Nucleophosmin Mutations Research Articles

Prognostic Impact of Nucleophosmin 1 (NPM1) Gene Mutations in Egyptian Acute Myeloid Leukemia Patients

Objective: Somatic mutations of the nucleophosmin gene (NPM1), which alter the subcellular localization of the product, are the most frequent mutations in patients with acute myeloid leukemia. The aim of the study was to assess the prevalence and prognostic impact of NPM1 gene mutations in adult AML patients.Materials and Methods: Polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) were used to screen 55 AML patients for mutations of NPM1 gene.Results: NPM1 mutations were found in 12 (21.8%) of 55 patients, significantly associated with higher total leukocytie count, marrow blast percentage (p=0.03 and p=0.02, respectively), and M5 subtype (p<0.001). Patients with NPM1 mutations had significantly higher complete remission rates (p=0.003) and a trend to lower rates of mortality, relapse and refractory disease (p=0.28, p=0.45 and p=0.08, respectively). Survival analysis showed significantly longer disease-free survival (mean 18.635±1.229 versus 11.041±1.250 months, p=0.044) and overall survival (mean 19.810±1.624 versus 12.063±1.244 months, p=0.041) in patients with NPM1 mutations compared with those without. Multivariate analyses confirmed NPM1 mutation as a significant independent predictor for disease-free survival (HR=0.066, p=0.001) and overall survival (HR=0.125, p=0.002).Conclusion: NPM1 mutation is a prognostic factor for a favorable outcome in Egyptian population. This finding is of major clinical importance since it strongly suggests that NPM1 mutations may allow one to divide the heterogeneous patient group of AML into prognostically different subgroups.Conflict of interest:None declared.

Detection of NPM1, FLT3 and C-KIT mutations in acute myeloid leukemia and their prognostic analysis

This study was aimed to evaluate the frequencies and prognostic significance of the nucleophosmin 1 (NPM1) mutation, the fms-like tyrosine kinase 3 (FLT3) mutation and c-KIT mutation in acute myeloid leukemia (AML) and to explore their relevance to clinical characteristics, cytogenetics and survival. Genomic DNA from 78 newly diagnosed AML from August 2010 to October 2012 was screened by PCR and sequencing or capillary electrophoresis (CE) for NPM1, FLT3 and c-KIT mutations. The results showed that the incidence of NPM1 mutation was 14.1% in AML patients and 26.7% in normal karyotype AML patients. NPM1 mutant cases were significantly associated with old age (P < 0.05), high peripheral white cell count and platelet counts (P < 0.05) and low expression of CD34 (P < 0.05), but no statistic difference was found in sex, percentage of bone marrow blasts, Hb, expression of CD117 and HLA-DR, complete remission rate, overall survival and relapse rate (P > 0.05). The prevalences of FLT3-ITD and FLT3-TKD mutations were 11.5% (9/78) and 3.8% (3/78) respectively, and no one patient has both of the two mutations. Patients with FLT3-ITD mutation had higher white blood cell counts and percentage of in bone marrow blasts (P < 0.05), and lower overall survival (P < 0.05), more relative to normal karyotype (P < 0.05), while no statistic difference was found in sex, age, platelet count, Hb level, complete remission rate and relapse rate (P > 0.05). No statistic analysis was performed due to the cases of less FLT3-TKD mutation. C-KIT mutation accounts for 7.7% (6/78). Patients with C-KIT mutation had a higher percentage in abnormal karyotype (P < 0.05), and higher relapse rate (P < 0.05), and lower overall survival, whereas no statistic difference was found in sex, age, percentage of bone marrow blasts, peripheral blood cell count, complete remission rate (P > 0.05). It is concluded that the detection of NPM1, FLT3 and C-KIT mutations may contribute to guiding treatment and evaluating prognosis of patients with AML.

A single center analysis of nucleophosmin in acute myeloid leukemia: value of combining immunohistochemistry with molecular mutation analysis

