- Top of page
- Patients and methods
The proto-oncogene C-KIT encodes a tyrosine kinase receptor that is expressed on mast cells and haematopoietic stem cells and can show somatic mutations in patients with mastocytosis. Only scattered information is available about mutations in C-KIT in patients with other myeloid neoplasms. Moreover, the prevalence of mutations in C-KIT in bone marrow specimens of individuals with systemic mastocytosis is largely unknown. Using sequence analysis, we have screened cDNAs of the C-KIT domain encompassing codon 510–626 and codon 763–858 in bone marrow (BM) mononuclear cells (MNCs) of patients with myelodysplastic syndromes (n = 28) and patients with systemic mastocytosis (n = 12) for the presence of mutations. Furthermore, restriction fragment length polymorphism analysis was applied for identification of the C-KIT 2468AT and the C-KIT 1700TG mutation, as well as the C-KIT 1642AC polymorphism. All 11 patients with systemic indolent mastocytosis tested positive for C-KIT 2468AT. In contrast, no mutation was identified in the case of aggressive mastocytosis. Among patients with myelodysplastic syndromes, no patient showed a somatic mutation in C-KIT. The allele frequency for C-KIT 1642AC among the entire patient population was 0·038 and was 0·125 among age- and sex-matched healthy controls. Our data demonstrate that myelodysplastic syndromes without histological or cytological evidence of mastocytosis do not exhibit somatic mutations in exons 10, 11, 12, 16, 17 and 18 of C-KIT. In contrast, BM MNCs of patients with systemic indolent mastocytosis were all positive for C-KIT 2468AT and negative for additional mutations in these exons. The C-KIT 1642AC polymorphism is not associated with myelodysplastic syndrome or systemic mastocytosis.
The human C-KIT proto-oncogene encodes a receptor tyrosine kinase (KIT) that is expressed on mast cells, immature haematopoietic progenitor cells, melanocytes and germ cells. The entire coding region has a length of 2·928 bp with a deduced amino acid sequence of 976 amino acid residues (Yarden et al, 1987). The KIT receptor is a member of the tyrosine kinase receptor family, subtype III (Ullrich & Schlessinger, 1990), which includes receptor tyrosine kinases sharing structural similarities. These include an extracellular ligand binding domain with five immunoglobulin-like repeats, a hydrophobic membrane spanning region and an intracellular cytoplasmic tail that contains a tyrosine kinase catalytic domain (Yarden et al, 1987). Furthermore, a juxtamembrane region exists that separates the transmembrane domain from the cytoplasmic domain and which is conserved among members of the same subclass of tyrosine kinase receptors. Within the KIT receptor, the structural domains are located between the amino acid residues of codon 1 and codon 520 (extracellular region), between codon 521 and codon 543 (transmembrane region), and between codon 544 and codon 976 (cytoplasmic region). The latter contains the tyrosine kinase enzymatic domain (Yarden et al, 1987).
Besides C-KIT 2468AT of the human cell line HMC-1, a corresponding mutation has been identified in the murine mastocytoma cell line P-815 (Tsujimura et al, 1994) and the rat tumour mast cell line RBL-2H3 (Tsujimura et al, 1995). Furthermore, mutations have been identified in canine (Ma et al, 1999) and murine mastocytomas (Tsujimura et al, 1996), suggesting a causal role of somatic C-KIT mutations in proliferation of mast cells.
Because mutations in C-KIT can result in ligand-independent clonal cell growth, the question arose whether or not disorders affecting mast cells and/or pluripotent haematopoietic progenitor cells are associated with C-KIT mutations. Nagata et al (1995) were the first to report on the presence of C-KIT 2468AT in peripheral blood mononuclear cells (MNCs) of a patient with myelofibrosis and in three patients with myelodysplastic syndrome and associated mastocytosis. Several investigators have screened samples of patients with stem cell disorders and/or mastocytosis for somatic mutations in C-KIT using single-strand conformation polymorphism (SSCP) analysis (Nagata et al, 1995, 1997; Nakata et al, 1995; Kimura et al, 1997). However, it has to be noted that SSCP does not always enable mutation detection. In particular, if polymerase chain reaction (PCR) products longer than 200 bp are analysed, the detection sensitivity decreases (Sheffield et al, 1993). Therefore, it is possible that mutations exist in C-KIT transcripts that could not have been identified by SSCP analysis in previous studies.
