic mastocytosis. These results support further consideration for SphK inhibitors as a cytoreductive strategy in proliferative MC disorders.

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells.

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells.

Mast cell leucemia - case report

Mastocytosis is a rare disease caused by the proliferation and accumulation of mast cells in places that have connective tissue and marrow. The term mast cell leukemia is used to define the proliferation of neoplastic mast cells in the bone marrow. A dog was treated at the Veterinary Hospital of the University of Santo Amaro - UNISA, with prostration, apathy, vomiting, and intense termofilia districao exhalation, the examination of the bone marrow was visualized hypercellularity 60% where 43.8% were mature mast cells. The animal died and sent to the Pathology Service to necropsy exam that shows severe hepatomegaly, splenomegalyand and presence of small ulcers and gastric erosion. It was carried out cytological and histological examination. therefore found a case of mast cell leukemia.

Diagnosis of Mastocytosis in Children and Adults in Daily Clinical Practice.

Mastocytosis comprises a heterogeneous group of disorders characterized by clonal, neoplastic proliferation of mast cells accumulating in one or multiple organs. In the majority of cases skin involvement is the first clinical manifestation of the disease. Clinical work-up consists of a combination of morphological, immunohistochemical, flow cytometric immunophenotyping and molecular examination. Cutaneous mastocytosis predominates in children, whereas systemic mastocytosis is the most common form of the disease in adults. Therefore, different diagnostic algorithms have to be applied in adult patients and children with suspected mastocytosis. This comprehensive review presents currently defined variants of the disease and recommendations to facilitate diagnostic work-up in children and adults with suspected mastocytosis in daily clinical practice.

Morphological Features and KIT Receptor Expression in Canine Cutaneous Mast Cell Tumor and Systemic Mastocytosis / Morfološke Karakteristike I Ekspresija KIT Receptora Kod Kutanih Mastocitoma I Sistemske Mastocitoze Pasa

Abstract Mast cell neoplasia in dogs can occur in two different forms: common as cutaneous tumor, or less common as a systemic form of neoplastic mast cell proliferation - systemic mastocytosis. The aim of this study was to compare the histological and cytological features, KIT receptor expression and presence of c-KIT proto-oncogene mutations in neoplastic cells of dogs with canine cutaneous mast cell tumor (CMCT) and systemic mastocytosis. Microscopical examination of the cytological smears obtained from all selected dogs revealed that cellular specimens were constituted mostly of round cells with a central nuclei and fine to coarse purple cytoplasmic granules. Histopathological examination of skin samples of dogs with CMCT and a dog with systemic mastocytosis showed proliferation of the neoplastic mast cells in the superficial and/or deep dermis. Similar findings were observed in tissue samples derived from lymph nodes, spleen, liver, myocardium and kidneys of a dog with systemic mastocytosis. Three dogs with high grade CMCT as well as one dog with systemic mastocytosis showed cytoplasmic CD117 expression, while 3 dogs with low grade CMCT, had membranous expression of CD117. Based on our study, histological features and cytoplasmic CD117 expression in neoplastic cells of dogs with systemic mastocytosis are similar to those in dogs with high grade CMCTs. Nevertheless, mutations of c-KIT proto-oncogene were not found in tumor samples either from dogs with CMCT or dog with systemic mastocytosis.

Subthreshold IKK activation modulates the effector functions of primary mast cells and allows specific targeting of transformed mast cells.

Mast cell differentiation and proliferation depends on IL-3. IL-3 induces the activation of MAP-kinases and STATs and consequently induces proliferation and survival. Dysregulation of IL-3 signaling pathways also contribute to inflammation and tumorigenesis. We show here that IL-3 induces a SFK- and Ca²⁺-dependent activation of the inhibitor of κB kinases 2 (IKK2) which results in mast cell proliferation and survival but does not induce IκBα-degradation and NFκB activation. Therefore we propose the term "subthreshold IKK activation".This subthreshold IKK activation also primes mast cells for enhanced responsiveness to IL-33R signaling. Consequently, co-stimulation with IL-3 and IL-33 increases IKK activation and massively enhances cytokine production induced by IL-33.We further reveal that in neoplastic mast cells expressing constitutively active Ras, subthreshold IKK activation is associated with uncontrolled proliferation. Consequently, pharmacological IKK inhibition reduces tumor growth selectively by inducing apoptosis in vivo.Together, subthreshold IKK activation is crucial to mediate the full IL-33-induced effector functions in primary mast cells and to mediate uncontrolled proliferation of neoplastic mast cells. Thus, IKK2 is a new molecularly defined target structure.

