Laboratory Assessment of Patients with Systemic Mastocytosis

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Abstract

The wast majority of patients with mastocytosis carry somatic KIT mutations. Mastocytosis associated with germline KIT activating mutations is exceedingly rare. We investigated the unique clinicopathologic features of a patient with systemic mastocytosis caused by a de novo germline KIT K509I mutation. We sought to investigate the effect of the germline KIT K509I mutation on human mast cell development and function. Primary human mast cells derived from CD34(+) peripheral blood progenitors were examined for growth, development, survival, and IgE-mediated activation. In addition, a mast cell transduction system that stably expressed the KIT K509I mutation was established. KIT K509I biopsied mast cells were round, CD25(-), and well differentiated. KIT K509I progenitors cultured in stem cell factor (SCF) demonstrated a 10-fold expansion compared with progenitors from healthy subjects and developed into mature hypergranular mast cells with enhanced antigen-mediated degranulation. KIT K509I progenitors cultured in the absence of SCF survived but lacked expansion and developed into hypogranular mast cells. A KIT K509I mast cell transduction system revealed SCF-independent survival to be reliant on the preferential splicing of KIT at the adjacent exonic junction. We concluded that germline KIT mutations associated with mastocytosis drive a well-differentiated mast cell phenotype distinct to that of somatic KIT D816V disease, the oncogenic potential of which might be influenced by SCF and selective KIT splicing. Within the mast cell compartment, both germline and somatic mutations in KIT have been identified in patients with rare familial presentations of mastocytosis. While seeking to characterize patients with unique, inherited allergic phenotypes among patients referred for evaluation of severe allergic skin, airway, or gastrointestinal disease or systemic mastocytosis (SM), we identified 9 atopic subjects with persistent increases in serum basal tryptase levels in the absence of evidence for a clonal mast cell disorder. Thorough clinical evaluations were undertaken, total and fractionated serum tryptase levels were obtained, in vitro basophil activation was assayed, and bone marrow biopsies were performed in 5 of these index patients. Increased basal serum tryptase levels were found to segregate with distinct clinical features, and it was noted that multiple family members of each index patient shared elements of this phenotype. An autosomal dominant inheritance pattern of increased basal total serum tryptase levels was revealed in all 9 families. The mean basal total serum tryptase level measured during a resting state was 21.6 ng/mL among subjects with inherited tryptasemia; however, tryptase fractionation in a subset of 18 affected patients further revealed that mature tryptase in serum was undetectable (<1 ng/mL), suggesting increase in protryptase levels. Prominent among features segregating with basal serum tryptase level increases were symptoms consistent with chronic and episodic mast cell degranulation, with 26 of 33 subjects reporting episodic urticaria, flushing, and/or cramping abdominal pain frequently associated with urgency, diarrhea, or both. A history of anaphylaxis was reported in 10 of 33 subjects from 6 different families. Gastrointestinal manifestations, whether chronic or episodic, were another prominent feature seen in 28 of 33 subjects with increased tryptase levels. Atopic symptoms were present in 31 of 33 tryptasemic subjects, with environmental allergies and asthma being reported among 28 of 33 affected subjects. Connective tissue abnormalities were present in 23 of 33 subjects with increased tryptase levels from 8 of 9 families, and chronic musculoskeletal pain was present among 11 of 33 tryptasemic subjects from 6 of 9 families. Autonomic dysfunction, manifesting as postural orthostatic tachycardia syndrome, was reported in 10 of 33 affected subjects, and a neuropsychiatric diagnosis was present in 17 of 33 affected subjects. These 5 clusters of clinical characteristics (cutaneous, connective tissue, gastrointestinal, atopic, and neuropsychiatric) were independently and significantly associated with increased tryptase levels. Because some of the reported symptoms in tryptasemic subjects could have been consistent with the familial presentation of a clonal mast cell disorder, index patients in 5 of 9 families underwent bone marrow biopsy. A significant increase in mast cell numbers was observed (P < .05; mean, 9.6 cells/high-power field hpf; range, 4-19.4 cells/hpf) compared with that seen in healthy volunteers (mean, 2.5 cells/hpf; range, 1-5 cells/hpf). However, none of the patients met the World Health Organization established criteria for a diagnosis of SM or monoclonal mast cell activation. Mast cell aggregates were not present, aberrant expression of CD2/CD25 was absent, and KIT D816V mutation test results were negative in all 5 patients. Spindled-shaped mast cells (>25%) were observed in a single patient. In vitro basophil activation was assessed in affected subjects from 6 of 9 families. Basophils from affected subjects demonstrated reduced activation, as measured based upon CD203c mean fluorescence intensity after stimulation with anti-IgE or N-formyl-methionylleucyl-phenylalanine. However, CD63 induction within the CD203c basophil population, although variable, was not significantly different between patients and paired control subjects. Additional genetic study is ongoing, and as the dysfunctional pathways in these families are elucidated, we might gain additional insights into the hypermobile connective tissue phenotype, atopic disease, autonomic dysfunction, and neuropsychiatric illness and their interface with mast cell mediators.