Abstract 1170: Correlation between cytidine deaminase single nucleotide polymorphisms andin vitrodrug sensitivity, DNA methylation and outcome in normal karyotype acute myelogenous leukemia.

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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