Oncogenic Kit signals on endolysosomes and endoplasmic reticulum are essential for neoplastic mast cell proliferation.

Kit is a receptor-type tyrosine kinase found on the plasma membrane. It can transform mast cells through activating mutations. Here, we show that a mutant Kit from neoplastic mast cells from mice, Kit(D814Y), is permanently active and allows cells to proliferate autonomously. It does so by activating two signalling pathways from different intracellular compartments. Mutant Kit from the cell surface accumulates on endolysosomes through clathrin-mediated endocytosis, which requires Kit’s kinase activity. Kit(D814Y) is constitutively associated with phosphatidylinositol 3-kinase, but the complex activates Akt only on the cytoplasmic surface of endolysosomes. It resists destruction because it is under-ubiquitinated. Kit(D814Y) also appears in the endoplasmic reticulum soon after biosynthesis, and there, can activate STAT5 aberrantly. These mechanisms of oncogenic signalling are also seen in rat and human mast cell leukemia cells. Thus, oncogenic Kit signalling occurs from different intracellular compartments, and the mutation acts by altering Kit trafficking as well as activation.

Systemic Mastocytosis with Associated Acute Myelogenous Leukemia

Systemic mastocytosis (SM) is a condition associated with a clonal neoplastic proliferation of mast cells. Approximately 40% of patients with SM present with an associated clonal hematological non–mast cell lineage disorder. Patients presenting with SM–acute myeloid leukemia (AML) have the worst prognosis. We present a case of a 62-year-old woman who was diagnosed with SM-AML. After initial treatment with a standard regimen of cytosine arabinoside (Ara-C)/idarubicin, her bone marrow showed residual blasts. She was subsequently treated with a second induction regimen of clofarabine and high-dose Ara-C, which resulted in remission of AML, although a residual mast cell infiltrate persisted in her bone marrow. After consolidation therapy with clofarabine/Ara-C, the patient received a stem cell allograft. A follow-up bone marrow showed no residual blasts but persistent mast cells occupying about 5% of the marrow volume.

Production of stem cell factor in canine mast cell tumors

Mast cell tumor (MCT) is the most common cutaneous tumor in dogs. We recently revealed that production of stem cell factor (SCF) contributes to the proliferation of neoplastic mast cells in an autocrine/paracrine manner. The aim of the present study was to determine the contribution of the mechanism in clinical MCTs. In consequence, high SCF expression (>10 times compared to HRMC cells) was observed in 5 of 7 MCT samples used in the study regardless of KIT mutation, which was confirmed in immunohistochemical analysis. In addition, production of SCF was observed in Ki-67-positive cells in the MCT xenograft. These results indicate the broad contribution of SCF autocrine/paracrine mechanism on clinical MCTs, providing the rationale for the clinical use of KIT inhibitors regardless of KIT mutation.

Stem cell factor contributes to tumorigenesis of mast cells via an autocrine/paracrine mechanism

Mastocytosis is a disease accompanied by the abnormal expansion and accumulation of mast cells. Although the D816V mutation is detected in most cases of systemic mastocytosis, the mutation is rarely observed in other forms of mastocytosis, such as cutaneous mastocytosis and mast cell leukemia/sarcoma, for which the mechanism of tumorigenesis remains unknown. In this study, we demonstrated a novel mechanism of mast cell tumorigenesis via SCF autocrine/paracrine release. SCF was highly expressed in a WT KIT-expressing HRMC line, contributing to the phosphorylation of KIT. Neutralization of external SCF using a neutralizing antibody or suppression of SCF production by RNA interference inhibited the growth of HRMC cells, indicating the essential role of SCF in cell proliferation. To the best of our knowledge, this is the first report to determine the significant contribution of SCF autoproduction to neoplastic proliferation of mast cells. These results indicate the possibility that targeting SCF production may become a novel treatment for mast cell malignancies.

