Allele specific qPCR is widely used to identify and assess KIT D816V in the peripheral blood of adults with mastocytosis in order to facilitate diagnosis, assessment of disease burden and response to therapy. To examine the value of this assay in children with cutaneous mastocytosis, in FY 2019, we completed and published the assessment of data on 65 children with pediatric-onset cutaneous and systemic mastocytosis by correlating KIT mutation status with clinical findings, serum tryptase levels and bone marrow histopathology. We concluded that KIT D816V in peripheral blood was exclusively found in children known to have both cutaneous and systemic disease; and correlated with serum tryptase and disease severity. These findings formed the basis of an algorithm which may be used by care-givers to assist in arriving at the decision to perform a bone marrow biopsy in children presenting with cutaneous mastocytosis. In FY 2019, we continued research on extracellular vesicles (EVs)that associate with hematologic disorders and are reported as vectors of molecular information that effect other cell types. In FY 2019, we thus completed and published an examination of blood samples from patients with systemic mastocytosis (SM) and found EVs with a mast cell signature including the presence of tryptase, FcepsilonRI, MRGPRX2 and KIT. The concentration of these EVs correlated with parameters of disease including levels of serum tryptase and hepatosplenomegaly. We then questioned if SM-EVs might affect hepatic stellate cells, given the abnormal liver pathology associated with mastocytosis. We found that mastocytosis EVs are taken up by hepatic stellate cells (HSCs) and this interaction altered the proliferation, cytokine production and differentiation of these cells. This data is consistent with the conclusion that SM-EVs have the potential to influence cells outside the hematological compartment, and that therapeutic approaches for treatment of SM should in part target inhibition of the effects of EVs on target tissues. In FY 2019, we also expanded these studies to examine the effect of EVs on bone formation, as osteoporosis is a major complication of systemic mastocytosis and correlates with the presence of mast cell infiltrates in the bone marrow. We found that EVs released by neoplastic mast cells and present in the serum of patients with SM (SM-EVs) block osteoblast differentiation and functionality in culture; and when injected into mice reduce the expression of osteoblast markers. We also found evidence that these EVs contain microRNAs linked to osteogenesis regulation; and were able to demonstrate that miRNA-30a and miRNA-23a mediate the inhibition of osteoblast differentiation/maturation by SM-EVs. We thus concluded that SM-EVs and neoplastic mast cell-derived EVs deliver miRNAs that epigenetically attenuate osteoblast function and alter bone homeostasis which results in reduction of bone mass. These findings reinforce the possibility of novel approaches targeted to EVs in the management of bone disease in patients with mast cell proliferative disorders. In FY 2019, we performed a study where we examined lymphocyte populations in patients with mastocytosis to explore whether an excess mast cell burden might influence specific subsets of lymphocytes. We found that patients with mastocytosis exhibited a significantly lower median frequency and absolute cell count of both circulating CD8+ T cells and Natural Killer cells accompanying a significantly increased ratio of CD4+/C8+ T cells. Stratification of patients according to clinical manifestations further revealed that CD19+CD21lowCD38low B cells were significantly higher in patients with a history of autoimmune disease and counts of terminally differentiated CD4+ T cells were significantly higher in patients with osteoporosis or osteopenia. These data suggest the need for further studies on abnormalities in lymphocyte subsets and the attendant clinical consequences in both mast cell proliferative and activation disorders. Unexplained anaphylaxis may occur in patients with mastocytosis and such episodes may be quite severe. Omalizumab has been proposed as a potential therapeutic strategy to reduce the number of anaphylactic episodes in patients with systemic mastocytosis and short-term studies seem to validate this approach. In FY 2019, we reported the success of the use of omalizumab in prevent recurrent episodes of anaphylaxis over a 12-year period in two patients with systemic anaphylaxis and where the drug was well tolerated without adverse events related to administration. In both patients, anaphylactic episodes were rare. There was also an improvement in cutaneous manifestations in both individuals, along with a significant reduction in serum tryptase and bone marrow mast cell burden in one patient. Our report supports the design of long-term controlled trials for the use of omalizumab as a therapeutic option for patients with clonal disorders of mast cells. Patients with indolent (non-aggressive) systemic mastocytosis are not candidates for cytoreductive therapy and are generally treated with symptomatic therapy that only partly decreases symptoms. There is, however, a documented association between severity of mastocytosis and elevated serum levels of interleukin IL-6. Furthermore, mast cells have been shown to double their rate of division and exhibit increased reactivity and release of mediators when cultured in the presence of IL-6. In addition, in an animal model of mastocytosis, anti-IL-6 has been shown to slow disease progression. In FY 2019 we thus initiated a clinical trial where adults with indolent systemic mastocytosis are randomized and treated with sarilumab which binds to the IL 6 receptor and inhibits IL 6 associated human mast cell signaling, proliferation and reactivity (decreased mediator release). Over a four-month period, evaluations at study visits will include quality of life and symptom assessments and measurement of serum tryptase levels. Bone marrow examinations will be performed at the onset and conclusion of the study.
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