Mastocytosis associated with a rare germline KIT K509I mutation displays a well-differentiated mast cell phenotype

Abstract

Background: Mastocytosis associated with germline KIT activating mutations is exceedingly rare. We report the unique clinicopathologic features of a patient with systemic mastocytosis caused by a de novo germline KIT K509I mutation.

Objectives: We sought to investigate the effect of the germline KIT K509I mutation on human mast cell development and function.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: K509I; KIT; germline; mast cells; mastocytosis; well differentiated.

FIG. 1

Clinicopathological features of germline KIT K509I well-differentiated systemic mastocytosis. A, Cutaneous presentation as an infant. B, Diffuse cutaneous presentation as an adult with comparison to typical KIT D816V urticarial pigmentosa. C, KIT and CD25 staining of bone marrow mast cells with comparison to typical KIT D816V morphology. D, Diagram of KIT and location of the heterozygous K509I mutation in relationship to GNNK splice site.

FIG. 2

Clinical reaction and response to imatinib. A, Erythematous rash exaggerated by the initiation of imatinib. B, Serum total tryptase levels upon discontinuation and reinitiation of imatinib. C, Tryptase staining of bone marrow biopsy demonstrating mast cell involvement pre and post imatinib.

FIG. 3

KIT K509I CD34⁺ progenitors display enhanced proliferation and develop into hypergranular HuMCs in the presence of SCF. A, Growth of K509I HuMCs in the presence of SCF. Data are the mean ± SEM of 3 independent experiments performed in duplicate. B, Light microscopy with toluidine blue staining (20x magnification) and electron microscopy. C, Side scatter (SSC) histogram. Representative of 3 experiments.

FIG. 4

SCF-depletion impacts KIT K509I HuMC proliferation, development and survival. KIT K509I HuMC survival (A) and apoptosis (B) with various SCF concentrations. Data are the mean ± SEM of 3 independent experiments performed in triplicate. C, Immunoblot of KIT phosphorylation pre and post SCF stimulation. Representative of 3 experiments. D, SCF independent KIT K509I HuMC growth. Data are the mean ± SEM of 3 independent experiments performed in duplicate. E, Light microscopy with toluidine blue staining (20x magnification).

FIG. 5

Activating potential of KIT K509I is dependent on the GNNK⁻ isoform. MTT assay of transduced IC2 cells cultured in the (A) presence or (B) absence of SCF. Data are the mean +/− SEM of 3 independent experiments performed in triplicate. Growth and survival of transduced IC2 cells cultured in the (C) presence or (D) absence of SCF. Data are the mean +/− SEM of 2 independent experiments performed in duplicate. Control: empty vector.

FIG. 6

KIT K509I HuMCs display enhanced antigen-mediated activation. β-hexosaminidase release (A and B), PGD2 release (C and D) and intracellular Ca²⁺ flux (E and F) in the presence or absence of SCF. Data are the mean +/− SEM of 3 independent experiments performed in triplicate. Ca²⁺ flux represents an experiment performed in triplicate. G, FcεR1 surface expression by flow cytometry. Representative of 3 experiments. H, CD226 immunoblotting of whole cell lysates. Representative of 2 experiments.