Targeting the KIT activating switch control pocket: a novel mechanism to inhibit neoplastic mast cell proliferation and mast cell activation

Activating mutations in the receptor tyrosine kinase KIT, most notably KIT D816V, are commonly observed in patients with systemic mastocytosis. Thus, inhibition of KIT has been a major focus for treatment of this disorder. Here we investigated a novel approach to such inhibition. Utilizing rational drug design, we targeted the switch pocket (SP) of KIT which regulates its catalytic conformation. Two SP inhibitors thus identified, DP-2976 and DP-4851, were examined for effects on neoplastic mast cell proliferation and mast cell activation. Autophosphorylation of both wild type (WT) and, where also examined, KIT D816V was blocked by these compounds in transfected 293T cells, HMC 1.1 and 1.2 human mast cell lines; and in CD34+-derived human mast cells activated by stem cell factor (SCF). Both inhibitors induced apoptosis in the neoplastic mast cell lines and reduced survival of primary bone marrow mast cells from patients with mastocytosis. Moreover, the SP inhibitors more selectively blocked SCF potentiation of FcεRI-mediated degranulation. Overall, SP inhibitors represent an innovative mechanism of KIT inhibition whose dual suppression of KIT D816V neoplastic mast cell proliferation and SCF enhanced mast cell activation may provide significant therapeutic benefits.

Ponatinib Exerts Growth-Inhibitory Effects on Neoplastic Mast Cells and Synergizes with Midostaurin in Producing Growth Arrest and Apoptosis,

Abstract 3497Aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL) have a poor prognosis. In these patients, neoplastic mast cells (MC) usually harbor the D816V-mutated variant of KIT and are resistant to conventional cytoreductive drugs and to several tyrosine kinase inhibitors (TKI) such as imatinib. More recently, various KIT kinase blockers including midostaurin (PKC412), have been described to overcome KIT D816V-mediated resistance in neoplastic MC. However, despite encouraging first results observed in clinical trials, these novel kinase blockers are unable to induce long-lasting complete remissions in all patients with ASM and MCL. One reason for the poor response in these patients may be the expression and activation of additional KIT-independent pro-oncogenic signalling molecules and pathways that trigger survival of neoplastic MC. Therefore, current research is seeking novel broadly acting drugs and drug combinations directed against the pro-oncogenic signaling machinery of neoplastic MC. Ponatinib (AP24534) is a broadly acting novel multikinase inhibitor that has been shown to exert major anti-leukemic effects in chronic myeloid leukemia. The aim of our current study was to evaluate the effects of ponatinib on growth and survival of neoplastic MC. Ponatinib was applied as single agent or in combination with midostaurin (PKC412). As assessed by Western blotting, ponatinib was found to inhibit KIT-phosphorylation in both subclones of the human MC leukemia cell line HMC-1, namely HMC-1.1 harboring KIT G560V but not KIT D816V, and HMC-1.2 cells harboring KIT G560V and KIT D816V. Interestingly, the D816V mutation of KIT was found to induce relative resistance against ponatinib. Ponatinib was also found to counteract the phosphorylation of Lyn, a Src-kinase that serves as a major KIT-independent signalling molecule and survival factor in neoplastic MC. Activated STAT5 in MC was also blocked by ponatinib in a dose-dependent manner. In a next step, we examined the effects of ponatinib on proliferation of neoplastic MC by 3H-thymidine uptake experiments. Ponatinib was found to induce dose-dependent growth inhibition in both HMC-1 subclones, with higher IC50-values in HMC-1 cells harbouring KIT D816V (IC50: 100–500 nM) compared to cells lacking KIT D816V (IC50: 1–10 nM). Furthermore, ponatinib was found to inhibit the proliferation of primary neoplastic MC isolated from patients with indolent SM (ISM, n=2) and ASM (n=1), with IC50-values ranging between 50 nM and 500 nM. Growth inhibitory effects of ponatinib on neoplastic MC were accompanied by induction of apoptosis as assessed by light microscopy, flow cytometry, and TUNEL assay. Finally, we were able to demonstrate that ponatinib synergizes with midostaurin in producing growth-inhibition and apoptosis in HMC-1.1 cells and HMC-1.2 cells. Synergistic effects obtained with suboptimal concentrations of single agents were accompanied by a complete blockage of all relevant kinase targets tested including KIT, Lyn, and STAT5. In conclusion, ponatinib exerts major growth-inhibitory effects on neoplastic MC. KIT D816V-expressing MC are less sensitive to ponatinib. This relative resistance of MC against ponatinib can be overcome by combining ponatinib with midostaurin in an in vitro assay. Whether the drug-combination also exerts major anti-neoplastic effects in vivo in patients with ASM and MCL remains to be determined. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.