Mutations of nucleophosmin 1 are frequently found in acute myeloid leukemia and lead to aberrant cytoplasmic accumulation of nucleophosmin protein. Immunohistochemical staining is therefore recommended as the technique of choice in front-line screening. In this study, we assessed the sensitivity and specificity of immunohistochemistry on formalin-fixed bone marrow biopsies compared with gold standard molecular analysis to predict nucleophosmin 1 mutation status in 119 patients with acute myeloid leukemia. Discrepant cases were further characterized by gene expression analyses and fluorescence in situ hybridization. A large overlap between both methods was observed. Nevertheless, nine patients demonstrated discordant results at initial screening. Five cases demonstrated nuclear staining of nucleophosmin 1 by immunohistochemistry, but a nucleophosmin 1 mutation by molecular analysis. In two cases this could be attributed to technical issues and in three cases minor subpopulations of myeloblasts had not been discovered initially. All tested cases exhibited the characteristic nucleophosmin-mutated gene expression pattern. Four cases had cytoplasmic nucleophosmin 1 staining and a nucleophosmin-mutated gene expression pattern without a detectable nucleophosmin 1 mutation. In two of these cases we found the chromosomal translocation t(3;5)(q25;q35) encoding the NPM-MLF1 fusion protein. In the other discrepant cases the aberrant cytoplasmic nucleophosmin staining and gene expression could not be explained. In total six patients (5%) had true discordant results between immunohistochemistry and mutation analysis. We conclude that cytoplasmic nucleophosmin localization is not always caused by a conventional nucleophosmin 1 mutation and that in the screening for nucleophosmin 1 abnormalities, most information will be obtained by combining immunohistochemistry with molecular analysis.

The prognostic impact of FLT3-ITD and NPM1 mutations in patients with relapsed acute myeloid leukemia and intermediate-risk cytogenetics

Internal tandem duplication of the fms-like tyrosine kinase-3 gene (FLT3-ITD) and nucleophosmin-1 (NPM1) mutations have prognostic importance in acute myeloid leukemia (AML) patients with intermediate-risk karyotype at diagnosis, but less is known about their utility to predict outcomes at relapse. We retrospectively analysed outcomes of 70 patients with relapsed, intermediate-risk karyotype AML who received a uniform reinduction regimen, with respect to FLT3-ITD and NPM1 mutation status and first complete remission (CR1) duration. CR1 duration, but not molecular status, was significantly correlated with CR2 rate. On univariate analysis, patients with mutated FLT3-ITD (FLT3+) had significantly worse overall survival (OS) compared with those with neither an NPM1 nor FLT3-ITD mutation (NPM1-/FLT3-). On multivariate analysis, shorter CR1 duration was significantly correlated with inferior OS at relapse (P<0.0001), while FLT3 and NPM1 mutation status and age were not significantly correlated with OS. Patients who subsequently underwent allogeneic stem cell transplant (alloSCT) had a superior OS regardless of CR1 duration, but outcomes were better in patients with CR1 duration>12 months. In intermediate-risk karyotype AML patients receiving reinduction, CR1 duration remains the most important predictor of OS at relapse; FLT3-ITD and NPM1 status are not independent predictors of survival.

Cytoplasmic Expression of Nucleophosmin 1 as a Marker for Diagnosing Residual Disease of Acute Myeloid Leukemia

Nucleophosmin 1 (NPM1) is a nuclear protein and in approximately 50% to 60% of cytogenetically normal acute myeloid leukemia (AML), NPM1 is mutated and localized in the cytoplasm. Both wild type and mutant NPM1 can be detected by immunohistochemistry (IHC). We set out to evaluate whether subcellular localization of NPM1, as detected by IHC, is stable during disease course in AML, and whether cytoplasmic expression of NPM1 (NPMc+) can be used to differentiate recovery versus residual disease in formalin-fixed and hydrochloric acid/EDTA decalcified specimens. IHC against NPM1 was performed on bone marrow biopsies of 31 patients with AML at initial diagnosis and on 40 follow-up biopsies. Immunostaining patterns of NPM1, defined as NPMc+, NPMc-, and indeterminate, were correlated with morphology, ancillary tests, and clinical follow-up information. Of the 20 (64.5%) cases with NPMc- at initial diagnosis, none of the 27 follow-up biopsies showed NPMc+, regardless of disease status. Of the 11 cases (35.5%) with NPMc+ at initial diagnosis, all of the 3 biopsies with persistent/relapsed disease, and none of the 7 benign biopsies had NPMc+ at follow-up. For the 3 biopsies with indeterminate follow-up pathologic diagnoses of recovery versus residual disease, all were NPMc- at follow-up, which was consistent with remission and was supported by clinical follow-up. Therefore, subcellular localization of NPM1 as detected by IHC is stable during the course of the disease, and NPMc+ at follow-up supports residual disease in cases with NPMc+ at initial diagnosis.

