Recent advances in the understanding of mastocytosis: the role of KIT mutations*


  • *

    All authors have contributed equally to this manuscript.

Alberto Orfao, MD, PhD, Centro de Investigación del Cáncer (IBMCC), University of Salamanca/CSIC, Campus Miguel de Unamuno, 37007 Salamanca, Spain. E-mail:


Mastocytosis is a heterogeneous disorder characterised by the expansion and accumulation of mast cells in different organs and tissues. Mast cell physiology is closely dependent on activation of the stem cell factor/Kit signalling pathways and accumulating evidences confirm the physiopathological key role of activating KIT mutations (typically D816V) in mastocytosis and their relationship with the clinical manifestations of the disease. This paper reviews the most recent advances in the understanding of the molecular mechanisms associated with KIT mutations in mastocytosis, including recent data about the use of new therapies targeting the Kit molecule and its associated downstream signalling pathways.

Mastocytosis is a relatively heterogeneous disorder characterised by the expansion and accumulation of mature-appearing mast cells (MC) in different organs and tissues such as the skin, gastrointestinal tract, liver, spleen, bone marrow (BM) and other lymphoid tissues. The disease was first described as a rare form of urticaria (Nettleship & Tay, 1869), prior to the description of MC by Paul Ehrlich in 1879 (Ehrlich, 1879). The link between MC and urticaria pigmentosa (UP) was quickly made (Unna, 1887). Since then, other forms of mastocytosis have been described, such as mast cell leukaemia (MCL), mastocytoma and systemic mastocytosis (SM), among others (Valent, 2004). Knowledge of the heterogeneous behaviour of the disease was further expanded by the demonstration of both childhood and adult forms of mastocytosis. Together, these observations called attention to the need for a classification of mastocytosis and led to a first proposal by Lennert and Parwaresch (1979). The Kiel classification (Lennert & Parwaresch, 1979) was followed by other attempts that tried to group mastocytosis into well-defined clinico-biological entities and, in 1991, a first consensus classification of mastocytosis was proposed (Metcalfe, 1991). Since then, important biological markers of the disease have been identified. Most relevant were the associations described between mastocytosis and increased serum tryptase levels (Schwartz et al, 1987), the presence of the D816V-activating KIT mutation (Furitsu et al, 1993; Longley et al, 1995; Nagata et al, 1995) and an aberrant CD25+ and CD2+ immunophenotype of BM MC (Escribano et al, 1998a).

The identification of these new biological markers has facilitated a better understanding of the molecular mechanisms of mastocytosis, contributed to improve the diagnosis and classification of the disease and promote the search for new molecular-targeted therapies. In line with this, in 2001 the World Health Organisation (WHO) proposed new criteria for the diagnosis and classification of mastocytosis (Valent et al, 2001).The WHO classification proposes a combination of several major and minor criteria for the diagnosis of SM. Accordingly, either one major (presence of dense infiltrates of >15 MC in the BM or in other extracutaneous organs detected by immunohistochemical analysis of tryptase expressing cells) and one minor (abnormal MC morphology, KIT mutation at codon 816, an aberrant CD25+ and/or CD2+ MC immunophenotype and/or serum total tryptase of >20 ng/ml) or at least three minor criteria, are required for the diagnosis of SM. In turn, in the new WHO classification, mastocytosis was grouped into seven different subtypes: cutaneous mastocytosis (CM), indolent SM (ISM), aggressive SM (ASM), SM associated with a clonal haematopoietic non-MC disorder (SM-AHNMD), MCL, MC sarcoma (MCS) and extracutaneous mastocytoma (ECM); within some of these subgroups, provisional variants were further described, e.g. isolated BM mastocytosis (BMM) and smouldering SM (SSM) are new provisional subvariants of ISM (Valent et al, 2001) (Table I). More recently, an International Working Conference was held in Vienna where a group of experts on mastocytosis proposed new standards for clinical evaluations and diagnostic assays (Valent et al, 2007a). In addition, this International Working Conference discussed the differential diagnosis of new poorly defined subgroups of patients with increased and/or altered MC. Among others, the differential diagnosis between well-differentiated SM (WDSM) and reactive MC hyperplasia, as well as between cases with a monoclonal MC population with undefined significance/monoclonal MC activation syndrome (MMAS) and reactive MC hyperplasia, were considered (Table I) (Valent et al, 2007a).

Table I.   Classification of mastocytosis: summarised description of the most relevant clinical and biological features of the different types of mastocytosis (Valent et al, 2001, 2007a).
TypeSkin lesionsExtracutaneous MC lesionsBM MC burdenAbnormal BM MC morphology (MC subtype)BM MC clusters (>15 MC)D816V KIT mutation CD25+/CD2+/− MC immunophenotypeSerum tryptase (>20 ng/ml)OrganomegaliesImpaired organ functionEosinophilia
  1. BM, bone marrow; MC, mast cells; CM, cutaneous mastocytosis; MCL, mast cell leukemia; ISM, indolent systemic mastocytosis; BMM, isolated BM mastocytosis; SSM, smouldering SM; ASM, aggressive systemic mastocytosis; SM-AHNMD, systemic mastocytosis associated with a clonal non-MC lineage hematological disease; WDSM, well-differentiated systemic mastocytosis; MMUS, monoclonal MC population with undefined significance; MMAS, monoclonal MC-activation syndrome; MCS, MC sarcoma; ECM, extracutaneous mastocytoma.

  2. Atypical MC I: Spindle shaped MC, MC with oval nucleus with or without an excentric position, or MC with hypogranulated cytoplasm and focal accumulation of granules but without signs of degranulation. Atypical MC II: MC with bi- or polylobulated nuclei and hypogranulated cytoplasm without signs of degranulation. Cytopathological score: High Grade: >20% of MC are ‘metachromatic blasts’ plus atypical MC II. Low Grade: <10% of MC are ‘metachromatic blasts’ plus atypical MC II. Data from Valent et al (2001, 2007a). Score: +, detected in most cases; −/+, detected in a subset of cases; −, not detected or detected rarely.

  3. *Subtypes of SM-AHNMD should be classified according to the type of AHNMD and of SM following the WHO/FAB criteria. †An atypical KIT mutation other than D816V is frequently detected.

MCL+High (>20%)+ (High grade)+−/+++−/++−/+
ISM++Low+ (Atypical MC I)++++−/+
BMM+Low+ (Atypical MC I)++++
SSM−/++Low/intermediate+ (Atypical MC I)+++++−/+
ASM−/++Intermediate/high+ (Atypical MC I/II)++++++
SM–AHNMD*++Low/high+ (Atypical MC I/II)+++−/+−/+−/+−/+
MMUS/MMAS+Low+ (Atypical MC I)−/+†−/+−/+
MCS+Low+ (High grade)ND−/+−/+
ECM+Low+ (Low grade)ND−/+−/+

On the other hand, the identification of the presence of the D816V KIT mutation, together with an extensive characterisation of the immunophenotype of clonal MC from patients with mastocytosis, represented a major step forward in the understanding of the molecular mechanisms of the disease and have accelerated the search for new therapies based on the use of Kit-specific tyrosine kinase (TK) inhibitors and other molecule-targeted agents, for those cases requiring cytoreductive therapy (Valent et al, 2005).

