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.

Endothelial cell damage has been reported to be associated with noninfectious transplant-related complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Among these, noninfectious transplant-related complications with endothelial cell damage (TRC-EC) include sinusoidal occlusive syndrome, transplant-associated microangiopathy, intestinal transplant-associated microangiopathy, capillary leak syndrome, idiopathic pneumonia syndrome, and diffuse alveolar hemorrhage. Because angiopoietin-2 (ANG2) plays an essential role in the endothelial cell damage of various inflammatory disorders, we hypothesized that ANG2 may also play a critical role in TRC-EC. We retrospectively estimated the incidence of TRC-EC and evaluated the association with ANG2 level at transplant. We studied 153 consecutive adult patients who underwent allo-HSCT at our institution between 2000 and 2012. Median patient age was 49 years (range, 16 to 68 years). With a median follow-up of 55 months, 3-year overall survival for all patients was 55%. The incidence of TRC-EC at day 100 was significantly higher in the high-ANG2 group (≥2000 pg/mL; n = 36) than in the low-ANG2 group (<2000 pg/mL; n = 117) (70% [95% confidence interval {CI}, 55% to 84%] versus 16% [95% CI, 11% to 24%]; P < .001). Multivariate analysis revealed that high ANG2 level at transplant was independently associated with higher risk of TRC-EC (hazard ratio, 6.01; 95% CI, 3.16 to 11.43; P < .001) and shorter overall survival (hazard ratio, 2.23; 95% CI, 1.66 to 4.48; P = .002). These results suggest that ANG2 level at transplant may be a useful marker for predicting the risk of TRC-EC after allo-HSCT. Prospective studies are warranted to validate our results.

Purpose: The aim of this study was to evaluate the antileukemia activity of a novel FLT3 kinase inhibitor, FI-700. Experimental Design:The antileukemia activity of FI-700 was evaluated in human leukemia cell lines, mutant or wild-type (Wt)-FLT3^expressing mouse myeloid precursor cell line, 32D and primary acute myeloid leukemia cells, and in xenograft or syngeneic mouse leukemia models. Results: FI-700 showed a potent IC 50 value against FLT3 kinase at 20 nmol/L in an in vitro kinase assay. FI-700 showed selective growth inhibition against mutant FLT3-expressing leukemia cell lines and primary acute myeloid leukemia cells, whereas it did not affect the FLT3 ligand (FL)d riven growth ofWt-FLT3^expressing cells.These antileukemia activities were induced by the significant dephosphorylations of mutant FLT3 and STAT5, which resultedin G 1 arrest of the cell cycle. Oral administration of FI-700 induced the regression of tumors in a s.c. tumor xenograft model and increased the survival of mice in an i.v. transplanted model. Furthermore, FI-700 treatment eradicated FLT3/ITD-expressing leukemia cells, both in the peripheral blood and in the bone marrow. In this experiment, the depletion of FLT3/ITD-expressing cells by FI-700 was more significant than that of Ara-C, whereas bone marrow suppression by FI-700 was lower than that byAra-C. Conclusions: FI-700 is a novel and potent FLT3 inhibitor with promising antileukemia activity.FLT3 (FMS-like receptor tyrosine kinase) also referred to as fetal liver kinase 2 or stem cell tyrosine kinase 1 is a class III receptor tyrosine kinase together with KIT, FMS, and platelet-derived growth factor receptor (1, 2). Class III receptor tyrosine kinases share several structural characteristics including five immunoglobulin-like domains in the extracellular region, a juxtamembrane domain, a tyrosine kinase domain separated by a kinase insert domain, and a COOH-terminal domain in the intracellular region (3). Several mutations of receptor tyrosine kinases, found in human and murine malignancies, have been implicated in the constitutive activation of kinases (4). In 1996, a unique mutation of the FLT3 gene was first identified in acute myeloid leukemia (AML) cells (5). This mutation (FLT3/ITD) is formed when a fragment of the juxtamembrane domain -coding sequence is duplicated in a direct head-to-tail orientation (6). Subsequently, a missense point mutation at the D835 residue and point mutations, deletions, and insertions in the codons surrounding D835 within a tyrosine kinase domain of FLT3 (FLT3/KDM) have been found (7). To date, a number of studies, involving >5,000 individuals, have shown that FLT3/ITD and FLT3/KDM occur in 15% to 35% and 5% to 10% of adults with AML, respectively (8). Mutations of the FLT3 gene are, therefore, the most frequent genetic alterations thus far reported as involved in AML. Several large-scale studies in well-documented patients published to date have shown the effect on the clinical outcome in patients with FLT3 mutations (8). Interestingly, FLT3...

Noninfectious transplantation-related complications (TRCs), such as graft-versus-host disease or TRC with endothelial cell damage (TRC-EC), remain as the major obstacle for successful allogeneic hematopoietic cell transplantation (allo-HCT). However, the diagnosis and prognosis for the emergence of these complications are difficult to define during the early post allo-HCT period. Here, we tried to generate a novel diagnostic system for TRC-EC by analyzing 188 adult patients who received allo-HCT. Our study found that the peripheral blood levels of angiopoietin 2 (ANG2), C-reactive protein (CRP), D-dimer, and thrombomodulin (TM) at the onset of TRCs were significantly associated with the development of TRC-EC. We next developed a composite biomarker panel incorporating the risk values of ANG2, CRP, D-dimer, and TM at the onset of TRCs, which classified these patients into 3 risk groups: low, intermediate, and high risk. As a result, the panel was useful not only for the diagnosis of TRC-EC with high specificity and sensitivity, but also for the prediction of the patients' long-term outcome. The 5-year overall survival (OS) rates of patients in the low-, intermediate-, and high-risk groups since the occurrence from TRCs were 76.2%, 54.9%, and 26.9%, respectively, and the high-risk score was significantly associated with both poor OS (hazard ratio [HR], 5.60; 95% confidence interval [CI], 2.81 to 11.20; P < .01) and frequent nonrelapse mortality (HR, 19.75; 95% CI, 5.59 to 69.77; P < .01). Thus, the composite panel proposed in this study provides a powerful tool for the diagnosis of TRC-EC and for the prediction of survival for patients with TRC-EC after allo-HCT.