P-250 Nucleophosmin cytoplasmic and nucleus immunohistochemical cell positivity of bone marrow biopsy sections in AML and MDS patients

Background Nucleophosmin (NPM) is phosphoprotein in nucleolus and shuttles continuously between nucleus and cytoplasm. Major role of NPM is to mediate nuclear export of ribosome components to the cytoplasm and to control centrosome duplication and it also interacts with oncosupressors p53 and p19Arf thus controlling cell proliferation and apoptosis. NPM mutations are involved in pathogenesis of some AML cases. Introduction The abnormal mutated NPM1 protein shows an aberrant cytoplasmic localization in leukemic cells (NPMc), whereas the wild-type protein is mainly located in the nucleolus and on the nuclear membrane. Approximately one-third of cases of denovo acute myeloblastic leukemia (AML) carry mutations in the c-terminal region of NPM (NPM1) gene. NPM1 mutations are rare in myelodisplasia (MDS) but in some reports NPM mutations and NPMc cell bone marrow cell positivity were also found in MDS. Purpose Aim of the study were to analyze cell NPM immunohistochemical expression in nuclei (NPMn) and cytoplasm (NPMc) in bone marrow (BM) sections of 7 patients with de novo AML and 9 MDS patients. Materials and Methods Immunohistochemical procedures were performed on paraffin sections cut from B5-fixed/EDTA-decalcified BM biopsies. Sections were subjected to micro waving/antigen retrieval, pH9 and immunostained with anti-NPM monoclonal antibody (clone 376) using immuno alkaline phosphatase technique. NPMc and NPMn cell positivity between AML and MDS patients were statistically compared with Mann-Whitney and Chi-square tests. From patients informed consent was obtained. Results NPMn cell positivity was seen in 6/7 AML and in 8/9 MDS patients. NPMc was detected in 5/7 AML and in 8 out of 9 MDS patients. Percentages of NPMn of BM cells were higher in MDS than in AML patients with borderline statistical significance. NPMn immunoexpression above 30% of BM positive cells was more frequently (with statistical significance) observed in MDS than in AML patients (p< 0.05). Conclusions Higher NPMn cell positivity (above 30% of nucleated BM cells) in MDS is probably linked to higher number of normal BM cells in MDS. Lower NPMn cell positivity in AML is maybe associated to higher number of leukemic cells and also to NPM functional loss in progression and transformation of MDS to AML. NPMc was also observed in most of our AML and MDS patients. This finding is in a concordance with some recent reports also implicating that that NPM1 mutations may be also seen in MDS, but further studies are needed to analyze correlation of NPMc cell positivity and NPM1 mutations in MDS patients

NPM new interacting partners and influence of NPM mutations on gene regulation

Nucleophosmin (NPM) is a multifunctional protein. NPM mutation is found in one third of all acute myeloid leukemia (AML), and in more than 60% of AML with normal karyotype. It is also a recurrent translocation partner in AML.We got interested in the NPM function as a co‐regulator of gene expression. We hypothesized that the modifications of NPM disrupt gene regulation and thereby, favour leukemia.With a combination of biochemistry and molecular biology techniques, we identified new partners of NPM involved in gene regulation. We found that NPM colocalise with different transcription regulators at specific genes. Based on our results, we propose that the effect of NPM modifications on gene regulation has a significant impact on the capacity of hematopoietic cells to receive extra‐cellular signals which favours abnormal hematopoiesis and potentially, leukemia.

Nucleophosmin mutations in acute myeloid leukemia: A tale of protein unfolding and mislocalization

Nucleophosmin (NPM1) is an abundant, ubiquitously expressed protein mainly localized at nucleoli but continuously shuttling between nucleus and cytoplasm. NPM1 plays a role in several cellular functions, including ribosome biogenesis and export, centrosome duplication, chromatin remodeling, DNA repair, and response to stress stimuli. Much of the interest in this protein arises from its relevance in human malignancies. NPM1 is frequently overexpressed in solid tumors and is the target of several chromosomal translocations in hematologic neoplasms. Notably, NPM1 has been characterized as the most frequently mutated gene in acute myeloid leukemia (AML). Mutations alter the C-terminal DNA-binding domain of the protein and result in its aberrant nuclear export and stable cytosolic localization. In this review, we focus on the leukemia-associated NPM1 C-terminal domain and describe its structure, function, and the effect exerted by leukemic mutations. Finally, we discuss the possibility to target NPM1 for the treatment of cancer and, in particular, of AML patients with mutated NPM1 gene.