The D816V KIT mutation was first described by Furitsu et al (1993) in the HMC-1 human MC line (Butterfield et al, 1988). Later, it was shown that the D816V KIT mutation was also present in SM patients (Nagata et al, 1995), where it was repeatedly observed (Longley et al, 1995). However, the exact frequency of the D816V KIT mutation has remained a matter of debate, particularly for ISM patients, where it has been reported to range from 31% (Pardanani et al, 2003a) to virtually all patients (Fritsche-Polanz et al, 2001). Recently, our group confirmed the presence of the D816V KIT mutation in the great majority of adult SM patients (102/113 cases; 93%) in fluorescence-activated cell sorted (FACS)-purified populations of immunophenotypically aberrant MC; interestingly, in around one quarter (3/11 cases; 27%) of those few cases lacking the D816V KIT mutation, other mutations in the tyrosine-kinase domain 2 (TK2) of KIT, were detected (Garcia-Montero et al, 2006). Although these results require further confirmation by other groups in large series of patients, they support previous findings suggesting that the D816V KIT mutation could represent a hallmark of the disease in adult patients.

The present paper reviews the most recent advances in the understanding of SM, focussing on the role of KIT mutations to dissect the pathogenetic mechanisms of the disease. Accordingly, the stem cell factor (SCF)/Kit signalling pathways and the impact of KIT mutations on their behaviour, are reviewed; then, the physiopathological role of the mutations of KIT in SM and their relationship with the clinical manifestations of the disease are discussed; the final section reviews the recent data regarding the use of new therapies targeting the Kit molecule and other associated signalling pathways.

The stem cell factor/kit signalling pathway

The structure of the Kit molecule

Human KIT is a proto-oncogene that encodes for a transmembrane receptor (Kit) with intrinsic TK activity (Yarden et al, 1987). Expression of the Kit protein has been reported in both normal cells [e.g. haematopoietic progenitors (Simmons et al, 1994), normal mature MC (Metcalfe, 2005), Cajal cells (Huizinga et al, 1995), melanocytes (Halaban et al, 1993), and germ cells (Strohmeyer et al, 1995)] and neoplastic cells from gastrointestinal stromal tumours (GIST) (Andersson et al, 2002), seminomas (Strohmeyer et al, 1995), small cell lung cancer (Sekido et al, 1991), colon cancer (Toyota et al, 1993), neuroblastoma (Beck et al, 1995), breast cancer (Hines et al, 1995), acute myeloid leukaemia (AML) (Bene et al, 1998), T-cell acute lymphoblastic leukaemia (ALL) (Bene et al, 1998), multiple myeloma (Escribano et al, 1998b), myelodysplastic syndromes (MDS) (Orfao et al, 2004), myeloproliferative disorders (MPD) (Nakata et al, 1995), B-cell non-Hodgkin lymphoma (Bravo et al, 2000) and B-cell precursor ALL (Bene et al, 1998). In normal cells, Kit has been shown to play a major role in haematopoiesis (in the differentiation of erythroid, lymphoid, megakaryocytic and myeloid precursors) (Nocka et al, 1989), gametogenesis (Kissel et al, 2000), MC development and function (Metcalfe, 2005; Valent et al, 2005), melanogenesis (Nocka et al, 1989; Halaban et al, 1993) and gastrointestinal function (Miettinen & Lasota, 2005).

In humans, the KIT gene is located at chromosome 4q12, in the pericentromeric region of the long-arm of chromosome 4 (Yarden et al, 1987), adjacent to the highly homologous PDGFRA gene (Spritz et al, 1994). Genomic DNA of human KIT spans approximately 89 kb and contains 21 exons which are transcribed/translated into a type III TK receptor with a molecular mass of 145 kD and 976 amino acids in length (Giebel et al, 1992). The five immunoglobulin-like loops of the extracellular domain of Kit are encoded by exons 1–9 (amino acid residues: 23–520), the transmembrane domain by exon 10 (amino acids: 521–543), the juxtamembrane autoinhibitory domain by exon 11 (amino acids: 544–581) and the TK domain is encoded by exons 13–21 (amino acids: 582–937). The first three immunoglobulin (Ig)-like loops of the extracellular domain form the binding site for SCF or Kit ligand (Lev et al, 1993; Lemmon et al, 1997; Longley et al, 2001), while the fourth and fifth loops play a role in stabilising the SCF-induced Kit dimer (Blechman et al, 1995; Zhang et al, 2000); in addition, it has been proposed that the fifth Ig-like Kit domain is also required for the proteolytic cleavage from the cell surface (Broudy et al, 2001). The autoinhibitory juxtamembrane domain contains alpha-helical elements whose proper configuration is essential for the downregulation of tyrosine phosporylation (Lev et al, 1993; Hubbard, 2004; Mol et al, 2004). In turn, the kinase portion of Kit is composed of two domains which are separated by a kinase insert: (1) the TK1 domain is constituted by the small N-terminal lobe that expands from amino acids 582–684 and contains the ATP binding site, and; (2) the TK2 domain is formed by the large C-terminal lobe containing the phosphotransferase site and the activation loop (amino acids: 810–839) (Fig 1).

Figure 1.

 Schematic representation of the structure of Kit and its binding to other proteins and adaptors through Kit phosphorylated tyrosine residues. A summary of the downstream pathways activated by these interactions and their major biological effects, is also provided in the two columns in the right. Plain lines (—–) represent the hypothetical docking site; arrows (→) indicate activation, while crossed lines (⊥) indicate inhibitory effects. The vertical arrows indicate increased (↑) and decreased (↓) effects on different cell functions. References: a (Wollberg et al, 2003); b (Tauchi et al, 1994); c (Kozlowski et al, 1998); d (Linnekin et al, 1997); e (Timokhina et al, 1998); f (Price et al, 1997); g (Roskoski, 2005b); h (Lennartsson et al, 1999); i (Weiler et al, 1996); j (Thommes et al, 1999); k (Shivakrupa & Linnekin, 2005); l (Lev et al, 1992); m (Blume-Jensen et al, 1998); n (Tanaka et al, 2005); o (Gommerman et al, 2000); p (Trieselmann et al, 2003); q (Lennartsson et al, 2003).

Kit signalling pathways

At present it is well known that activation of the SCF/Kit signalling pathway is associated with multiple biological effects depending on the activated cell. Among others, these effects include cell proliferation, maturation/differentiation, suppression of apoptosis, degranulation and changes in the adhesion properties and motility of the activated cells (Blume-Jensen et al, 1991). To date, numerous interactions of Kit with different adaptor proteins have been described. Such interactions lead to Kit-mediated activation of several signal transduction pathways in common to many other growth factor receptors, such as those involving the phosphatydylinositol triphosphate (PI3)-kinase, protein kinase C (PKC), Ras/mitogen-activated protein kinase (MAPK), and Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathways, that are responsible for the ultimate effects of binding of SCF to Kit.

