Cytoplasmic Nucleophosmin in Acute Myelogenous Leukemia with a Normal Karyotype

Nucleophosmin (NPM1) exon-12 gene mutations are the hallmark of a large acute myelogenous leukemia (AML) subgroup with normal karyotype, but their prognostic value in this AML subset has not yet been determined. We screened 401 AML patients with normal karyotype treated within the German AML Cooperative Group Protocol 99 (AMLCG99) study for NPM1 mutations. Results were related with partial tandem duplications within the MLL gene (MLL-PTD), Fms-like tyrosine kinase 3–length mutations (FLT3-LM), the tyrosine kinase domain of FLT3 (FLT3-TKD), NRAS, KIT, and CEBPA mutations and with clinical characteristics and outcome. NPM1 mutations were detected in 212 (52.9%) of 401 patients. Fourteen mutations, including 8 new variants, were identified. NPM1-mutated cases associated frequently with FLT3 mutations but rarely with other mutations. The NPM1-mutated group had a higher complete remission (CR) rate (70.5% vs 54.7%, P = .003), a trend to a longer overall survival (OS; median 1012 vs 549 days, P = .076), and significantly longer event-free survival (EFS; median 428 vs 336 days; P = .012). The favorable impact of NPM1 mutations on OS and EFS clearly emerged in the large group (264 [66.8%] of 395 cases) of normal-karyotype AML without FLT3-LM. This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM1+/FLT3-LM+ double positive was similar to NPM1–/FLT3-LM+ cases. In conclusion, this study demonstrates that NPM1+/FLT3-LM– mutations are an independent predictor for a favorable outcome in AML with normal karyotype.

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The Residual Malignant Cells Could Be Masked by Therapeutic Use of Granulocyte-Colony Stimulating Factor in the Patients with AML1/ETO+ Acute Myelogenous Leukemia.

Blood ◽

10.1182/blood.v106.11.4512.4512 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4512-4512

Author(s):

Hee Won Moon ◽

Ho Young Kim ◽

Young Ree Kim ◽

Han Ik Cho ◽

Sung-Soo Yoon ◽

...

Keyword(s):