A powerful molecular synergy between mutant Nucleophosmin and Flt3-ITD drives acute myeloid leukemia in mice

Acute myeloid leukemia (AML) is the commonest myeloid malignancy, yet there has been little therapeutic progress for this disease in decades, and only 25–30% of patients survive long term.1 This reflects its pathogenetic complexity and the fact that the molecular basis of its largest cytogenetic subgroup, AML with a normal karyotype (AML-NK), was obscure until recently. Recent advances in DNA sequencing have revealed that AML-NK is molecularly heterogeneous with >30 genes recurrently targeted by somatic mutations in this disease.2 What is also evident is that each individual case of AML-NK appears to harbor only a small number of coding driver mutations, often as few as three and usually no more than five.2, 3 Furthermore, it is manifest that the precise combination of driver mutations in the genome of each AML impacts on its salient features, including responsiveness to treatments and prognosis.3

Mutation of the NPM1 gene contributes to the development of donor cell–derived acute myeloid leukemia after unrelated cord blood transplantation for acute lymphoblastic leukemia

Donor cell leukemia (DCL) is a rare but severe complication after allogeneic stem cell transplantation. Its true incidence is unknown because of a lack of correct recognition and reporting, although improvements in molecular analysis of donor-host chimerism are contributing to a better diagnosis of this complication. The mechanisms of leukemogenesis are unclear, and multiple factors can contribute to the development of DCL. In recent years, cord blood has emerged as an alternative source of hematopoietic progenitor cells, and at least 12 cases of DCL have been reported after unrelated cord blood transplantation. We report a new case of DCL after unrelated cord blood transplantation in a 44-year-old woman diagnosed as having acute lymphoblastic leukemia with t(1;19) that developed acute myeloid leukemia with normal karyotype and nucleophosmin (NPM1) mutation in donor cells. To our knowledge, this is the first report of NPM1 mutation contributing to DCL development.

Acute myeloid leukemia with translocation t(3;5): new molecular insights

Acute myeloid leukemia with translocation t(3;5): new molecular insights

Nucleophosmin mutations alter its nucleolar localization by impairing G-quadruplex binding at ribosomal DNA

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex–binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex–binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.

A Knock-In Npm1 Mutation in Mice Results in Myeloproliferation and Implies a Perturbation in Hematopoietic Microenvironment

Somatic Nucleophosmin (NPM1) mutation frequently occurs in acute myeloid leukemia (AML), but its role in leukemogenesis remains unclear. This study reports the first “conventional” knock-in mouse model of Npm1 mutation, which was achieved by inserting TCTG after nucleotide c.857 (c.854_857dupTCTG) to mimic human mutation without any “humanized” sequence. The resultant mutant peptide differed slightly different from that in humans but exhibited cytoplasmic pulling force. Homozygous (Npm1c+/c+) mice showed embryonic lethality before day E8.5, wheras heterozygous (Npm1wt/c+) mice appeared healthy at birth and were fertile. Approximately 36% of Npm1wt/c+ mice developed myeloproliferative disease (MPD) with extramedullary hematopoiesis. Those Npm1wt/c+ mice that did not develop MPD nevertheless gradually developed monocytosis and showed increased numbers of marrow myeloid precursors. This second group of Npm1wt/c+ mice also showed compromised cobblestone area formation, suggesting pathology in the hematopoietic niche. Microarray experiments and bioinformatic analysis on mice myeloid precursor cells and 227 human samples revealed the expression of CXCR4/CXCL12-related genes was significantly suppressed in mutant cells from both mice and humans. Thus, our mouse model demonstrated that Npm1 mutation can result in MPD, but is insufficient for leukemogenesis. Perturbation of hematopoietic niche in mutant hematopoietic stem cells (implied by underrepresentation of CXCR4/CXCL12-related genes) may be important in the pathogenesis of NPM1 mutations.

Mutant NPM1 is a Reliable marker of Minimal Residual leukemia in patients with Acute Myeloid Leukemia.