Upon non-covalent binding of a dimer of SCF to the second and third immunoglobulin loops of the extracellular domain, Kit undergoes dimerisation (Blume-Jensen et al, 1991; Zhang et al, 2000), followed by transphosphorylation of two tyrosine residues in the autoinhibitory juxtamembrane segment (Y568 and Y570) (Hubbard, 2004; Mol et al, 2004) (Fig 1). These molecular changes lead to a conformational modification of the activation loop from a compact, inactive structure to an extended and active conformation. Subsequent transphosphorylation of Y823 in the activation loop stabilises the enzyme in its most active form (Mol et al, 2004) (Fig 1). In turn, the activated intrinsic TK activity of Kit leads to auto-phosphorylation of other tyrosine residues that serve as docking sites for signal transduction molecules containing Src homology 2 (SH2) domains and other phosphotyrosine-binding domains.

The presence of multiple phosphorylation sites in the Kit sequence reflects the existence of several regulatory and catalytic domains. Based on the localisation of the phospho (p)-tyrosine binding sites, three preferential regulatory sites of Kit have been identified with activating and/or inhibitory effects on one or more downstream signalling transduction pathways: (1) the juxtamembrane domain; (2) the tyrosine kinase insert; and (3) the activation loop in the TK2 domain (Fig 1). Despite all the information accumulated in recent years about the different Kit-associated signalling pathways, it should be noted that most reported results derive from the study of cell line models and that some discrepancies exist regarding the exact interactions associated with the activity of the phosphorylated Kit molecule, which could be related to the specific cell type analysed (Boissan et al, 2000). Figure 1 summarises the interactions that have been established between different domains of Kit and other regulatory adaptor proteins, and the downstream activated/inhibited cell signalling pathways regulated by these proteins [for a more detailed description of these interactions see (Ronnstrand, 2004) and (Roskoski, 2005a)].

KIT activating mutations

Occurrence of different point mutations and in frame deletions/insertions of KIT have been shown to cause alterations of the downstream Kit signalling pathways that convert the KIT proto-oncogene into an active, dysregulated (ligand-independent) oncoprotein capable of inducing neoplastic transformation of normal Kit expressing cells (Kitamura et al, 1995). Since the first description of the activating KIT mutation in the HMC-1 human MC cell line (Furitsu et al, 1993), multiple KIT mutations have been reported in patients with mastocytosis; many of these mutations are associated with constitutional Kit phosphorylation and downstream activation, independent of SCF binding (Table II; Fig 2). In order to better understand the impact of KIT mutations, a few years ago Longley et al (2001) proposed that the activating KIT mutations be classified into two major groups based on their topological localisation: the ‘regulatory type’ and the ‘enzymatic pocket type’ mutations. The former KIT mutations typically affect regulation of the kinase activity of the Kit molecule by disrupting the autoinhibitory α-helix (Ma et al, 1999), affecting the binding of signal transducing or regulatory molecules to Kit and/or inducing ligand-independent dimerisation and activation; most frequently these ‘regulatory type’ mutations occur at the juxtamembrane domain of Kit. In turn, the ‘enzymatic pocket type’ mutations directly affect the enzymatic site at the TK2 activation loop and induce activation of Kit in the absence of dimerisation of the receptor.

Table II. KIT mutations that have been reported in patients with mastocytosis in comparison with other non-mast cell neoplasias also carrying KIT mutations.
DiseaseDomainExonMutationConsequence of mutationFrequency (%)CommentsReferences
  1. The following KIT mutations have also been found in piebaldism (Murakami et al, 2004): C136R, A178T, M318G, Q347X, M541L, W557X, E583K, F584L, F584C, G601R, V620A, A621T, H650P, G664R, C788R, R791G, R796G, G812V, W835R, T847P, E861A, P869S, Y870C.

  2. CM, cutaneous mastocytosis; SM, systemic mastocytosis; AML, acute myeloblastic leukaemia; ISM, indolent systemic mastocytosis; UP, urticaria pigmentosa; CML, chronic myeloid leukaemia; MF, myelofibrosis; MPD, myeloproliferative disorder; ASM, aggressive systemic mastocytosis; WDSM, well-differentiated systemic mastocytosis; TK1, Kit tyrosine kinase domain 1; MCL, mast cell leukaemia; GIST, gastrointestinal stromal tumour; NK, natural-killer.

MastocytosisExtracellular8del D419Unknown<5Familial SMHartmann et al (2005)
9K509IUnknown<5Familial SMZhang et al (2006)
Transmembrane10F522CActivating<5SMAkin et al (2004)
10A533DActivating<5Familial CMTang et al (2004)
Juxtamembrane11V559IActivating<5ASMNakagomi and Hirota (2007)
11V560GActivating<5ISM, MCLFuritsu et al (1993); Buttner et al (1998)
Activation loop17R815KUnknown<5Paediatric UPSotlar et al (2003)
17D816VActivating>90Adult SMGarcia-Montero et al (2006)
17D816YActivating<5SMLongley et al (1999)
17D816HUnknown<5SM-AMLPullarkat et al (2003)
17D816FActivating<5SMLongley et al (1999)
17I817VUnknown<5WDSMGarcia-Montero et al (2006)
17insV815_I816Unknown<5SMGarcia-Montero et al (2006)
17D820GUnknown<5ASMPignon et al (1997)
17E839KInactivating<5UPLongley et al (1999)
GISTExtracellular8del D419Unknown<5FamilialGISTHartmann et al (2005)
9insA502_Y503Unknown<5Lasota et al (2000)
Juxtamembrane11del in region K550_E561Activating25–50Hirota et al (1998); Nakahara et al (1998); Taniguchi et al (1999);  Andersson et al (2002)
11V559DActivating16Hirota et al (1998)
11V560DActivating40Andersson et al (2002)
11D579delActivating Nakahara et al (1998)
TK113K642EActivating<5Isozaki et al (2000); Lasota et al (2000)
Kinase insert14del K704_N705Unknown<10Andersson et al (2002)
15del S715Unknown>50Andersson et al (2002)
MPDExtracellular2D52NUnknown10Nakata et al (1995)
AMLExtracellular8D419delUnknown30Inv(16)Gari et al (1999)
8del+ins 416–419Unknown9Inv(16)Beghini et al (2004)
Transmembrane10V530IUnknown14Inv(16)Gari et al (1999)
Activation loop17D816VActivating20t(8;21) and inv(16)Beghini et al (2004)
17D816YActivating10 Beghini et al (2004)
17D816HActivating<5 Beghini et al (2004)
17N822KActivating Kasumi-1 cells. t(8;21)Beghini et al (2002)
Nasal and nasal-type NK/T-cell lymphomaJuxtamembrane11V559IUnknown<5 Hongyo et al (2000)
11E561KUnknown8 Hongyo et al (2000)
Activation loop17D816NUnknown<5 Hongyo et al (2000)
17V825AUnknown30 Hongyo et al (2000)
SeminomasJuxtamembrane11W557CUnknown6Intracranial germinomaSakuma et al (2004)
11W557RActivating<5 Sakuma et al (2003)
11L576PActivating<5Willmore-Payne et al (2006)
Activation loop17D816VActivating3–10Sakuma et al (2003, 2004); Kemmer et al (2004); Willmore-Payne et al (2006)
 D816HActivating≤5Sakuma et al (2003); Kemmer et al (2004)
 D816YActivating<5Willmore-Payne et al (2006)
17D816EActivating<5Willmore-Payne et al (2006)
17D820HUnknown<5Willmore-Payne et al (2006)
17D820VUnknown6Intracranial germinomaSakuma et al (2004)
17N822YUnknown6Intracranial germinomaSakuma et al (2004)
17N822KActivating<5 Kemmer et al (2004)
17Y823CActivating<5 Kemmer et al (2004)
17Y823DActivating<5 Kemmer et al (2004)
17Y823NActivating<5 Willmore-Payne et al (2006)
MelanomaJuxtamembrane11L576PActivating<5 Willmore-Payne et al (2005)
Figure 2.

