Mastocytosis encompasses a group of rare mast cell (MC) neoplasms classified as MC sarcoma, cutaneous mastocytosis (CM), or systemic mastocytosis (SM). Gain-of-function (GOF) variants in the KIT oncogene, encoding the receptor tyrosine kinase c-KIT (KIT), are linked to most cases of CM and SM. However, our understanding of if and how different KIT variants might distinguish morphologic SM phenotypes remains incomplete. We reviewed a large cohort of 454 patients diagnosed with SM based on bone marrow biopsy analysis and application of 2022 WHO diagnostic criteria. Established GOF KIT variants at codon 816 (D816V and D816Y) were detected in biopsy samples from >96% of SM cases (436/454), all of which showed spindled morphology in >25% of MCs. In 7 of the remaining 18 cases, all bone marrow MCs exhibited a uniformly round, slightly enlarged morphology consistent with SM with well-differentiated phenotype (SMWD). Next generation sequencing discovered germline missense KIT variants in 5/7 patients with SMWD, including two patients with p.Lys509Ile (K509I), two with p.Ala533Asp (A533D), and one with novel compound heterozygous variants p.Met541Leu (M541L) and p.Phe681Leu (F681L). We then developed a novel transfection assay to compare how each variant affected KIT expression, cellular localization, and autophosphorylation -/+ stimulation with the KIT ligand stem cell factor (SCF). Consistent with prior reports and in contrast to wild-type KIT, D816 KIT variants remained largely intracellular and displayed a strong, SCF-independent autophosphorylation of multiple tyrosine residues in the cytoplasmic tail. Conversely, SMWD-derived KIT variants displayed variable surface expression with distinct patterns of glycosylation and tyrosine phosphorylation relative to wild type. Baseline phospho-KIT levels were far lower than D816V/Y but higher than wild type, with additional enhancement observed after acute SCF stimulation. Immunohistochemical analysis further distinguished SMWD based on exclusive intracellular expression of CD25 on MCs in 6/7 cases. Overall, these results define unique molecular features of SMWD based on restricted intracellular CD25 expression and detection of milder, ligand-dependent hypomorphic KIT variants relative to strong D816 GOF mutations. Our findings provide new molecular insights into SMWD pathogenesis that should improve diagnostic accuracy and inform therapeutic application of (and response to) tyrosine kinase inhibitors targeting KIT.