Abstract 2484 Background:Mutations in NPM1 (Nucleophosmin-1) have been described in about 35% of adult patients with de novo AML and 45–60 % of AML patients with a diploid karyotype. NPM1 mutations predict for achieving higher complete remission (CR) rates and better outcomes in AML. Few studies have reported on the reliability of mutated NPM1 as a marker for minimal residual disease (MRD) detection in patients with AML. Methods:We conducted a retrospective analysis of patients (n=360) with newly diagnosed AML treated at our institution between 2008 and 2012. The study was approved by the Institutional Review Board. NPM1 mutation status was determined from DNA from unsorted bone marrow (BM) aspirate samples by a PCR-based method at baseline, remission, and relapse. Genomic DNA from bone marrow samples was isolated using the Autopure extractor (QIAGEN/Gentra, Valencia, CA). Mutations in exon 12 of NPM1 were assessed by a DNA-based semi-quantitative polymerase chain reaction capillary electrophoresis (PCR-CE) assay with analytical sensitivity of approximately 2.5%. Results:Data on remission and relapse samples from 360 newly diagnosed and previously untreated patients with AML, with available NPM1 analysis on their BM at the time of initial diagnosis, was collected (see flow chart below). Median age was 60 years (range 21 – 81 years). 262 patients (72%) had de novo AML, and 98 (27%) secondary AML. Cytogenetics was diploid in 137 (38%) patients, t(8;21) in 17 (5%), inversion 16 in 26 (7%), deletion 5 alone in 27 (7.5%), del 7 and 5 in 26 (7%), deletion 7 alone in 21 (5.8%), trisomy 8 in 17 (5%) and miscellaneous in 89 (24.7%) patients, respectively. Overall, 60 (16.6%) patients including 46 of the 137 (33.5%) diploid patients had NPM1 mutation at baseline. Secondary leukemia was more common in the NPM1 wild type (30%) than in the NPM1 mutated (13%) category.When analysed by age, in patients < 60 years (n=175), OS (overall survival), EFS (event free survival) and response rates were significantly superior in NPM1 mutated subgroup (p=0.001, 0.007, 0.02 respectively), while among patients ≥ 60 years (n=185) EFS and response rates were significantly higher in the NPM1 mutated subgroup (p=0.008, 0.03 respectively). Among the patients with diploid cytogenetics who were younger than 60 years (n=60) OS, EFS and CR duration was significantly better in the NPM1 mutated subset (p=0.007, 0.007 and 0.02 respectively), while in those ≥60 years (n=77) there was no statistically significant difference in the outcomes for the NPM1 mutated and wild-type subsets.Among the 60 NPM1 mutated patients 54 (90%) including 41/46 (89%) of those with diploid cytogenetics achieved complete response (CR) or CR without platelet recovery. Thirty nine patients (including 30 with diploid karyotype) had available NPM1 status at the time of CR and all (100%) were negative for NPM1 mutation.Among the patients with mutated NPM1 at baseline who have achieved a NPM1 negative status at CR, 10/39 overall (25%) and 7/30 diploid (23%) patients relapsed. NPM1 status was available for 6 patients overall including 4 with diploid karyotype at the time of relapse. Among them, 5/6 overall (83%) and 3/4 diploid (75%) patients had mutated NPM1, while 1/6 overall (16%) and 1/4 diploid (25%) patients remained NPM1 wild type. This patient relapsed with extramedullary disease (leukemia cutis) without any BM involvement.Among the 300 patients (including 91 with diploid karyotype) with wild type NPM1 at diagnosis, none acquired a mutated NPM1 clone, either at CR or at the time of relapse. Conclusions:These data suggest that mutated NPM1 is a reliable and stable marker for the detection of MRD at the time of CR. Hence, NPM1 mutations can be used to detect MRD and their recurrence may predict pending relapse. Disclosures:Cortes:Celgene: Research Funding. Ravandi:Johnson and Johnson: Honoraria; Celgene: Research Funding.

Mutated Nucleophosmin 1 (NPM1) Is an Immunogenic Target and Patients with NPM1mut Acute Myeloid Leukemia (AML) Showed High Expression of Different Leukemia-Associated Antigens (LAAs)