 Schematic representation of the structure of Kit, illustrating the known function of its domains and the localisation of the more frequently observed mutations in the KIT sequence, in association to a specific disease or group of diseases represented by round-circled symbols (A, acute myeloid leukaemia; G, gastrointestinal stromal tumor; L, nasal and nasal-type NK/T-cell lymphoma; M, mastocytosis; Me, melanoma; P, myeloproliferative disorder; S, seminoma/germinoma). Asterisks (*) indicate point mutation sites and underlined amino acids represent either in frame deletion or insertion sites.

KIT mutations cluster in relatively small regions – most frequently at exon 11 and 17 – leading to aminoacid changes at the juxtamembrane and TK2 domain of Kit, respectively. Less frequently, KIT mutations are detected at exons 2, 8, and 9 or at exons 13 and 14, coding for the extracellular and TK1 Kit domains (Table II; Fig 2). Although single point KIT mutations or in frame deletions/insertions are found in most patients (Taniguchi et al, 1999; Beghini et al, 2004; Garcia-Montero et al, 2006), more than one mutation has been also reported in a few cases (Furitsu et al, 1993; Buttner et al, 1998; Andersson et al, 2002; Willmore-Payne et al, 2006). It is currently well established that, apart from mastocytosis, KIT mutations can also be frequently observed in other neoplastic disorders. Interestingly, a careful analysis of the KIT mutations shows a clear association between the type of KIT mutation and specific disease groups (Table II; Fig 2). Accordingly, in the great majority (>90%) of adult cases with SM, mutations in the activation loop of KIT (most frequently D816V) are detected in MC in association with an aberrant CD25+ phenotype (Garcia-Montero et al, 2006), except among those few patients with WDSM whose BM MC are typically negative for both the D816V KIT mutation and cell surface CD25/CD2 (Garcia-Montero et al, 2006). Interestingly, D816V-negative SM patients frequently carry other KIT mutations in the activation loop involving codons 815, 816, 817, 820 and 839 (Pignon et al, 1997; Longley et al, 1999; Sotlar et al, 2003; Garcia-Montero et al, 2006). Despite this, a few KIT mutations at exon 11 have been also reported in individual SM patients in association with the typical D816V KIT mutation (Furitsu et al, 1993; Buttner et al, 1998). In contrast, the D816V KIT mutation has only been sporadically found among AML with Inv(16) and AML with t(8,21) (Beghini et al, 2004) – where coexistence of two independent diseases (AML and SM) appears to be relatively frequent (Pullarkat et al, 2003; Escribano et al, 2004) – and in rare cases of seminomas (Sakuma et al, 2003; Kemmer et al, 2004) and germinomas (Sakuma et al, 2004) but not in GIST, which are typically D816V-negative and commonly show activating mutations at the regulatory juxtamembrane region of Kit (exon 11) (Hirota et al, 1998; Nakahara et al, 1998; Taniguchi et al, 1999; Andersson et al, 2002) (Table II; Fig 2). Of note, mutation of KIT in mastocytosis has also been associated with decreased expression of Kit (CD117) on the cell surface, which could probably be due to an increased cleavage and release of the mutated Kit molecule into the extracellular compartment, leading to increased soluble levels of CD117.

At present, the exact mechanisms leading to the association between specific KIT mutations and unique groups of diseases remain unknown. Overall, such association could be related either to upregulation of different signal transduction pathways by distinct activating KIT mutations and/or to the existence of cell-type specific Kit-associated downstream signalling pathways and transcription factors. In line with the first hypothesis, recent results show the occurrence of mastocytosis-associated germ line KIT mutations (F522C, K509I and D816V) in patients that developed severe forms of mastocytosis (Akin et al, 2004; Garcia-Montero et al, 2006; Zhang et al, 2006). Nevertheless, the observation that most familial forms of mastocytosis show an indolent clinical course (Longley et al, 1999; Tang et al, 2004) would indicate that the type of mutation, rather than its pattern of expression (germline versus somatic KIT mutations), could be responsible for the clinical behaviour of the disease. In turn, mutation of the PDGFRA gene has been found in the eosinophilic compartment of some of those few cases diagnosed with either SM associated with chronic eosinophilic leukaemia (SM-CEL, a variant of SM-AHNMD) or GIST, who do not have KIT mutations (Heinrich et al, 2003; Pardanani et al, 2004), pointing out the involvement of common downstream Kit/PDGFRA activating pathways in the development of different diseases and the potential influence of the cell type-specific activated pathways and transcription factors on determining their nature. Of note, among SM-CEL cases, FIP1L1/PDGFRA mutation is a molecular marker of CEL, but not for the MC component (Valent et al, 2007b). In line with this later hypothesis, Kissel et al (2000) showed in a knock-in mouse model that receptor-mediated PI3-kinase signalling is critical for spermatogenesis and oogenesis, but not for haematopoiesis, melanogenesis and primordial germ cell development.

In turn, the potential association of specific KIT mutations with specific subtypes of mastocytosis remains to be elucidated. Despite the fact that the D816V KIT mutation is present in >90% of SM, with the exception of rare cases of WDSM and MCL (Garcia-Montero et al, 2006), the exact frequency of this mutation in patients with CM remains unknown. Accordingly, while a significant proportion of CM cases with a childhood onset do not show the D816V KIT mutation (Verzijl et al, 2007) and KIT mutations at codons 509, 533, 815, 816 and 839 have been reported in adult CM patients (Longley et al, 1999; Sotlar et al, 2003; Tang et al, 2004; Zhang et al, 2006), the greatest frequency of KIT mutation is still found at codon 816 (Sotlar et al, 2003; Yanagihori et al, 2005).

Altogether these observations support the notion that genetic examination of the KIT mutational status of purified MC from BM or other extracutaneous organs (peripheral blood, spleen, liver, lymph nodes and pleural fluid), in addition to lesional skin, is of great help for the differential diagnosis of cutaneous versus SM.

Impact of KIT mutation/activation in SM

In normal mature MC, activation of Kit signalling through SCF leads to an increased cell proliferation and survival, changes MC migration and adhesion, MC degranulation and mediator release. In SM patients such effects are typically enhanced by the occurrence of activating KIT mutations.