Apart from somatic mutations associated with proliferation of mast cells, five polymorphisms exist in C-KIT. These include C-KIT 1642AC (Nagata et al, 1996), C-KIT 1659AG (Gari et al, 1999), C-KIT 2415CT (Bowen et al, 1993; Nagata et al, 1995), C-KIT 2607GC (Nagata et al, 1995) and C-KIT 3169GA (Bowen et al, 1993) respectively. One of these polymorphisms (C-KIT 1642AC) results in the substitution of a leucine for a methionine residue at codon 541 (Met541Leu) (Nagata et al, 1996). Because this mutation did not result in disease manifestation in a two-generation family, this polymorphism is assumed not to be associated with haematological disorders (Nagata et al, 1996).
Systemic mastocytosis is a disease characterized by abnormal growth and accumulation of mast cells in the skin, bone marrow and/or visceral organs (Lennert & Parwaresch, 1979; Horny et al, 1985; Travis et al, 1988; Horan & Austen, 1991; Metcalfe, 1991; Valent, 1996). At diagnosis, patients frequently show an associated haematological disorder such as a myelodysplastic syndrome or a myeloproliferative disorder (Parwaresch et al, 1985; Metcalfe, 1991; Valent, 1996). These cases of mastocytosis presenting with other haematological disorders can be associated with a mutated C-KIT transcript (Boissan et al, 2000). Moreover, patients with systemic mastocytosis may develop haematological abnormalities that may resemble the picture of a myeloproliferative or myelodysplastic disorder (Horny et al, 1985; Lawrence et al, 1991).
- Top of page
- Patients and methods
In the present study, BM MNCs of patients with a myelodysplastic syndrome did not show mutations in the C-KIT coding region spanning nucleotides 1549–1898 and nucleotides 2309–2596 that included the exons 10, 11, 12, 16, 17 and 18 respectively. In contrast, all MNC samples obtained from patients with systemic indolent mastocytosis were positive for C-KIT 2468AT. In the single case of aggressive mastocytosis, no mutation was detectable. Furthermore, the allele frequency for the C-KIT 1642AC polymorphism was no higher among patients than among healthy age- and sex-matched control subjects.
Systemic mastocytosis frequently presents with an associated haematological disorder such as a myelodysplastic syndrome and can be related to activating mutations in C-KIT (Boissan et al, 2000). In contrast, only limited information is available about somatic mutations in C-KIT in BM MNCs of patients with myelodysplastic syndromes without concomitant mastocytosis. We have therefore screened cDNA samples of 28 cases of myelodysplastic syndrome for C-KIT mutations and evaluated BM sections for the presence of mast cell infiltrates. No evidence of an associated mastocytosis was found. Thus, our patient population consisted of individuals with myelodysplastic syndromes not associated with mastocytosis. In this context it is worth noting that Nagata et al (1995) have investigated the peripheral blood MNCs of four cases with mastocytosis and concomitant myelodysplastic syndrome (including two patients with RA and two patients with CMML) and identified the C-KIT 2468AT mutation in three patients while one case of CMML tested negative. Moreover, Worobec et al (1998a) detected this mutation in peripheral blood MNCs of three cases of myelodysplastic syndrome (including two patients with RA and one patient with CMML) and two cases of unclassifiable myelodysplastic syndrome. However, in contrast to our patients, all the patients in these two studies (Nagata et al, 1995; Worobec et al, 1998a) had evidence of diffuse mast cell lesions in the BM. In contrast, the patient with CMML that was negative for C-KIT 2468AT did not show BM mastocytosis (Nagata et al, 1995), which is in line with our observations. These latter data suggest that myelodysplastic syndromes without evidence of concomitant proliferation of BM mast cells are negative for the C-KIT 2468AT mutation. In contrast, myelodysplastic syndromes with BM mastocytosis can show the C-KIT 2468AT mutation. Finally, Bowen et al (1993) reported the absence of mutations in several regions of C-KIT in peripheral blood MNCs of patients with myelodysplastic syndromes. However, this observation has to be interpreted with caution because the authors investigated genomic DNA samples that do not enable the sensitive analysis of somatic mutations in C-KIT.