Abstract 3592Nucleophosmin gene 1 mutations (NPM1mut) are one of the most frequent molecular alterations in AML and distinct immune responses might contribute to the favorable prognosis of AML patients with NPM1mut. Recently, we showed specific T cell responses of CD4+ and CD8+ T cells against epitopes derived from mutated regions of NPM1 (Greiner et al., Blood. 2012 May 16, Epub). In the present study, we investigated clinical parameters and the clinical outcome of NPM1mut AML patients in accordance to their immune responses against different NPM1 epitopes. Moreover, we examined the quantitative expression of different leukemia-associated antigens (LAAs) in NPM1mutAML patients.In ELISpot analysis of 33 healthy volunteers and 27 AML patients, we detected T cell responses of CD4+ and CD8+ T cells against epitopes derived from the mutated region of NPM1. We performed further tetramer assays against the most interesting epitopes and chromium release assays to show the cytotoxicity of peptide-specific T cells. Microarray analysis was performed to analyze the expression of different LAAs in NPM1mut and NPM1wtAML patients.Two epitopes (peptide #1 and #3) derived from NPM1mut induced CD8+ T cell responses. 33% of the NPM1mut AML patients showed immune responses against peptide #1 and 44% against peptide #3. NPM1mut AML patients showed a significantly higher frequency of T cell responses against peptide #3 in contrast to HVs (p=0.046), whereas for peptide #1 the frequency of T cell responses of AML NPM1mut patients and HVs was not significantly different. Specific lysis of pulsed T2 cells but also NPM1mut leukemic blasts was detected in chromium release assays. Therefore, overlapping peptides (OL) were analyzed in ELISpot assays and the peptide called OL8 showed favorable results to activate both CD8+ and CD4+ T cells.We performed survival analysis for these 33 NPM1mut patients analyzed by ELISpot comparing cases with or without specific T cell responses. Our data suggest a trend to a better overall survival (OS) for patients with specific T cell responses against peptide #1 or #3. However, the patient numbers are small and the data have to be interpreted carefully. Analyses with material from larger controlled clinical trials with a high number of patients with NPM1mut AML have to be performed. Our microarray analysis of 30 AML patients showed a high expression of different LAAs like RHAMM, WT-1 and BCL-2 in all subtypes of cells of NPM1mutAML patients, also in leukemic progenitor cells. This demonstrates that NPM1 is an AML subtype suitable for poly-targeted immunotherapeutic trials.Taken together, NPM1mut might constitute an interesting target structure for individualized immunotherapeutic approaches in NPM1mut AML patients. We hypothesize that immune responses to NPM1 mutation may contribute to the favorable prognosis. Disclosures:No relevant conflicts of interest to declare.

Clonal heterogeneity in patients with cytogenetically normal acute myeloid leukemia with NPM1 mutations

The nucleophosmin 1 (NPM1) gene is one of the most commonly mutated genes in acute myeloid leukemia (AML), occurring in approximately 60% of adult cytogenetically normal AML (CN-AML). To date, these mutations have only been detected in cells of the myeloid lineage, whereas the potential clonal involvement of the lymphoid lineage is controversial. In our study, NPM1 mutations were analyzed using the highly sensitive real-time quantitative polymerase chain reaction (RQ-PCR) method on fluorescence-activated cell-sorted (FACS) purified different circulating mature cell populations in patients with NPM1-mutated CN-AML. As expected, NPM1 mutations were found in myeloid blood cells, including CD14+ monocytes and CD66b+ granulocytes. However, we were also able to detect NPM1 mutations in CD19+ B cells and CD3−14−16+56+ natural killer (NK) cells, albeit at lower levels. Surprisingly, mutations were also detected in CD3+ T cells from all analyzed patients. Our data demonstrate that NPM1-mutated CN-AML originates in an early stem cell with both lymphoid and myeloid differentiation potential.

MiR-10a is aberrantly overexpressed in Nucleophosmin1 mutated acute myeloid leukaemia and its suppression induces cell death

BackgroundAcute myeloid leukaemia (AML) with nucleophosmin-1 (NPM1) mutation is a major subtype of AML. The NPM1 mutation induces a myeloproliferative disorder, but evidence indicates that other insults are necessary for the development of AML. We utilised microRNA microarrays and functional assays to determine if microRNA dysregulation could be involved in the pathogenesis of in NPM1 mutated (NPM1mut)-AML.ResultsWe used a stringent locked nucleic acid (LNA) based microRNA microarray platform to profile bone marrow samples of patients with normal karyotype AML. A panel of five microRNAs dichotomised AML patients according to their NPM1 mutational status. miR-10a, let-7b and let-7c were significantly over-expressed, while miR-130a and miR-335 were under-expressed in NPM1mut-AML when compared to NPM1wildtype-AML. Of these, miR-10a is the most differentially expressed in NPM1mut-AML versus NPM1wildtype-AML (> 10 fold higher as confirmed by qRT-PCR). To investigate the functions of miR-10a, the OCI-AML3 cell line was utilised, which is the only commercially available cell line bearing NPM1mut. OCI-AML3 cells were firstly demonstrated to have a similarly high miR-10a expression to primary NPM1mut-AML patient samples. Inhibition of miR-10a expression by miRCURY LNA Inhibitors (Exiqon) in these cells resulted in increased cell death as assessed by MTS, cell cycle and Annexin-V assays and reduced clonogenic capacity, indicative of an involvement in leukaemic cell survival. In silico filtering of bioinformatically predicted targets of miR-10a identified a number of potential mRNA targets with annotated functions in haematopoiesis, cell growth and apoptosis. Lucferase reporter assays confirmed a number of these putative tumorogenic genes that are miR-10a suppressible including KLF4 and RB1CC1. This provides a potential mechanism for the pathogenic role of miR-10a in NPM1mut-AML.ConclusionsThis study provides, for the first time, in vitro evidence of a pro-survival role of miR-10a in NPM1mut-AML, that it may contribute to the pathogenesis of NPM1mut-AML and identifies putative tumorogenic targets.