Increased mast cell proliferation and survival  One of the most frequent and evident clinical manifestations of mastocytosis is the increased expansion of the MC compartment and the accumulation of neoplastic MC in different organs and tissues. This could be related to both an increase in MC proliferation and MC survival due to constitutive activation of Kit. In line with this, studies performed in murine BM-derived cultured MC have shown that activation of the PI3-kinase, p21Ras and MAPK pathways is essential for SCF-induced MC proliferation, the former two pathways (but not MAPK) being dependent on the presence of Kit p-Y719 (corresponding to human Y721) (Serve et al, 1995). Furthermore, the presence of p-Y821 (corresponding to human Y823) (Serve et al, 1995), which stabilises Kit in its most active form (Mol et al, 2004), is essential for Kit-mediated mitogenesis and survival, but it is independent of PI3-kinase, p21Ras and MAPK activation (Serve et al, 1995). In turn, in vitro studies performed with murine MC also show that cell cycle progression in SCF-induced MC is mediated by expression of cyclin D3 and pRb phosphorylation (Itakura et al, 2001). More recently, Tanaka et al (2005) have demonstrated constitutive activation and translocation to the nucleus of NFκB, a PI3-kinase downstream protein, in the SCF-independent HMC-1V560G,D816V cell line model. These observations would support the role of the PI3-kinase/NFκB pathway in neoplastic MC transformation, which could occur because of an altered cell cycle regulation due to an abnormally higher expression of cyclin D3 and pRb phosphorylation. Moreover, the same authors showed that SCF-independent HMC-1V560G,D816V cell proliferation is also mediated by the PKC pathway, while it was apparently independent of MAPK activation (Tanaka et al, 2005).

It is known that in vitro-derived (Mekori et al, 2001) or ex vivo-isolated (Akin et al, 2003) and cultured normal human MC undergo rapid apoptosis, if SCF is omitted from the culture medium. This is due to the fact that MC survival is also maintained by activation of SCF/Kit-associated signalling pathways. Accordingly, PI3-kinase plays a key role in promoting cell survival through activation of the Akt serine/threonine kinase, which in turn leads to phosphorylation and inhibition of Bad, a pro-apoptotic protein that promotes MC death (Blume-Jensen et al, 1998). Regulation of the anti-apoptotic activity of Kit by the PI3-kinase/Akt pathway is controlled by phosphorylation of Y721 at its kinase insert domain (Blume-Jensen et al, 1998; Shivakrupa & Linnekin, 2005); thus, conformational changes caused by mutations at the Kit activation loop (e.g. D816V) that cause activation of the PI3-kinase pathway, may also contribute to MC transformation through an enhanced cell survival. In addition, the mammalian target of rapamycin (mTOR), a downstream serine/threonine kinase target of Akt in the PI-3K pathway, is also constitutively activated in HMC-1 cells carrying the D816V KIT mutation (Gabillot-Carre et al, 2006). Of note, D816V MC isolated from SM patients, but not normal human MC, are sensitive to rapamycin (Gabillot-Carre et al, 2006), highlighting the role of the PI3K/Akt/mTOR pathway in the abnormal cell growth, proliferation and survival of neoplastic MC. Furthermore, Stat-5, a Jak-2 downstream regulator of MC proliferation and survival (Shelburne et al 2003, Ikeda et al 2005), is also constitutively activated in multiple cell lines and MC from patients carrying different activating KIT mutations (Growney et al, 2005; Pan et al, 2007).

Despite the overall increased numbers of MC found in the skin, BM and other tissues in patients with SM, a great variation in the overall MC burden exists among individual patients, even in cases carrying the same KIT mutation. In line with this, KIT mutations have been identified not only in MC from SM patients but they are also frequently detected in other BM haematopoietic cell compartments, particularly among CD34+ haematopoietic progenitor and precursor cells (HPC), eosinophils and, to a lower extent, within the CD34 neutrophil and monocytic precursors (Garcia-Montero et al, 2006). Interestingly, the frequency of cases showing involvement of KIT mutation in BM cell compartments other than MC is significantly lower among patients included within those types of mastocytosis associated with a good prognosis, in comparison with cases of ASM, MCL and SM-AHNMD. Altogether, these results could suggest that SM patients showing multilineage involvement of BM haematopoietic cells could represent more advanced stages of the disease. However, the relatively stable course of the disease in most SM patients and the observation that the same KIT mutation (e.g. D816V) can associate with indolent (good-prognosis) and malignant tumours (Garcia-Montero et al, 2006) highlight the potential role of other genetic and/or epigenetic factors in determining the progression/outcome of the disease; further studies are required in this regard.

Altered mast cell migration and adhesion  The pattern of MC involvement in SM typically reflects the tissue distribution of normal MC (Valent et al, 2005). However, significant differences can be observed between distinct forms of the disease. Accordingly, while skin involvement represents a hallmark of CM and ISM, ASM and MCL patients frequently show involvement of BM, spleen, liver, lymph nodes and/or peripheral blood in the absence of cutaneous lesions (Valent et al, 2001). Similarly, some ISM patients also show recurrent anaphylaxia episodes together with BM infiltration by MC, in the absence of cutaneous lesions (Akin & Metcalfe, 2003). Altogether, these findings point out the occurrence of variable patterns of involvement of different tissues in mastocytosis.

It is currently well-established that MC migration to peripheral tissues is also mediated by SCF/Kit signalling. SCF alone or in combination with interleukin-3, is a potent attractant for MC (Meininger et al, 1992). In addition, locally produced SCF may also exert an inhibitory effect on the chemotactic migration of MC induced by IgE-specific antigens, contributing to the accumulation of MC at the peripheral sites in allergic and non-allergic conditions (Sawada et al, 2005). SCF-independent activating D816V KIT mutation may induce alterations in this finely regulated mechanism, enhancing chemotaxis of CD117+ (Kit+) cells (Taylor et al, 2001) and inducing an abnormal accumulation of MC in different tissues (e.g. BM and/or peripheral blood of ASM and MCL patients).

Activation of Kit has been also shown to mediate MC adhesion to the extracellular matrix via fibronectin, through the activation of fibronectin receptors on MC (Dastych & Metcalfe, 1994); this effect is mediated by activation of PI3-kinase (Serve et al, 1995). The key role of PI3-kinase in cell adhesion has been shown to be dependent on p-Y719 (equivalent to human Y721) in the Kit kinase insert domain of normal MC differentiated from murine BM haematopoietic precursor cells, while residual adhesion activity is also partially due to an Src-dependent PI3-kinase activation mechanism (Serve et al, 1995). Surprisingly, Kit-mediated cell adhesion appears to be independent of the presence of mutations at Y821 (equivalent to human Y823) (Serve et al, 1995).

Augmented mast cell degranulation  Over a decade ago, Costa et al (1996) showed that the injection of SCF induced MC degranulation and increased levels of MC tryptase and histamine in normal subjects. However, recent findings indicate that human MC degranulation is driven through binding of IgE/antigen immunocomplexes to the high-affinity IgE receptor (FcɛRI) on the surface of MC (Tkaczyk et al, 2004). SCF acts in synergy with antigens (Tkaczyk et al, 2004) to markedly enhance degranulation and production of cytokines by MC. SCF activates phospholipase C-γ and induces calcium mobilisation, leading to MC degranulation when added together with the antigen (Hundley et al, 2004), which in turn induces an FcɛRI-mediated activation of PKC. This synergy in MC degranulation is mediated by tyrosine phosphorylation of non-T-cell activation linker (NTAL) which acts as a pivotal link between the signalling cascades following Kit activation and cross-linking of FcɛRI (Tkaczyk et al, 2004). Moreover, the Stat-5 molecule, a critical factor in IgE-induced MC activation (Barnstein et al, 2006), is constitutively phosphorylated in cells carrying activating KIT mutations (Pan et al, 2007). Accordingly, constitutive, ligand-independent Kit mutations would favour an enhanced MC response against antigen and/or physical stimuli present in the MC environment, leading to the release of different MC mediators and the associated clinical symptoms (e.g. pruritus, severe anaphylactic episodes and abdominal pain). In fact, increased serum tryptase levels is a minor diagnostic criteria for SM (Valent et al, 2001) and elevated serum tryptase levels are associated with different subtypes of SM (Table I) (Valent et al, 2001; Garcia-Montero et al, 2006). In addition, in SM patients with recurrent anaphylaxia, serum tryptase levels also show a significant increase during the anaphylactic episodes. Although a clear relationship has been found between some allergens and the occurrence of mastocytosis-associated anaphylactic episodes (e.g. wasp venom), in many patients, the stimuli responsible for massive MC degranulation remains to be identified.

