Flt3 mutations and leukaemia

It is now 20 years since a molecular classification of acute myeloid leukaemia (AML) was initiated through the recognition of a number of leukaemia-specific cytogenetic abnormalities and their role as independent prognostic factors (Bloomfield et al, 1984). The intensive study of some of these markers, in particular the fusion transcripts from chromosomal translocations, has provided considerable insight into the underlying disease pathogenesis through characterization of the resulting aberrant gene products and their biological and clinical consequences. This has now been incorporated into the recent World Health Organization (WHO) classification of haemopoietic and lymphoid neoplasms in which AML patients with four well-defined recurring cytogenetic abnormalities have been put together as a subgroup (Harris et al, 1999). In general, however, patients are divided into three different risk groups based on cytogenetics: those with favourable, intermediate or standard, and poor risk disease. In some centres, the response to initial therapy is also taken into account (Wheatley et al, 1999). This classification has paved the way for a move from the indiscriminate use of high-dose chemotherapy for all patients to a more risk-adapted treatment approach. Patients with favourable cytogenetics, i.e. t(15;17), t(8;21) and inv(16), have particularly benefited from an improved understanding of the molecular pathology of their disease through identification of potential therapeutic targets. For example, the addition of all-trans retinoic acid (ATRA) during induction chemotherapy for acute promyelocytic leukaemia (APL) patients with the PML/RARα (promyelocytic leukaemia/retinoic acid receptor alpha) fusion gene has increased the 5-year survival a further 20–30% compared with chemotherapy alone (Tallman et al, 1997; Burnett et al, 1999). Patients with poor risk disease have adverse survival, which hardly exceeds 20% at 5 years (Grimwade et al, 1998). They often have complete or partial loss of genetic material, e.g. −7, −5, del(5q), del(3q) or complex karyotypes, abnormalities that are currently less suitable candidates for targeted therapy. Approximately two-thirds of newly diagnosed AML patients, however, have intermediate/standard risk disease; their remission rate is similar to that of patients with favourable disease, but their outcome is hampered by an increased relapse rate (Grimwade et al, 1998). Although some of these patients have identifiable cytogenetic abnormalities that may allow the development of novel therapies, the majority of patients, ≈ 50% of all patients, have a normal karyotype. There has therefore been much interest in identifying further molecular markers of leukaemia that could help to improve the prognostic stratification of patients. In addition, with clear evidence for a multistep pathogenesis from both mouse models of leukaemia and the variable outcome for patients within defined cytogenetic groups, knowledge of co-operating mutations may further assist in improving therapy. Mutations in growth factor receptors and their downstream signalling molecules have long been obvious candidates for causing dysregulation of the delicate balance between proliferation and differentiation in haemopoietic cells. Many years of searching have now started to bear fruit with the demonstration that acquired mutations in the tyrosine kinase receptor gene, FLT3, are common in AML and have a major impact on prognosis. This review will outline the current knowledge on these mutations and their biological and clinical significance in leukaemia. FLT3 (fms-like tyrosine kinase 3), also known as stem cell tyrosine kinase-1 (STK1) or fetal liver tyrosine kinase-2 (FLK-2), is one of the class III tyrosine kinase (TK) receptors that share sequence homology and structural characteristics. The latter include five immunoglobulin-like domains in the extracellular region, an intracellular juxtamembrane (JM) domain, two TK domains interrupted by a kinase insert and a C-terminal tail (Agnes et al, 1994) (Fig 1). The gene is located at chromosome 13q12 and consists of 24 exons (previously reported as 21) (Rosnet et al, 1991; Abu-Duhier et al, 2001a). Schematic representation of FLT3 and the different types of activating mutations detected in patients with AML (Ig, immunoglobulin). FLT3 is predominantly expressed on haemopoietic progenitor cells in the bone marrow, thymus and lymph nodes (Rosnet et al, 1993), but is also found on other tissues such as placenta, brain, cerebellum and gonads (Maroc et al, 1993). Interaction with its ligand (FL) results in receptor dimerization, autophosphorylation and the subsequent phosphorylation of cytoplasmic substrates that are involved in signalling pathways regulating the proliferation of pluripotent stem cells, early progenitor cells and immature lymphocytes (Lyman, 1995). This interaction is influenced by other cytokines such as Kit ligand (KL). In fact, when primitive human progenitor cells are stimulated in vitro with either FL or KL alone, they show little or no proliferative response, but both ligands together synergistically enhance growth (Hannum et al, 1994). Further evidence for the importance of FLT3 in early haemopoiesis