Acute myeloid leukemia and transcription factors: role of erythroid Krüppel-like factor (EKLF)

We have investigated the role of erythroid transcription factors mRNA expression in patients with acute myeloid leukemia (AML) in the context of cytogenetic and other prognostic molecular markers, such as FMS-like Tyrosine Kinase 3 (FLT3), Nucleophosmin 1 (NPM1), and CCAAT/enhance-binding protein α (CEBPA) mutations. Further validation of Erythroid Krüppel-like Factor (EKLF) mRNA expression as a prognostic factor was assessed.We evaluated GATA binding protein 1 (GATA1), GATA binding protein 2 (GATA2), EKLF and Myeloproliferative Leukemia virus oncogen homology (cMPL) gene mRNA expression in the bone marrow of 65 AML patients at diagnosis, and assessed any correlation with NPM1, FLT3 and CEBPA mutations. EKLF-positive AML was associated with lower WBC in peripheral blood (P = 0.049), a higher percentage of erythroblasts in bone marrow (p = 0.057), and secondary AMLs (P = 0.036). High expression levels of EKLF showed a trend to association with T-cell antigen expression, such as CD7 (P = 0.057). Patients expressing EKLF had longer Overall Survival (OS) and Event Free Survival (EFS) than those patients not expressing EKLF (median OS was 35.61 months and 19.31 months, respectively, P = 0.0241; median EFS was 19.80 months and 8.03 months, respectively, P = 0.0140). No correlation of GATA1, GATA2, EKLF and cMPL levels was observed with FLT-3 or NPM1 mutation status. Four of four CEBPA mutated AMLs were EKLF positive versus 10 of 29 CEBPA wild-type AMLs; three of the CEBPA mutated, EKLF-positive AMLs were also GATA2 positive. There were no cases of CEBPA mutations in the EKLF-negative AML group. In conclusion, we have validated EKLF mRNA expression as an independent predictor of outcome in AML, and its expression is not associated with FLT3-ITD and NPM1 mutations. EKLF mRNA expression in AML patients may correlate with dysregulated CEBPA.

Assessment of Clonal Evolution in 42 AML with NPM1 Mutations by Molecular Characterization of Paired Diagnosis and Relapse Samples