Kit-targeted therapy in mastocytosis

In the last decade major advances have been achieved in the field of molecular-targeted therapy, in which drugs are selected on the basis of specific molecular abnormalities causing individual diseases. Among the new drugs developed, the STI571 TK inhibitor (Imatinib mesylate or Gleevec) has been considered as ‘a paradigm of targeted therapies’ (Druker, 2004) representing a novel molecular approach to the treatment of BCR/ABL+, PDGFR- and KIT-mutated malignancies.

Imatinib was first identified as a potent inhibitor of the c-abl protein kinase and it was shown to have similar activity against v-abl and both the p210 and p190 forms of bcr/abl (Druker et al, 1996; Carroll et al, 1997; Beran et al, 1998). Moreover, imatinib was found to inhibit the kinase activity of PDGFR α and β chains (Druker et al, 1996; Carroll et al, 1997).

Imatinib mesylate and Kit

In vitro studies have proven that imatinib inhibits wild type Kit (wtKit) (Zermati et al, 2003) and suppresses proliferation of the HMC-1V560G cell line, while it is ineffective on inhibiting the growth of HMC-1V560G,D816V cells (Akin et al, 2003). Apart from wtKit, Kit molecules carrying mutations in the extracellular, transmembrane and juxtamembrane domains, such as V560G (Akin et al, 2003), F522C (Akin et al, 2004) and K509I (Zhang et al, 2006), remain sensitive to imatinib. In contrast, several experiments have provided compelling evidence regarding the resistance against the growth-inhibitory effects of imatinib on cells carrying the D816V KIT mutation (Ma et al, 2002; Akin et al, 2003). In fact, imatinib did not show preferential ex vivo cytotoxicity against neoplastic BM MC obtained from patients with mastocytosis who carried the D816V KIT mutation (Ma et al, 2002; Akin et al, 2003); in addition, structural changes in the Kit kinase domain induced by the D816V KIT mutation have been identified as responsible for preventing binding of imatinib to Kit (Mol et al, 2004).

Clinical studies using imatinib mesylate in mastocytosis

To date, 31 adult SM cases treated with imatinib mesylate have been reported in the literature (Table III) and mutational studies of KIT were performed in 27 of these patients. Of these, 13 corresponded to good-prognosis categories (10 ISM and 3 SSM), 12 were ASM (two were mastocytosis with a pediatric-onset and either transmembrane or juxtamembrane KIT mutations associated with an aggressive clinical course), one case corresponded to a SM-AHNMD, another to a WDSM, three cases were SM-CEL and the remaining case was SM with FIP1L1/PDGFRA gene rearrangement [data on eosinophilia was not provided by authors (Droogendijk et al, 2006)]. Such a lack of homogeneous criteria in patients’ selection leads to increased difficulty in adequately evaluating the response to therapy. In any case, in line with the results of in vitro analyses, these studies showed significant clinical responses to imatinib in cases lacking the D816V mutation as well as in SM-AHNMC carrying another imatinib-target, such as SM-CEL with FIP1L1/PDGFRA gene rearrangements (Pardanani et al, 2003b; Elliott et al, 2004). However, overall complete response (CR) was obtained in only 4/31 cases (13% of the cases revised), corresponding to one-third of all cases lacking the D816V KIT mutation (Pardanani et al, 2003b,c) and SM-CEL patients carrying the FIP1L1/PDGFRA fusion gene (Elliott et al, 2004). An additional CR was reported in a case of SM associated with chronic myeloid leukaemia (Agis et al, 2005); nevertheless, this patient had been previously treated with hydroxycarbamide and, based on the effectiveness of hydroxycarbamide in SM associated with MPD or MDS (Sheikh et al, 2006), the role of imatinib in inducing CR in this case could not be accurately established (Agis et al, 2005). Finally, sustained response to imatinib has also been obtained in a rare case of WDSM carrying the F522C transmembrane KIT mutation, associated with an aggressive course of the disease (Akin et al, 2004) and in a case of familial mastocytosis carrying the K509I juxtamembrane KIT mutation (Zhang et al, 2006). In the WDSM patient, a dramatic improvement in clinical symptoms, bone pain, and quality of life, together with a decrease in both BM MC infiltration (from 50% to <10%) and serum tryptase levels (from 173 to 20 ng/ml), was noted; at present, she remains alive under imatinib therapy, showing good clinical condition in the absence of an increase in BM MC and serum tryptase levels of 5–17 μg/l [J. Robyn, Laboratory of Allergic Diseases (NIH/NIAID), Bethesda, MD, USA, personal communication, December 2006]. Similar results were obtained with imatinib therapy in the second case. Interestingly, a clear predominance of BM MC showing a ‘round-shape’ morphology – in the absence of CD25 expression in one of them (Akin et al, 2004) – was observed in these two patients, suggesting that a careful examination of both the morphology and immunophenotype of BM MC may provide valuable criteria for the identification of this subtype of mastocytosis, where mutational analysis of KIT could be of great utility for predicting response to imatinib (Akin et al, 2004). In line with this, we have recently reported that most WDSM patients do not carry the D816V KIT mutation (Garcia-Montero et al, 2006).

Table III.   Clinical characteristics and outcome of patients with mastocytosis treated with imatinib.
Case numberAge (years)CategoryMutationPrevious treatment/sConcomitant therapyBest response/Follow-upReference
  1. H, hepatomegaly; S, splenomegaly; HC, hydroxycarbamide; IFN, interferon-α; ND, not done; CHOP, cyclophosphamide, doxorubicin, vincristine and prednisone; CR, complete remission; MR, major response; NR, no response; PR, partial response; PCR, pure partial response; IR, incomplete remission; BM, bone marrow; GPR, good partial response; ISM, indolent systemic mastocytosis; CEL, chronic eosinophilic leukaemia; ASM, aggressive systemic mastocytosis; WDSM, well-differentiated systemic mastocytosis; CE, corticosteroids; 2CdA, cladribine.

  2. *Prednisone treatment at 30 mg/d the first 2 weeks. Response criteria follow the guidelines proposed by Valent et al (2003).