Cell Line ◽

Acute Myelogenous Leukemia ◽

Normal Karyotype ◽

Myelogenous Leukemia ◽

Leukemic Cells ◽

P Value ◽

Trisomy 8 ◽

Morphological Examination ◽

Suspected Infection ◽

Acute Myelogenous

Abstract Among 200 AML cases for the past 7 years, we observed 6 cases with acute myelogenous leukemia(AML) which showed remission by morphologic criteria in BM examination, but revealed clonal changes in most cells by FISH after G-CSF administration. Remarkably, 5 of 6 cases were AML with AML1/ETO rearrangement, and FISH study revealed that most of AML1/ETO+ cells were mature neutrophils, suggesting differentiation of leukemic cells. A true remission of leukemia was probably never achieved with G-CSF alone and 4 of 6 cases have relapsed and 3 have died. Most cases of present study had infections or were suspected as infection, thus G-CSF which was administered and endogenously produced by infection seems to bring synergic effect (Table 1). To elucidate the mechanism involved in this finding, we measured the numbers of G-CSF receptor (G-CSFr) in AML1/ETO positive (Kasumi-1) and negative AML cell lines (CTV-1), and in leukemic cells from 8 patients with AML1/ETO positive and negative AML by flow cytometry. The number of G-CSFr was 2,673/cell in AML1/ETO+ Kasumi-1 cell line and 522/cell in AML1/ETO− CTV-1 cell line(Table 2). In 8 patients with AML, the number of baseline G-CSFr in AML1/ETO+ AML cells was significantly higher than that in AML1/ETO− AML cells (mean number 446.2 VS 226) (p value =0.0029). We assume that therapeutic G-CSF administration could result in differentiation and proliferation of AML1/ETO+ leukemic cells due to higher expression of G-CSF receptor. In conclusion, we strongly recommend that complete remission should be confirmed by FISH test, because malignant clone can be differentiated and masked in morphological examination or conventional cytogenetic test, especially for AML1/ETO+ AML. Table 1. Clinical and laboratory summary of cases Case No. Initial diagnosis Follow-up* Cytogenetics FISH % Blast in BM Cytogenetics FISH Other findings Outcomes * when showing discrepancy between morphologic examination and FISH test 1 t(8;21)(q22;q22) AML1/ETO: 94.5% 0%(PB) - AML1/ETO:95.5%(PB) Pneumonia Relapse 2 t(8;21)(q22;q22) AML1/ETO: 98% 39% - AML1/ETO:99% Suspected infection, G-CSF administration Relapse, death 3 t(8;21)(q22;q22) AML1/ETO: 47% 0.5% t(8;21)(q22;q22) AML1/ETO:55% Suspected infection, G-CSF administration Alive 4 t(8;21)(q22;q22) AML1/ETO: 94.5% 3.9% t(8;21)(q22;q22) AML1/ETO:77.5% Fever Relapse, death 5 t(8;21)(q22;q22) AML1/ETO: 93.5% 0.3% Normal karyotype AML1/ETO:33.5% G-CSF administration Relapse 6 Trisomy 8 Trisomy 8: 98.5% 28.1% Trisomy 8 Trisomy 8:96.5% Candidiasis, G-CSF administration Death Table 2. Quantitation of G-CSF receptors in Kasumi-1 and CTV-1 cell line G-CSF receptor (PE molecule per cell) Baseline After G-CSF administration Kasumi-1 cell line 2,673 1,953 CTV-1 cell line 522 556

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Mechanism of Altered Nucleo-Cytoplasmic Traffic of Nucleophosmin in Acute Myelogenous Leukemia Carrying Exon-12 NPM Mutations (NPMc+ AML).

Blood ◽

10.1182/blood.v106.11.4396.4396 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4396-4396

Author(s):

Brunangelo Falini ◽

Niccolo Bolli ◽

Jing Shan ◽

Maria Paola Martelli ◽

Ildo Nicoletti ◽

...

Keyword(s):

Acute Myelogenous Leukemia ◽

Nuclear Export ◽

Mutational Analysis ◽

Nuclear Export Signal ◽

Normal Karyotype ◽

Myelogenous Leukemia ◽

Wild Type ◽

Nuclear Localization Signals ◽

Tryptophan Residues ◽

Acute Myelogenous

Abstract We recently found that about one-third of adult AML (60% of all AML with normal karyotype) display aberrant cytoplasmic expression of nucleophosmin (NPM) which is due to mutations occurring at the exon-12 of NPM gene (Falini B, et al., Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype..N Engl J Med2005; 352: 254–266). Hereby, we clarify the molecular mechanism underlying cytoplasmic NPM accumulation that yet remained to be elucidated. AML-associated mutated NPM alleles encode abnormal NPM proteins (25 mutants so far identified) which have acquired at the C-terminus a nuclear export signal-(NES) motif and lost at least one of tryptophan residues 288 and 290 which determine nucleolar localization. Both alterations are crucial for mutant NPM export from nucleus to cytoplasm. In fact, the cytoplasmic NPM accumulation is blocked by leptomycin-B and ratjadones which are specific inhibitors of exportin-1/CRM1, and by re-insertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. Thus, for cytoplasmic accumulation of NPM to occur, the NES motif and tryptophan mutations must act in concert. Possibly, when NPM mutans enter the nucleus by virtue of their nuclear localization signals (NLS), their capability to bind nucleoli must be hindered at least partially to become a CRM-1 target. Specific antibodies anti-NPM mutant proteins showed that the mutants localized exclusively in the cytoplasm and recruited in that site the wild-type NPM protein which is physiologically located in the nucleoli. These findings suggest that the NPM mutants may interfere with the functions of wild-type NPM and possibly contribute to leukemogenesis. Immunostaining of 393 AML cases using anti-NPM monoclonal antibodies predicted the presence of NPM exon-12 mutations in all 191 NPM-cytoplasmic positive cases. This finding is consistent with the fact that, despite genomic heterogeneity, all NPM mutants contain a NES motif which, in the presence of altered tryptophans, promotes their rapid export from the nucleus to the cytoplasm. The immunohistochemical test is diagnostically relevant since it can be used as simple first-step procedure in molecular-genetic characterization of AML and as a surrogate for mutational analysis in selected cases. These findings are also clinically relevant since cytoplasmic NPM/NPM mutations are predictors of good response to induction therapy and favourable prognosis in AML with a normal karyotype.