While mutation analysis in patients with myelodysplastic syndromes has been limited to a few cases, individuals with systemic indolent mastocytosis have been more intensively studied. To date, peripheral blood MNCs (Nagata et al, 1995; Afonja et al, 1998; Beghini et al, 1998; Worobec et al, 1998a, 1998b) or lesional skin (Büttner et al, 1998; Reinacher-Schick et al, 1998; Longley et al, 1999) have been investigated in the great majority of studies, while almost no information is available about the prevalence of a mutated C-KIT transcript in the BM MNCs of these patients. Interestingly, C-KIT 2468AT was present in 100% of BM MNC samples of our patients with systemic indolent mastocytosis. In contrast, in peripheral blood MNCs this mutation was only found in 17·1% (Worobec et al, 1998a) and was not detectable in a further case of adult systemic indolent mastocytosis (Worobec et al, 1998b). In this context, it is notable that one of our 11 patients initially tested negative but was clearly positive for C-KIT 2468AT in four of five additional RFLP analyses using further aliquots of the same cDNA preparation. It is therefore possible that RFLP analysis of peripheral blood MNCs using five cDNA aliquots per patient may increase detection sensitivity. Nevertheless, the fact that 10 of our 11 patients (91%) with systemic indolent mastocytosis were positive for C-KIT 2468AT in a single cDNA aliquot suggests that BM MNCs more frequently bear cells expressing the C-KIT 2468AT mutation and, thus, should be investigated at diagnosis to establish or exclude clonality in patients with suspected systemic mastocytosis.
In addition to systemic indolent mastocytosis and myelodysplastic syndromes with concomitant BM mastocytosis, the C-KIT 2468AT mutation has been detected in several cases of mastocytosis associated with other haematological disorders (Boissan et al, 2000). These cases comprised two patients with myelofibrosis (Nagata et al, 1995; Worobec et al, 1998a), one case of acute myeloid leukaemia M4 (Sperr et al, 1998), one case of unclassifiable myeloproliferative syndrome (Worobec et al, 1998a) and one case of polycythaemia vera (Worobec et al, 1998a). Alternatively, cases with proven BM mastocytosis showing an associated haematological disorder can also have mutations other than C-KIT 2468AT, as has been described by Beghini et al (1998).
Apart from haematological diseases showing BM mastocytosis, some haematological disorders not involving the mast cell lineage can be associated with other mutations in C-KIT. These cases included two patients with primary myelofibrosis and one case of chronic myelogenous leukaemia showing the C-KIT 154GA mutation (Asp52Asn) (Kimura et al, 1997). Furthermore, Gari et al (1999) reported three in frame deletions plus insertion mutations in exon 8 of C-KIT in three out of seven patients with acute myeloid leukaemia associated with inv(16) and in one of two cases with t(8;21). In this study, one of the patients with inv(16) testing negative for an in frame deletion plus insertion mutation showed a C-KIT 1588GA mutation replacing a valine with an isoleucine (Val530Ile) residue (Gari et al, 1999). However, the authors did not report the presence or absence of BM mastocytosis, which is also the case in another study that reported on the detection of a mutation in codon 816 of C-KIT in a patient with acute myeloid leukaemia M2 (Asp816Tyr) (Beghini et al, 2000). Interestingly, in the latter study the C-KIT 2468AT mutation was also detected among three patients suffering from acute myeloid leukaemia M2 with t(8;21) and in two patients with acute myeloid leukaemia M4Eo with inv(16) (Beghini et al, 2000).