 146ISMNoneNoneNoneMR (CR) 10 monthsPardanani et al (2003b,c)
 231ASMNoneHC, IFNNoneMR (CR) 19 monthsPardanani et al (2003b,c)
 372ASMNoneHCNoneMR (CR) 1 monthPardanani et al (2003b,c)
 461ASMNoneCHOPNoneNR 3 monthsPardanani et al (2003b,c)
 545ASMNoneIFN, Pred
NoneMR (IR)
MR 10·5 months
Pardanani et al (2003b,c)
 670ASMNoneIFNNoneMR (IR) 8 monthsPardanani et al (2003b,c)
 730ISM-CELFIP1L1-PDGFRAHC, IFNNoneMR (CR) 19 monthsElliott et al (2004)
 850SMFIP1L1-PDGFRANone in the previous 6 monthsPrednisone*MR (CR) 3 monthsDroogendijk et al (2006)
FIP1L1-PDGFRANoneNoneResponse of SM not evaluableMerante et al (2006)
1051ISM-CELFIP1L1-PDGFRAHC, CE, IFNNoneMR 24 monthsFlorian et al (2006)
1126Familial ASMK509IIFNNonePR (GPR) 24 monthsZhang et al (2006)
1225WDSMF522CIFNNoneMR (IR) 39 monthsAkin et al (2004); J. Robyn, personal communication (12/10/2006)
1343SM -AHNMD (CML)D816V
HUNoneMR (CR) 6 monthsAgis et al (2005)
1478ASMD816VIFNNoneNR 9 monthsPardanani et al (2003b,c)
1585ASMD816VHUNoneNR 5 monthsPardanani et al (2003b,c)
1633ASMD816VNone in the previous 6 monthsNoneNR 4 monthsMusto et al (2004)
1749SSMD816VNone in the previous 6 monthsPrednisone*MR (IR) 3 monthsDroogendijk et al (2006)
1845ISMD816VNone in the previous 6 monthsPrednisone*NR 3 monthsDroogendijk et al (2006)
1945ISMD816VNone in the previous 6 monthsPrednisone*MR (PCR) 3 monthsDroogendijk et al (2006)
2045ISMD816VNone in the previous 6 monthsPrednisone*MR (PCR) 3 monthsDroogendijk et al (2006)
2158ISMD816VNone in the previous 6 monthsPrednisone*MR (IR) 3 monthsDroogendijk et al (2006)
2245SSMD816VNone in the previous 6 monthsPrednisone*MR (IR) 3 monthsDroogendijk et al (2006)
2345SSMD816VNone in the previous 6 monthsPrednisone*MR (IR) 3 monthsDroogendijk et al (2006)
2473ASMD816VIFNPrednisone*NR 3 monthsDroogendijk et al (2006)
2548ISMD816VNone in the previous 6 monthsPrednisone*MR (IR) 3 monthsDroogendijk et al (2006)
2646ISMD816VNone in the previous 6 monthsPrednisone*MR (PCR) 3 monthsDroogendijk et al (2006)
2743ISMD816VNone in the previous 6 monthsPrednisone*MR (PCR) 3 monthsDroogendijk et al (2006)
2842ASMNDIFNNoneNR 72 monthsHennessy et al (2004)
2980ASMNDIFN, prednisoneNoneNR 72 months, deadHennessy et al (2004)
3059ISMNDNone in the previous 6 monthsPrednisone*PR 3 monthsDroogendijk et al (2006)
3160ISMNDNone in the previous 6 monthsPrednisone*MR (IR) 3 monthsDroogendijk et al (2006)

Recently, Droogendijk et al (2006) reported on the occurrence of different degrees of response to imatinib administered in combination with glucocorticoids in a group of D816V-positive mastocytosis patients; the combined use of these two drugs makes it difficult to evaluate response to imatinib, as glucocorticoids alone may also decrease the neoplastic MC burden, as well as the MC-mediator related symptoms – headache, pruritus, flushing and mainly, abdominal discomfort. Thus, caution should be taken when considering the effectiveness of imatinib in such cases.

These results, together with the good life expectancy and quality of life of patients (see below), indicate that a risk-benefit based therapy should be used in mastocytosis, even when targeted-therapies are considered. As a consequence, in vitro studies on the effectiveness of the drug, as well as on its short- and long-term in vivo toxicity, are required. As mentioned above, the D816V KIT mutation is found in the vast majority of adult patients with sporadic SM (Garcia-Montero et al, 2006) and thus, imatinib therapy will not be appropriate for most of these patients. Furthermore, the life expectancy of 161 patients suffering from pure CM, ISM, BMM and WDSM, after a median follow-up of 152 months (range 6–476 months, with 45 cases having a follow up of >20 years), was similar to that observed among individuals who have not mastocytosis [Spanish Network on Mastocytosis (REMA), unpublished data). This is particularly relevant because of the adverse effects described after imatinib therapy, including cardiotoxicity (Kerkela et al, 2006; Park et al, 2006), as well as the development of different clonal abnormalities in CML patients treated with imatinib (Bumm et al, 2003; Herens et al, 2003; Medina et al, 2003; Meeus et al, 2003; O'Dwyer et al, 2003; Alimena et al, 2004; Gozzetti et al, 2004; Guilbert-Douet et al, 2004) including trysomy 8 (O'Dwyer et al, 2003; Bernardeschi et al, 2004; Terre et al, 2004; Tunca & Guran, 2005) and exceptional cases of MDS and acute leukaemia (Alimena et al, 2004; Kovitz et al, 2006). In this sense, the experience gained in the long-term follow-up of non-haematological malignant diseases treated with imatinib, in which haematopoietic stem cells are not involved, such as GIST, could provide some light on the real risk of developing secondary haematological malignancies in patients treated with imatinib mesylate.

Taking all these findings and considerations together, the current REMA recommendations regarding the use of imatinib therapy in mastocytosis only include (1) those exceptional cases of ASM and MCL who are negative for the D816V KIT mutation; (2) SM patients carrying juxtamembrane KIT mutations (e.g. K509I and F522C) associated with an aggressive course of the disease; and (3) aggressive cases of SM-AHNMD associated with FIP1L1/PDGFRA gene rearrangements (SM-CEL). Of note, in these latter cases, imatinib therapy should be prescribed to bring eosinophil counts and thus the CEL-component under control, whereas the SM component of SM-CEL typically behaves as an indolent disease that does not require imatinib or any other targeted or cytoreductive therapy.

Other tyrosine kinase inhibitors and mastocytosis

On the basis of experimental data, other TK inhibitors, such as PKC412 (Gotlib et al, 2005; Gleixner et al, 2006) and dasatinib (Schittenhelm et al, 2006; Shah et al, 2006), have also been used to treat patients with mastocytosis (Table IV). Accordingly, PKC412 was initially used on a compassionate basis in a case of MCL, resulting in a good partial response associated with marked improvement in the patient performance status, resolution of organ dysfunction and a dramatic decrease in both BM MC infiltration and circulating MC; however, after 3 months of therapy, progression to AML was observed (Gotlib et al, 2005). A phase II study designed to assess PKC412 efficacy and safety in ASM/MCL patients is currently in progress (Gotlib et al, 2006); preliminary results of this study showed partial (but not complete) responses in six of nine ASM cases. In order to improve the efficacy of PKC412, synergistic interactions with AMN107 therapy have been evaluated in vitro showing induction of apoptosis and downregulation of CD2 and CD63 in both HMC-1V560G,D816V cells and in primary neoplastic MC (Gleixner et al, 2006).

Table IV.   Recently developed molecular-targeting drugs capable of inhibiting wild-type and/or mutated Kit and their downstream signal transduction pathways.
DrugTargetTumour/cell lineKIT mutationsReferences
  1. BM, bone marrow; TK, tyrosine kinase; Hsp90, heat shock protein 90; MC, mast cell; GIST-T1, gastrointestinal stromal tumour T1 cell line; HMC-1, human mast cell-derived cell line; MCL, mast cell leukaemia.

