Recently, somatic mutations of the nucleophosmin gene (NPM1), which alter the subcellular localization of the product, have been reported in acute myeloid leukemia (AML). We analyzed the clinical significance of NPM1 mutations in comparison with cytogenetics, FLT3, NRAS, and TP53 mutations, and a partial tandem duplication of the MLL gene (MLL-TD) in 257 patients with AML. We found NPM1 mutations, including 4 novel sequence variants, in 64 of 257 (24.9%) patients.NPM1 mutations were associated with normal karyotype and with internal tandem duplication (ITD) and D835 mutations in FLT3, but not with other mutations. In 190 patients without the M3 French-American-British (FAB) subtype who were treated with the protocol of the Japan Adult Leukemia Study Group, multivariate analyses showed that the NPM1 mutation was a favorable factor for achieving complete remission but was associated with a high relapse rate. Sequential analysis using 39 paired samples obtained at diagnosis and relapse showed that NPM1 mutations were lost at relapse in 2 of the 17 patients who had NPM1 mutations at diagnosis. These results suggest that the NPM1 mutation is not necessarily an early event during leukemogenesis or that leukemia clones with NPM1 mutations are sensitive to chemotherapy.

IntroductionThe prevalence and significance of several genetic abnormalities in patients with acute myeloid leukemia (AML) have been reported. The most powerful prognostic factor in AML has been the karyotype of the leukemia cells. 1 Three cytogenetic risk groups (favorable, intermediate, and poor) are widely accepted, but there is a practical limitation to the definition of cytogenetic risk, especially in patients falling in the intermediate group. Additional prognostic factors are therefore required. It has been reported that abnormalities in the RAS and p53 genes as well as the FLT3 gene are implicated in the pathogenesis of AML. [2][3][4][5][6][7] Mutations in FLT3, RAS, and p53 have been found in approximately 30%, 20%, and 5% to 10% of adult AML cases, respectively, indicating that mutations in these 3 genes are the most frequent genetic alterations in AML.We and several groups have demonstrated that FLT3 mutations are a strong prognostic factor in AML. [8][9][10][11][12][13][14][15][16][17][18] To date, several large-scale analyses have revealed that FLT3 mutations are essentially found in myeloid-lineage leukemia cells. 16,19,20 However, FLT3 mutations within an activation-loop were found in 5 of 30 acute lymphoblastic leukemia (ALL) cases with mixed-lineage leukemia (MLL) gene-rearranged ALL. 21 It is notable that FLT3 was highly expressed in MLL gene-rearranged ALL, leading to the constitutive activation of wild-type FLT3 kinase, and that primary ALL cells and an ALL cell line SEMK2-M1, which strongly expressed FLT3 but did not carry FLT3 mutations, had the same sensitivity to a potent FLT3 inhibitor as leukemia cells and a cell line with FLT3 mutations. 21,22 FLT3 is preferentially expressed on hematopoietic stem cells as well as in the brain, placenta, and liver. 23,24 The ligand to FLT3 (FL) is expressed as a membrane-bound or soluble form by bone marrow stroma cells and stimulates the stem cells alone or in cooperation with other cytokines. 25-32 FL-FLT3 interaction, therefore, plays an important role in the survival, proliferation, and differentiation of stem cells. In FLT3-expressing leukemia cells, FL stimulation enhances proliferation and reduces apoptosis.Although FLT3 is expressed on the surface of a high proportion of AML cells as well as B-lineage ALL cells, [33][34][35][36][37] little is known about the clinical significance of the FLT3 expression level in acute leukemia. In this study, we analyzed the expression level of the FLT3 transcript quantitatively in comparison with several gene alterations in 181 de novo AML cases. Because the prevalence of the MLL gene rearrangement is lower in adult de novo AML than in therapy-related AML and in infant or childhood acute leukemia, the From the Department of Infectious Diseases and the Department of Hematology, Nagoya University School of Medicine, Japan; the Department of Medicine, Japanese Red Cross Nagoya First Hospital; the Department of Medicine, Saiseikai Maebashi Hospital, Japan; the Research and Development Center for Higher Education, N...

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Nucleophosmin (NPM) is a nucleolar protein with multi-functions including centromere duplication, nuclear-cytoplasmic shuttling, ribosomal biogenesis, p53 stability. NPM1 mutations were found in a large number of patients with acute myeloid leukemia (AML) especially with normal karyotype. The mutations lead to the aberrant subcellular localization of NPM protein. However, their impacts on clinical outcome remain controversial. We screened the mutations of NPM1 in 257 AML patients and analyzed the clinical significance. NPM1 mutations were found in 64 of 257 patients (24.9%). Seven types of mutations, including four novel mutations, were identified. NPM1 mutations were associated with normal karyotype, FLT3 mutations (both FLT3-ITD and D835 mutation) but not with other gene alterations such as N-RAS, p53 mutations and partial tandem duplication of the MLL gene. In 190 patients except the M3 subgroup, who were treated according to the protocol of Japan Adult Leukemia Study Group, the multivariate analysis revealed that NPM1 mutation was a favorable factor for achieving complete remission, but significantly associated with relapse. A sequential analysis, using paired samples obtained at diagnosis and relapse in 39 patients, revealed that NPM1 mutations were lost at relapse in 2 of the seventeen patients who had NPM1 mutations at diagnosis and none of the patients, who did not have NPM1 mutations at diagnosis, gained NPM1 mutations at relapse. Our results suggest that NPM-mutated AML should be a distinct subgroup with specific clinical characteristics and outcome. Loss of NPM mutations at relapse implies that NPM mutation is not necessarily a primary genetic alteration and that these leukemic clones could be sensitive to chemotherapy.

The Rho family of small GTPases, including Rho, Rac and Cdc42, has been well characterised as a molecular switch that transduces signals from plasma membrane to the downstream effectors. RhoH gene, a member of the Rho family, is specifically expressed in haematopoietic cells. The known function of RhoH is antagonising Rac and mediating activation of ZAP-70 in T lymphocytes; however, biological roles of RhoH in myeloid cells remain unknown. Here, we analysed the prognostic implication of the expression level of the RhoH gene transcript in bone marrow samples from 90 newly diagnosed acute myeloid leukaemia (AML) patients using a real-time fluorescence detection method. Kaplan-Meier analysis demonstrated that low expression of the RhoH transcript was a predictor of worse prognosis in both overall and disease-free survival. Multivariate analysis demonstrated that low expression of RhoH was an independent unfavourable prognostic factor for both overall and disease-free survival of AML patients. Overexpression of RhoH leads to dephosphorylation of Bad at Serine 75 residue possibly through deactivation of Rac. It is possible that low expression of RhoH (i.e. high GTP-Rac) contributes to chemotherapy resistance in leukaemia cells. Our result suggests that inhibition of Rac and its signalling components might provide a useful anti-leukaemic strategy.