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Genome-Wide Analysis of Microdeletions and Identification of NF1 Inactivation in Acute Myelogenous Leukemia.

Blood ◽

10.1182/blood.v114.22.165.165 ◽

2009 ◽

Vol 114 (22) ◽

pp. 165-165

Author(s):

Sami Malek ◽

Peter Ouillette ◽

Yin Wang ◽

Yan Liu ◽

Whitney Wright ◽

...

Keyword(s):

Acute Myelogenous Leukemia ◽

Copy Number ◽

Research Funding ◽

Normal Karyotype ◽

B Cell Lymphoma ◽

Myelogenous Leukemia ◽

Acute Myelogenous ◽

Copy Number Changes ◽

Induced Apoptosis ◽

High Level

Abstract Abstract 165 Genomic aberrations are of dominant importance to the biology and clinical outcome of patients with acute myelogenous leukemia (AML). To further our understanding of such aberrations in AML, we analyzed DNA from highly purified AML blasts and paired buccal cells from 95 patients for subchromosomal copy number changes and allele identities using ultra-high-density Affymetrix SNP 6.0 array-based genomic profiling. A total of 358 somatically acquired copy number changes were detected in 95 AML genomes. We detected 16 losses and 22 gains of entire chromosomes, 285 subchromosomal losses and 35 subchromosomal gains. No recurrent high-level amplifications or recurrent homozygous deletions were identified. Eight of the 34 AML cases (24%) with normal karyotype each had one lesion detected through 6.0 array profiling, all but one of which was less than 4Mb in length. Focusing on microdeletions as potential indicators of the locations of novel tumor suppressor genes or genes with importance to AML biology, we identified 60 deletions that were less than 1 Mb in length and 158 deletions of less than 5 Mb, the vast majority of which were undetectable by conventional cytogenetics. Through fine mapping of microdeletions on 17q, we identified Neurofibromin 1 (NF1) null states due to mutations or absent expression in ∼7% of AML. NF1 mutations were present in the hematopoetic stem cell compartment (CD34+/CD38- cell population) and siRNA-mediated NF1 suppression using recombinant lentiviruses significantly increased colony formation of primary AML blasts in methylcellulose. Further, AML blasts without functional NF1 displayed sensitivity to rapamycin-induced apoptosis, thus identifying a dependence on mTOR signaling for survival. As an additional validation of using microdeletions to guide pathogenetic gene discovery, we identified deletions involving RUNX1, IRF8, Core Binding Factor Beta (CBFB) and Casitas B-cell lymphoma B (CBLB), genes known to be altered in AML. IRF8 expression was found to be absent in ∼30% of all AML but sequencing of all coding exons of IRF8 of 48 AML cases did not disclose somatically acquired mutations. In summary, this comprehensive description of subchromosomal copy number changes and microdeletions in adult AML substantially adds to our knowledge of the pathological anatomy of the AML genome and should inform future searches for novel genes with importance to AML biology. Disclosures: Malek: Cephalon: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Affymetrix: Research Funding. Erba:Lilly: Research Funding; Antisoma: Research Funding; Wyeth: Research Funding; Cephalon: Honoraria, Research Funding; MGI Pharma: Honoraria; Pharmion: Honoraria; Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gemin-X: Research Funding; Kanisa: Research Funding.

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