  2. *Mouse KIT mutation corresponding to human D816V.

ImatinibKitGIST-T1del (exon 11)Nakatani et al (2005)
ImatinibKitBM MCF522CAkin et al (2004)
ImatinibKitHMC-1V560GMa et al (2002)
17-AAGHsp90Kasumi-1, HMC-1
N822K, D816V, V560GFumo et al (2004); Yu et al (2006)
GeldanamycinHsp90GIST-T1del (exon 11)Nakatani et al (2005)
IMD-0354NF-κBHMC-1D816V, V560GTanaka et al (2005)
PKC412TKHMC-1 and neoplastic MC (MCL)D816VGotlib et al (2005); Gleixner et al (2006)
DasatinibSrc, KitMC and leukemic cell linesD816Schittenhelm et al (2006)
AP23464Kit (Akt, Stat-3)HMC-1D816VCorbin et al (2005)
AMN107Kit, Abl, PDGFRHMC-1V560G, D816VGleixner et al (2006)
SU5416 (Semaxinib)TKErythroleukemic cells (from Spi-1/PU.1 transgenic mouse)D814V mouse*Kosmider et al (2006)
EXEL-0862Kit, Stat-3, Stat-5HMC-1
V560G, D816VPan et al (2007)
D816VGabillot-Carre et al (2006)

In a pilot Phase II trial for SM with dasatinib – primarily an abl/src inhibitor with other TK activities (Verstovsek et al, 2006) – in which response was assessed after a minimum of 3 months (three cycles) of therapy, a total of 24 cases were evaluated for response and toxicity; these included six patients with ASM, four with SM-AHNMD – [two with chronic myelomonocytic leukaemia, one with myelofibrosis (MF), one with an hypereosinophilic syndrome (SM-HES)] and 14 with ISM with uncontrolled symptoms despite optimal supportive care measures. Only two patients (8%) showing a low MC burden achieved CR – (one case each of SM-MF and SM-HES), with a relatively limited overall response rate of 37%.

Other TK inhibitors that have shown cytotoxic activity in vitro in both cell line and mouse models include semaxinib (SU5416) and EXEL-0862; both compounds have been shown to be effective in blocking the activity of Kit carrying the D814V mouse mutation, equivalent to the human D816V KIT mutation (Table IV). Accordingly, Kosmider et al (2006) have shown that semaxinib is capable of inducing growth arrest and apoptosis in murine cells carrying the D814V KIT mutation, by inhibiting Kit autophosphorylation and activation of Akt, Erk1/Erk2 and Stat-3 downstream signalling pathways. In turn, AP2346, a new potent ATP-based inhibitor that targets the activation-loop of Kit mutants, would selectively inhibit proliferation of human D816V-positive cell lines without disrupting normal haematopoietic progenitor-cell growth (Corbin et al, 2005).

Other alternative molecular targeted-therapies in mastocytosis

Proteins of the Kit signalling pathway, other than the Kit receptor itself, have been also evaluated as potential targets for the treatment of patients with SM and D816V KIT mutation. Accordingly, current in vitro observations suggest a therapeutic potential for some compounds that interfere in the Kit signalling pathways. Among these compounds, the novel IMD-0354 NF-κB inhibitor, together with the ansamycin antibiotic derivatives 17-AAG and geldanamycin, which target heat shock protein 90 (Hsp90), appear to be of particular interest. Accordingly, IMD-0354 has been shown to induce a complete suppression of proliferation of HMC-1D816V,V560G cells, but not of cord blood-derived normal human MC, in which NF-κB is not activated (Tanaka et al, 2005). In turn, the use of some Hsp90 inhibitors (e.g. 17-AAG) which may also target tyrosine phosphorylation of Kit (e.g. geldanamycin) has been associated with an apoptotic effect and a decline in Kit protein levels (Yu et al, 2006), as well as with an inhibition of the interaction between Hsp90 and Kit in GIST-T1 cells (Nakatani et al, 2005), respectively. Furthermore, encouraging results have been obtained in the ex vivo treatment of neoplastic BM MC from patients with SM with 17-allylamino-17-demethoxygeldanamycin (Fumo et al, 2004), the levels and activity of both Kit and other downstream signalling molecules (e.g. Akt and Stat-3) being downregulated in HMC-1 cells after 17-AAG treatment (Fumo et al, 2004). These results are in line with recent pharmacological evidences supporting the notion that Hsp90 may contribute to the stabilisation of Kit (Nakatani et al, 2005; Yu et al, 2006) through an inhibitory effect on its proteosomal degradation.

In addition, several humanised monoclonal antibody (hMAb) derivatives are being tested as potential targeted drugs against neoplastic MC. The anti-CD44 hMAb A3D8 decreased proliferation of both MC-derived cell lines and primary neoplastic MC obtained from patients with MCL and SSM (Boehm et al, 2005). Alemtuzumab, an anti-CD52 hMAb antigen, has shown selective reduction in eosinophil counts associated with clinical benefit in HES patients (Sefcick et al, 2004) and has been also tested in MC disorders (Santos et al, 2006). Mylotarg, an anti-CD33 hMAb conjugated to an antitumoral antibiotic (calicheamicin) has been also effective in vitro against MC (Krauth et al, 2007). In turn, LMB-2, an anti-CD25 hMAb Fv fragment fused to truncated Pseudomonas aeruginosa exotoxin A, produced reduction in CD25+ clonal cell numbers from several haematological malignancies (Kreitman & Pastan, 2006), including MC in BM cultures obtained from patients with mastocytosis (Escribano et al, 2006). Despite their in vitro proven efficacy, some of these targeted immunotoxins counteract growth of both normal and neoplastic MC (Krauth et al, 2007), while others produce significant side effects including, infusion syndrome (Krauth et al, 2007), lymphopenia (Klastersky, 2006), hepatotoxicity and vascular leak syndrome (Escribano et al, 2006). Therefore, the exact benefit of most of these hMAb derivative drugs for the treatment of severe MC disorders remains to be determined.

Concluding remarks

Overall, it can be concluded that mutation-associated constitutive activation of Kit in mastocytosis may contribute to a better understanding of the clinical manifestations of the disease. Despite this, a significantly high variability in MC burden and clinical symptoms and signs of the disease can not be explained solely on the basis of the KIT mutation. Other factors, such as the specific type of mutation, the presence of antigen/IgE immunocomplexes in the MC microenvironment, the natural history of the disease in individual patients, MC burden and associated/secondary genetic alterations, could contribute to such heterogeneity and their real relevance deserves further investigation. Independent of these factors, the recent development of new drugs targeting Kit and other proteins involved in its downstream activation pathways, have opened new perspectives in the treatment of SM patients requiring cytoreductive therapy.

Grants and financial support

This work was supported by grants from the Instituto de Salud Carlos III (ISCIII), Fondo de Investigaciones Sanitarias (FIS) of the Ministerio de Sanidad y Consumo, Spain (grants PI050726, PI061377, PI05769, PI06529, REMA G03/007 and RETICS RD06/0020/0035-FEDER) and from the Fundación MMA. ACG-M and LS are recipients of grants from FIS (CP03/00035 and CMO3/0043, respectively).