has come from FLT3 knockout mice. They are healthy with normal peripheral blood counts, but have reduced numbers of bone marrow early B-cell precursors, plus a defect in primitive cells as measured by long-term competitive repopulation assays and a reduced ability to reconstitute B-cell, T-cell and myeloid lineages when transplanted into irradiated hosts (Mackarehtschian et al, 1995). The expression of FLT3 has been reported at the mRNA and/or protein level in 93% of AML patients, 87% of T-cell acute lymphoblastic leukaemia (ALL) and up to 100% of B-cell ALL patients (Drexler, 1996). It has not been detected in chronic myeloid leukaemia in the chronic phase, but appears to be highly expressed during disease transformation, irrespective of the phenotype (Birg et al, 1992). In leukaemic cell lines, it is expressed in ≈ 90% of preB-cell lines, but less frequently (40–80%) in myeloid and monocytic cell lines (Drexler, 1996). Studies have also demonstrated very high levels of FLT3 mRNA and protein in AML patients, leading to the suggestion that its overexpression may play a role in the survival and proliferation of leukaemic cells (Carow et al, 1996). This is in line with the finding that FL can induce proliferation in some FLT3-expressing primary leukaemic cells and cell lines (Dehmel et al, 1996). Furthermore, the chronic exposure of mice to FL by transplantation with primary haemopoietic cells constitutively expressing the FL gene has been shown to induce leukaemia with a long latency period, indicating a possible autocrine mechanism for maintaining the survival of a leukaemic clone (Hawley et al, 1998). The first FLT3 mutations to be identified were serendipitously detected during an investigation into the incidence and distribution of FLT3 mRNA in samples from adult AML and childhood ALL patients (Nakao et al, 1996). Unexpectedly long fragments were detected in the polymerase chain reaction (PCR) products of the JM domain in five out of 30 AML patients. They were also found using genomic DNA from the same patients, excluding the possibility of aberrant alternative splicing. Further analysis showed that they all contained a tandemly duplicated sequence, sometimes with insertion of additional nucleotides. The duplicated region was variable in both size and location in different individuals but always fell within the JM domain encoded by exons 14 and 15 (previously 11 and 12) (Fig 1). The resulting transcripts were always in frame and would therefore be expected to produce functional FLT3 chains. Since that first description, numerous other studies have confirmed and extended these findings to the extent that FLT3/ITDs are currently the most frequent single mutation described in AML, with a reported incidence between 13·2% and 32% in adult patients (Table I). They have also been detected in 3% of patients with myelodysplastic syndromes (Horiike et al, 1997; Yokota et al, 1997; Xu et al, 1999) and occasional patients with ALL (Xu et al, 1999; Nakao et al, 2000), although some of the latter patients had biphenotypic characteristics. They have not been found in patients with chronic myeloid leukaemia, chronic lymphoid leukaemia, non-Hodgkin's lymphoma or multiple myeloma (Yokota et al, 1997), or in normal individuals (Ishii et al, 1999; Kottaridis et al, 2001). Preliminary in vitro analysis of FLT3/ITDs transfected into Cos7 cells showed that they induced ligand-independent receptor dimerization and phosphorylation, irrespective of the location and length of the ITD, and led to phosphorylation of wild-type (WT) FLT3 expressed in the same cell (Kiyoi et al, 1998). They have been shown to confer growth factor independence on factor-dependent cell lines such as Ba/F3 and 32D cells, and to induce constitutive activation of downstream signalling molecules such as signal transducer and activation of transcription 5 (STAT5), mitogen-activated protein kinase (MAP kinase), Akt, Src homology 2 domain-containing (SHC) transforming protein 1, Cbl, Vav and SH2-containing protein tyrosine phosphatase 2 (SHP2) (Hayakawa et al, 2000; Mizuki et al, 2000; Kiyoi et al, 2002; Tse et al, 2002). However, comparable constitutive activation is not always observed in primary leukaemic blasts. Of 27 AML samples studied by Fenski et al (2000), 18 had ligand-dependent FLT3 phosphorylation, and three of these had a FLT3/ITD. Conversely, three samples had constitutive FLT3 phosphorylation, but only one had a FLT3/ITD. Similarly, Birkenkamp et al (2001) reported that, of 12 samples with both FLT3 and STAT5 constitutive phosphorylation, only eight had a FLT3/ITD and, of five samples with constitutive STAT5 but no FLT3 phosphorylation, two had a FLT3/ITD. It is likely that, at least to some extent, these results indicate the redundancy of signalling pathways within the cell and the multiple ways in which they can become activated. A striking feature of the FLT3/ITDs identified in AML patients is their diversity in both size and location. In general, the length of the duplication varies between 12 and 204 bp, but it has been reported to be as short as 3 bp and as long as > 400 bp (Schnittger et al, 2002a). Most ITDs occur at the 5′ end of the JM domain, in exon 14, and the TK1 domain is minimally involved, but they