Elsevier

Leukemia Research

Volume 30, Issue 4, April 2006, Pages 373-378
Leukemia Research

A novel K509I mutation of KIT identified in familial mastocytosis—in vitro and in vivo responsiveness to imatinib therapy

https://doi.org/10.1016/j.leukres.2005.08.015Get rights and content

Abstract

KIT mutation has been implicated in sporadic mastocytosis, yet clusters in only a few sites in the molecule. For those malignancies associated with KIT mutation or over-expression, imatinib offers a specific therapeutic option, yet it has no effect on D816V mutation commonly seen in sporadic mastocytosis. The majority of cases of familial mastocytosis seem to lack KIT mutation. We report a kindred with mastocytosis in whom in vitro and in vivo sensitivity to imatinib was demonstrated. Mutation analysis of the KIT coding region in this family identified a novel A > T mutation at nucleotide 1547 [K509I] in exon 9 in both of the affected patients.

Introduction

KIT is the cellular homologue of v-kit, which is found in the Hardy-Zuckerman 4 feline sarcoma virus [1] and is a Class III transmembrane receptor tyrosine kinase (RTK). Following ligand binding to the Ig domains, dimerisation occurs thus activating intrinsic kinase activity by transphosphorylation of specific residues in the JM and TK domains. KIT mutation has been implicated in a wide range of tumour types and although many KIT mutations have been identified to date, they appear to cluster in only a few sites in the molecule.

Exon 11 mutations affect the juxtamembrane (JM) domain, in particular the tyrosine residues 550–560 that form an inhibitory alpha helix, which suppresses autophosphorylation. Mutations of this region disrupt the binding of regulatory proteins to the JM region, result in autophosphorylation and constitutive activation of the tyrosine kinase. Such activating mutations have been seen in mast cell lines [2], [3] as well as small numbers of adults with mast cell disease [4]. Gastrointestinal stromal tumours (GISTs) are also commonly associated with exon 11 mutations.

Exon 17 mutations affect the TK domain with the most commonly observed mutation being an A > T substitution at nt 2648 resulting in a D816V substitution. This stabilises the kinase loop in its active configuration and hence induces activation of STAT3 and STAT1 and up-regulates downstream targets of STAT3 such as Bcl-xL and c-myc [5]. The mast cell line HMC-1 contains the D816V mutation resulting in ligand-independent activation and cellular transformation [3]. TK mutations are frequent in cases with mast cell expansion and the incidence of KIT mutation is higher in patients with systemic mastocytosis compared to those with cutaneous mastocytosis.

The expanding variety of identified KIT mutations is of importance with the advent of novel tyrosine kinase inhibitors. Imatinib is a tyrosine kinase inhibitor that targets Bcr-Abl, PDGFR-α and -β and Kit. Imatinib inhibits wild type Kit and JM mutants but has no effect on D816V [6] due to conformational changes of the drug's binding site. Imatinib has been used in 12 cases of mast cell disease [7] resulting in 5 complete remissions. Notably the failures were in the two patients with the D816V mutation. A proportion of cases of mastocytosis with eosinophilia are now known to have the FIP1L1-PDGFRA fusion that is imatinib sensitive [8]. Imatinib sensitivity has additionally been recently demonstrated in a case of sporadic mastocytosis bearing a novel germline F522C transmembrane mutation of exon 10 [9]. This is at difference to the previous cases discussed. In this paper we report a case of familial mastocytosis where in vitro and in vivo sensitivity to imatinib was demonstrated. Subsequent genetic study demonstrated the absence of the D816V mutation, consistent with imatinib responsiveness, and further identified a novel K509I mutation in exon 9 in both of the affected patients.

Section snippets

Patients

Two related patients are currently under the care of the Department of Haemato-Oncology at St. Bartholomew's Hospital with aggressive and indolent mastocytosis. The family tree is demonstrated in Fig. 1.

Patient II2 presented to our department in July 1981, aged 26, with hepatosplenomegaly. She had previously been well, apart from an undiagnosed childhood skin complaint, an appendicectomy in 1968 and an axillary vein thrombosis in 1971. Examination of her skin revealed dermatographism and

Imatinib sensitivity testing

Sensitivity testing to imatinib for patient II2 revealed that in liquid culture 1 μM induced no change in viability as compared to that of the control; however, 5 μM resulted in a reduction in viability from day 12 onwards (Fig. 2(a)). For semisolid culture, reduction in colony counts for both CFU-GM and CFU-Baso was noted in the presence of imatinib for the BM-MNC from patient II2 as compared to control MNC cells from healthy individuals (Fig. 2(b)).

Clinical response to imatinib

The results of in vitro work suggested some

Discussion

Familial mastocytosis is a very rare condition with perhaps only 50 families recorded in the literature [15], [16]. It is usually inherited in an autosomal dominant manner, presenting with cutaneous disease in infancy or childhood that may then persist. Variable systemic involvement is noted. Discordance has been noted for monozygotic twins so additional factors may be necessary [17].

In this study we have identified a K509I change in the juxta-membrane domain of KIT present in the affected

Acknowledgements

NC and LYZ were supported by the Leukemia Research Fund and the Wessex Cancer Trust, JVM and BS were supported by the Leukemia Research Fund and MS, JF and TAL were supported by Cancer Research UK.

Contributions. LYZ and NC provided detailed mutational screening of KIT and developed the ARMS assay; BS and JVM provided in vitro imatinib sensitivity data; MS, JF, TAL and JDC contributed to the clinical care of the patients and their initial molecular evaluation; MS wrote paper and coordinated

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