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Partial tandem duplication (PTD) of the MLL gene and internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor tyrosine kinase gene have been described in acute myeloid leukemia (AML) patients, preferentially in those with normal cytogenetics. These alterations have been associated with a poor prognosis. In our study, we analyzed the prevalence and the potential prognostic impact of these aberrations in a large unselected and well-defined cohort of 956 patients with AML. Results were correlated with cytogenetic data and clinical outcome. MLL PTD was detected by RT-PCR, subsequent nucleotide sequencing, and Southern blotting. The overall incidence was found to be 5.0% (48/956), whereas FLT3 ITD was detected in 19.2% (184/956). Sixteen cases were positive for both alterations. The rate of MLL PTD in FLT3 ITD positive patients was significantly higher than that in FLT3 ITD negative patients [16/184 (8.7%); 32/772 (4.1%); P = 0.025]. However, both aberrations were highly increased in patients with normal karyotype (MLL PTD 35/431, P = 0.004; FLT3 ITD 132/334, P < 0.001). When restricted to this subgroup, the rate of MLL PTD in patients with FLT3 mutations was not significantly increased. No statistically significant differences were detected between patients positive for MLL PTD and patients negative for MLL PTD in the rate of complete remissions or the overall survival, although we did see a significantly shorter disease-free survival in patients age 60 or younger. In conclusion, although there is an overlap in the mutational spectrum in AML with FLT3 ITD and MLL PTD mutations, our data do not support a common mechanistic basis. Although associated with inferior disease-free survival, the results of this study do not unequivocally support the notion that MLL PTD mutations represent an independent prognostic factor.

IntroductionThe receptor tyrosine kinase (RTK) FLT3 belongs to the class III RTK subfamily that also includes KIT, FMS, and platelet-derived growth factor receptor (PDGF-R). 1,2 These class III RTK members are characterized by an extracellular domain consisting of 5 immunoglobulin-like domains, a juxtamembrane domain, and 2 kinase domains (KDs) interrupted by a kinase insert. 3 Ligand binding to the extracellular domain results in dimerization of the receptor followed by autophosphorylation on specific intracellular tyrosine residues. Subsequently, multiple downstream signaling pathways are activated. [4][5][6] Activation of the FLT3 receptor with its ligand (FL) plays an important role in proliferation and differentiation of early hematopoietic progenitors. [7][8][9] The FLT3 receptor tyrosine kinase is expressed on blast cells in most patients with acute myelogenous leukemia (AML), and activating mutations of FLT3 have been detected in approximately 30% of these patients. 10 Two distinct groups of FLT3 mutations are most common: internal tandem duplications (ITDs) of the juxtamembrane coding sequence in 20% to 27% of patients with AML 11-13 and point mutations at codon 835 (Asp835) within the second kinase domain in about 7% of patients with AML. 12,14 Patients carrying the FLT3 ITD mutation seem to have a significantly worse prognosis, whereas the effect of point mutations on the prognosis of patients with AML has not yet been defined. 11,12 Both types of mutations constitutively activate the FLT3 receptor, leading to activation of downstream signaling proteins, including STAT5 (signal transducer and activator of transcription 5) and MAP (mitogen-activated protein) kinase, and result in factorindependent proliferation of growth factor-dependent murine lymphoid and myeloid cells. [14][15][16] Additional evidence in support of an oncogenic role of FLT3 mutations stems from studies demonstrating that mice receiving transplants of bone marrow retrovirally infected with FLT3 ITD develop a myeloproliferative disease. 17 Recently, a series of novel mutations within the activation loop were identified in patients with AML. In that study, deletion of isoleucine 836 (Ile836del) occurred in 13 of 87 patient samples carrying point mutations in the kinase domain, and one sample was found to contain a novel point mutation, changing isoleucine 836 to methionine with an additional arginine inserted after codon 836 (Ile836MetϩArg). 12 The effect of these mutations on FLT3 receptor signaling activity has not yet been investigated.Because the FLT3 receptor is expressed in blast cells of most patients with AML and a significant fraction of these patients carry an activating mutation conferring a worse prognosis, targeting the molecule by specific inhibitors may establish new treatment PKC412 is a Novartis compound. [18][19][20] In this study, we investigated the role of newly described FLT3 receptor mutations on receptor activation and cell growth. Our results indicate that both deletion and insertion mutants are sufficient to ...