differ in the precise sequence duplicated and its starting point. Several inserted sequences of unknown origin varying between 9 and 36 bp have also been detected (Kiyoi et al, 1998; Frohling et al, 2002; Thiede et al, 2002a). There is no sequence that is common to all reported duplications, but the involved region does contain a tyrosine-rich stretch of sequence, and most ITDs include at least one of the tyrosines 589, 591, 597 or 599, which form part of the motifs YFYV and YEYDLK. As the latter are homologous to autophosphorylation sites in the JM domains of other TK receptors, e.g. the platelet-derived growth factor beta receptor (PDGFβR), it was initially thought that the mechanism by which duplication might lead to enhanced growth was through the gain of additional Src homology binding domain-2 (SH2) binding domains (Yokota et al, 1997). However, neither substitution of all four tyrosines with phenylalanine nor deletion of between one (ΔY599) and four (ΔY589–599) tyrosines alters the in vitro constitutive phosphorylation state and ability to induce growth factor independence of a FLT3/ITD, although these residues are clearly important for ligand-dependent activation of the WT receptor (Kiyoi et al, 2002). Instead, current models suggest that the JM domain has a negative regulatory role that is disrupted by the ITD elongation (Gilliland Kiyoi et al, 2002). In the WT receptor, the JM domain takes up an α-helical conformation, which blocks activation of the kinase and may inhibit self-dimerization. Ligand binding overcomes the inhibitory effect by inducing a conformational change and/or phosphorylation of key tyrosine residues. The ITDs may therefore prevent the protective association between the JM domain and kinase, exposing the latter to constitutive activation. They may further allow recruitment of molecules that could stabilize this conformation or alter downstream signalling. The in vivo tumorigenic potential of a FLT3/ITD has been demonstrated by injection of 32D cells carrying a FLT3/ITD into syngeneic mice, which led to the rapid development of a leukaemia-like disease (Mizuki et al, 2000). Furthermore, AML cells with an ITD showed an increased ability to repopulate bone marrow in non-obese diabetic (NOD)/severe combined immunodeficient (SCID) mice (Rombouts et al, 2000). However, although transplantation of bone marrow cells retrovirally transduced with FLT3/ITDs into recipient mice led to an oligoclonal myeloproliferative disorder, it was insufficient to cause leukaemia (Kelly et al, 2002a). This indicates the requirement for additional co-operating mutations for a fully transformed phenotype in this model. Further evidence for this co-operation has come from transplantation of cells carrying a FLT3/ITD into PML/RARα transgenic mice, which considerably shortened the latency and increased the penetrance for developing an APL-like disease (Kelly et al, 2002b). It can be hypothesized that, in such murine models, only 'two hits' are required to generate leukaemia, the mutation in the transcription factor (e.g. PML/RARα) producing a block in differentiation, and the mutant growth factor receptor providing a proliferative or survival signal (Deguchi conversely, low frequencies have been found in M6 and M7 et al, et al, 2002; et al, Thiede et al, (Table I). Although studies of patients are much the incidence is ≈ overall excluding patients with M3, range As with adult patients, a FLT3/ITD is associated with leucocytosis, and the frequency is in M3, particularly variant M3 et al, 1999; et al, 2003). A of patients have been reported to have more one ITD with most frequently two but up to five additional detected et al, et al, 2002; et al, et al, Thiede et al, 2002a). The independently as they in the sequence and may an underlying genetic There is also evidence for partial or complete loss of the WT in a number of patients in the mutant was of unselected patients, of patients with normal et al, et al, et al, Thiede et al, 2002a). of genetic was not observed using in analysis in patients with > and it is more likely that this has from et al, 2002a). The overall frequency of TKD mutations reported in four studies of unselected patients is range (Table II). was in only one study et al, and a high percentage of bone marrow blast cells in two studies (Frohling et al, 2002; Thiede et al, 2002a). levels were reported in FAB types and M5, and a level in (Yamamoto et al, et al, 2002b). Although the latter two studies found no the different cytogenetic risk groups, found levels of and in patients with normal (Frohling et al, 2002; Thiede et al, 2002a). The association of FLT3 mutations with cytogenetic or other acquired mutations is of considerable biological interest as it may to co-operating abnormalities leading to the transformed As a high frequency of FLT3/ITDs has been observed in patients with and, when combined with TKD mutations, the patients studied a FLT3 mutation et al, 2002; et al, and The ITDs in particular to be associated with M3 variant are together with and the of the short region 3 PML/RARα et al, 2002; et al, 2002; et al, 2002; et al, et al, and indicating that these may have a common A recent study found no in the incidence of FLT3/ITDs between patients with or a