C-FMS mutational analysis in acute myeloid leukaemia.

Abstract

The human c-FMS, located on chromosome 5 at band 5q33.3, encodes a 972 amino acid transmembrane glycoprotein which functions as the receptor of the monocyte-colony stimulating factor (M-CSF or CSF-1). Early studies suggested a leukaemogenic role, as overexpression of c-FMS in mice leads to the development of myeloblastic leukaemia. In addition, studies employing hybridization to allele-specific oligonucleotide (ASO) probes identified activating point mutations in approximately 18% of acute myeloid leukaemia (AML) and 15% of myelodysplastic syndrome (MDS) patients (Ridge et al, 1990; Tobal et al, 1990), with codon 969 being more frequently involved than codon 301. Mutations were more common in leukaemias characterized by monocytic differentiation, namely acute myelomonocytic leukaemia (M4-AML) and chronic myelomonocytic leukaemia (CMML). However, Shepherd et al (1990), utilizing direct sequencing, failed to find codon 969 and 301 mutations in either AML or MDS, a finding that has been supported by subsequent studies (Springall et al, 1993; Misawa et al, 1997). The role of c-FMS mutations in leukaemogenesis, therefore, remains controversial and the explanation for the discrepancy may lie in the fact that the early studies used ASO hybridization techniques where probe specificity, if not carefully controlled, may yield misleading results. In order to clarify the pathogenetic relevance of c-FMS mutations in AML, we have screened, for the first time, the entire coding sequence and intron/exon boundaries of exons 2–22 of the gene, using conformation sensitive gel electrophoresis (CSGE) as previously described (Abu-Duhier et al, 2003). Genomic DNA was obtained from the marrow at presentation of 60 cases of AML entered into the Medical Research Council (MRC) AML X trial (32 males, 28 females, mean age 41.6 years, range 15–59 years). The cases were classified, according to the French–American–British (FAB) criteria, as M0 (n = 4), M1 (n = 8), M2 (n = 10), M3 (n = 10), M4 (n = 12), M5 (n = 10) and M6 (n = 6). Standard cytogenetic analysis demonstrated inv(16) (n = 7), t(8;21) (n = 2), t(15;17) (n = 10), other abnormalities (n = 25) and a normal karyotype (n = 16). Genomic DNA was also prepared from the peripheral blood of 70 normal individuals using the Nucleon Biosciences BACC II kit. Genomic DNA was amplified using the polymerase chain reaction. Three patients were found to have mutations, including two with a novel exon 6 mutation (nucleotide 10906 G > T) that is predicted to result in an alanine to serine substitution at codon 245. These mutations were detected in a 52-year-old male (46XY, inv(16)(p13;q22), FAB group M1) and a 49-year-old male (46XY, FAB group M6). Interestingly, both cases lacked FLT3 (internal tandem duplication and Asp835) and c-KIT (exon 8 and Asp816) mutations. The third case, a 32-year-old male with acute promyelocytic leukaemia, possessed an exon 9 mutation (nucleotide 18073 G > A) that is predicted to result in a glycine to serine substitution at codon 413. The latter case also possessed an FLT3 Asp835 mutation. The Gly413Ser change has recently been reported in 5% of patients with idiopathic myelofibrosis (Abu-Duhier et al, 2003). Neither of these changes was detected in 70 normal control subjects. We were not able to detect the previously reported c-FMS codon 301 and 969 mutations. Therefore, our data suggests that c-FMS codon 301 and 969 mutations are rare events in AML and that, as a result, they are unlikely to play a significant role in leukaemogenesis. This conclusion is in agreement with recent studies that employed similar screening methods (Misawa et al, 1997; Meshinchi et al, 2003). Using the same techniques, we have previously identified several mutations in the c-KIT and FLT3 genes (Abu-Duhier et al, 2003). However, in this study, we have identified novel c-FMS mutations involving exons 6 and 9 in a small number of AML patients. The biological consequences of these changes remain unclear and further study is required. Nevertheless, both mutations are located in the extracellular domain of c-FMS, with Gly413 being a highly conserved amino acid. We have previously reported mutations affecting the nearby residue Asp419 of c-KIT, a fact that highlights the likely importance of this region of class III receptor tyrosine kinases (RTKs). It is possible that these changes could lead to constitutive activation of the receptor, as random mutagenesis of c-FMS has revealed multiple sites for activating mutations within the extracellular domain. Finally, mutually exclusive RTK class III mutations appear to be characteristic of AML with inv(16), occurring in approximately 40% of cases (Care et al, 2003). It is of interest, therefore, that the c-FMS exon 6 mutation that occurred in the AML with inv(16) was not associated with either an FLT3 or c-KIT mutation.