The Midostaurin (PKC412) Metabolite CGP52421 Shows Little Growth-Inhibitory Activity Against Against Neoplastic Mast Cells but Retains Inhibitory Effects on IgE-Dependent Activation and Histamine Release

Abstract 1417The multikinase inhibitor midostaurin (PKC412) is currently tested in clinical trials in patients with advanced systemic mastocytosis (SM). Although clinical symptoms improve in many patients and sometimes the proliferation of neoplastic mast cells (MC) can be kept under control for some time, most patients progress after a variable latency period, even if their mediator-related symptoms are still completely suppressed. In vivo, midostaurin is metabolized to two major and active metabolites, namely CGP62221 and CGP52421. Whereas the in vitro effects of midostaurin on growth and activation of MC are well documented, only little data on effects of midostaurin-metabolites are available. We examined the effects of midostaurin and its pharmacologically relevant metabolites CGP52421 and CGP62221, on IgE-dependent mediator secretion in basophils as well as growth of primary neoplastic MC and the human MC leukemia cell line HMC-1. All three compounds were found to inhibit IgE-dependent secretion of histamine in blood basophils, with comparable IC50 values (<1 μM). Midostaurin and CGP62221 were also found to inhibit the proliferation in HMC-1.1 cells and HMC-1.2 cells with IC50 values ranging between 50 and 250 nM. However, the second metabolite of PKC412, CGP52421, did not produce comparable anti-proliferative effects, even when applied at concentrations up to 1 μM. Corresponding results were obtained when analyzing drug-effects on primary neoplastic MC obtained from two patients, one with indolent SM and one with aggressive SM. Furthermore, whereas midostaurin and CGP62221 induced apoptosis in HMC-1.1 and HMC-1.2 cells as evidenced by light microscopy, caspase 3 staining and TUNEL assay, CGP52421 did not induce apoptosis in neoplastic MC. Finally, midostaurin and CGP62221 were found to induce dephosphorylation of KIT V560G and KIT D816V in HMC-1 cells, whereas no effects of CGP52421 on KIT activation were seen. In drug competition experiments, the metabolite CGP52421 did not interfere with midostaurin- or CGP62221-induced growth inhibition in HMC-1 cells. In conclusion, the pharmacologically relevant midostaurin metabolite CGP52421 inhibits IgE-mediated histamine release but does not efficiently block KIT-dependent proliferation of neoplastic MC. This observation may help to explain clinical responses including improvement of mediator-related symptoms seen in midostaurin-treated patients with advanced SM, and would favor the development of new treatment concepts employing midostaurin as an inhibitor of both MC activation and MC growth in patients with SM. Disclosures:Dutreix:Novartis: Employment. Gross:Novartis: Employment. Roesel:Novartis Pharma AG: Employment. Valent:Novartis: Consultancy, Honoraria, Research Funding.