mutation et al, 2002). A possible association of FLT3 mutations with abnormalities of the gene has also been Although are only observed in of de novo AML patients (Schnittger et al, 2000), of both and FLT3 was first in two AML patients out of studied by et al Frohling et al found no in FLT3 mutant ITD or between patients. However, two studies reported in form have that may be an et al found a FLT3/ITD in et al found a FLT3 mutation ITD or in 50% of patients with an and in with a but in only of patients with an are more frequent in patients with a normal karyotype. The mechanism of duplication of gene is likely to be for is no evidence that homologous thought to be for the of et al, a role in the of their may indicate exposure to leading to DNA et al, 2002). Of although FLT3/ITDs are in AML patients with a high level of expression of FLT3 mRNA has been reported in ALL patients with et al, 2002), and a high level of TKD mutations has been found in with an et al, 2002). A number of associations have also been for the incidence of FLT3/ITD mutations is low in patients with t(8;21) and et al, Frohling et al 2002; et al, Thiede et al, (Table I). these are thought to lead to a block in differentiation by causing aberrant recruitment of the complex to of in myeloid and lymphoid development (Deguchi et al, 2002; et al, 2002; et al, et al, Thiede et al, mutations and FLT3/ITDs are only found together in ≈ of patients (Kiyoi et al, 1999; et al, 1999; et al, et al, 2002). results suggest that is no biological in both mutations as alone a growth Kiyoi et al were the first to a study of adult patients with de novo AML, excluding M3, and to an adverse outcome in those with a FLT3/ITD. Furthermore, analysis showed that a FLT3/ITD was the most prognostic factor for survival in patients the age of by Since many studies have the impact of these mutations on clinical As a the of an ITD in adult patients to have little or no impact on the ability to complete remission and only one study found a reduced rate (Table In however, reduced has been reported in three studies et al, 1999; et al, et al, (Table The most impact of an ITD is its association with increased relapse risk survival and overall survival which has been reported in most studies of and less years of age III and In addition to Kiyoi et al other groups have found that an ITD is the most factor an adverse outcome in analysis et al, et al, Frohling et al, 2002). The incidence of a mutation in AML is low (Xu et al, and, will be to the potential clinical impact in this In a single study of patients years of a FLT3/ITD had no impact on clinical outcome et al, although this is not as patients in this have disease, with an at 5 years no and effect of the mutation would be to Two studies are of particular interest as they to show a FLT3/ITD as an independent prognostic factor for disease In a study of patients, Thiede et al found that, an increased and reduced was only a for reduced with an ITD Similarly, et al reported that an ITD in their study of patients, but not The in outcome in these two studies may have been influenced by a number of such as the short of less and the of patients studied with to age and the type of disease novo and AML and They however, the possibility that different treatment might have a outcome in their patients, especially the of very high-dose In study of patients to the with a of up to a FLT3/ITD was highly of increased reduced and et al, 2001). In in the two studies the was up to from to (Schnittger et al, Thiede et al, 2002a). this may on the subgroup of AML, as Frohling et al found that intensive up to of was of no in FLT3/ITD patients with normal This also in the study by et al three different induction although they some suggestion of improving outcome in FLT3/ITD patients. It is clear that of treatment in patients with a FLT3/ITD further within in to further risk-adapted therapy. outcome in cytogenetic In patients with FAB type M3, an ITD does not remission but widely results have been reported for the impact on Several studies observed no in either or between patients with and an ITD (Kiyoi et al, 1997; et al, 2002; et al, Thiede et al, although in one was a for survival in patients et al, 2002). However, two other studies reported in form found that an ITD reduced although in one study was not different of early in the patients et al, 2002; et al, 2002). In patients with intermediate risk disease or normal cytogenetics, studies have shown that does not differ between those with and a mutation et al, et al, Thiede et al, however, all studies that or and/or is in patients carrying a FLT3/ITD (Rombouts et al, 2000; Kottaridis et al, et al, Frohling et al, 2002; et al, Thiede et al, 2002a). In study of patients with intermediate risk disease, for example, at 5 years was for FLT3/ITD patients compared with for patients et al, 2001). differences the patients with a FLT3/ITD and intermediate risk disease should be with poor risk disease patients and for more therapy. outcome in to the number and level of In the one study the of more one FLT3/ITD, this to further the adverse outcome of ITDs in AML and was associated with a a and percentage of bone marrow at and a et al, 2001). However, studies have shown that patients