Abstract 237▪▪This icon denotes a clinically relevant abstractMutations in the nucleophosmin 1 (NPM1) gene represent one of the most frequent gene mutations in acute myeloid leukemia (AML), in particular in cytogenetically normal (CN)-AML. NPM1 mutations (NPM1mut) are considered as an early genetic event in the pathogenesis of AML. To address the role of clonal evolution from diagnosis to relapse in NPM1mut AML, we applied high-resolution genome-wide single nucleotide polymorphism (SNP) array analysis using the Affymetrix 6.0 platform to detect copy number alterations (CNAs) and uniparental disomies (UPDs) in paired samples from 42 patients. In addition, we determined NPM1 and FLT3 [internal tandem duplication (ITD) and tyrosine kinase domain (TKD)] mutation status in all samples. Blood or bone marrow samples obtained at complete morphologic remission were available for all patients to exclude germline copy number variations.At diagnosis, 29 cases (69%) had a normal karyotype by cytogenetics and no CNAs and UPDs by SNP analysis. In the 13 remaining cases, we found a total of 10 CNAs in 7 cases (19%), and 6 UPDs in 6 cases (14%): deletions of 9q21 (size range 0.9 to 17 Mb) were detected in 5 cases and were the only recurrent CNA; the only recurrent UPD affected the long arm of chromosome 13 in 4 cases, all resulting in homozygous FLT3-ITD mutations with FLT3-ITD/wildtype ratios >1; heterozygous FLT3-ITD and –TKD mutations were detected in 9 and 7 patients, respectively.At the time of relapse, the number of CNAs increased (34 CNAs in 16 cases, 38%) while the frequency of UPDs remained unchanged (6 UPDs in 6 cases, 14%). Of note, in 6 patients (14%) the NPM1 mutation was no longer detectable at the time of relapse; SNP analysis showed completely distinct CNAs/UPDs in 4 of these patients; 3 of these 4 cases had a small gain at 11q23 corresponding to MLL partial tandem duplications as confirmed by PCR. These findings suggest that these 4 cases were therapy-related AMLs (t-AML) rather than relapsed AML. The median interval from diagnosis to relapse/tAML in these 4 cases was 65 months compared with 9 months for the relapsed cases still having the NPM1 mutation. In the two remaining cases, genetic alterations were neither present at diagnosis nor at relapse. Analysis of other gene mutations (eg, IDH1 and 2, DNMT3A, ASXL1, p53) is currently under way to further elucidate the clonal origin of these cases.Of the 36 NPM1mut positive relapse samples, 15 maintained a “normal karyotype”, and 2 showed the CNAs already present at diagnosis; 19 relapse samples (53%) displayed clonal evolution with acquiring new (n=15) and/or loosing single aberrations (n=4): Acquired recurrent alterations comprised deletions of tumor suppressor genes [ETV6 (n=2), TP53 (n=2), NF1 (n=2), WT1 (n=2)], most of which are uncommon in de novo NPM1mut AML. All 6 UPDs detected in relapse samples affected 13q, of which 3 were already present at diagnosis. One patient with initial heterozygous FLT3-ITD mutation developed a homozygous state by acquiring UPD13q at relapse. Two cases with wild-type FLT3 at diagnosis acquired UPD13q at relapse. Of note, one UPD13q was not present in the corresponding relapse sample anymore.In conclusion, almost half (45%) of NPM1mut AML showed evolution to a more aberrant karyotype at relapse, including acquisition of high-risk genetic changes that may account for the adverse prognosis of relapsed patients. Conversely, other alterations such as UPD13q or del(9q) detected at diagnosis were not always present in relapse samples, implying that relapse had evolved from a more ancestral clone. In addition, our data suggest that in a proportion of cases t-AML rather than relapse had developed. Further analysis, such as gene mutation studies of paired diagnosis/ relapse samples, will provide more detailed information on clonal evolution events in the pathogenesis of NPM1mut AML. Disclosures:No relevant conflicts of interest to declare.

Upregulation of eIF4E in Nucleophosmin 1 (NPM1) Haploinsufficient Cells Alters CCAAT Enhancer Binding Protein Alpha (C/EBPα) Activity: Implications for MDS and AML

Abstract 2432NPM1, is a highly conserved, ubiquitous nucleolar phosphoprotein that belongs to the nucleoplasmin family of nuclear chaperones. NPM1−/− mice die at mid-gestation (E11.5) from anemia, underscoring the gene’s role in embryonic development. NPM1 is one of the most frequently mutated genes in AML. Mutations in NPM1 are found in 50% of normal karyotype AML patients, and mutant NPM1 (NPMc+) is aberrantly located in the cytoplasm of leukemic blasts in about 35% of all AML patients. Furthermore, NPM1 maps to a region on chromosome 5q that is the target of deletions in both de novo and therapy-associated human MDS. NPM1 thus acts as a haploinsufficient tumor suppressor in the hematological compartment, although the mechanism of its contribution to dysmyelopoiesis remains unknown. NPM-1+/− mice develop a hematological syndrome similar to that observed in human MDS, and develop AML over time. The NPM1 deficient model therefore provides a platform to interrogate the molecular basis of MDS.We identified nucleophosmin (NPM1) in a screen for protein binding partners of C/EBPα. C/EBPα is a single exon gene, but is expressed as two isoforms that arise by alternate translation start sites to yield a full length C/EBPα p42 and a truncated dominant negative C/EBPα p30 isoform. Translational control of isoform expression is orchestrated by a conserved upstream open reading frame (uORF) in the 5’ untranslated region (5’UTR) and modulated by the translation initiation factors eIF4E and eIF2. We generated factor-dependent myeloid cell lines from the bone marrow of Npm1+/+ and Npm1+/− mice. These lines are IL-3-dependent and inducible toward neutrophil maturation with GM-CSF and/ or all- trans retinoic acid (ATRA). Neutrophils derived from MNPM1+/− cells display defective neutrophil-specific gene expression, including a cassette of C/EBPα-dependent genes. These observations led us to postulate that myeloid abnormalities in NPM1 deficiency reflect an aberrant NPM1-C/EBPα axis. We show that NPM1 haploinsufficiency upregulates eIF4E (eukaryotic initiation factor 4E) (but not eIF2), which binds the mRNA-Cap (m7-GTP) as part of the mRNA translation initiation complex, eIF4F. Increased eIF4E is observed in about 30% of all malignancies. Initial increased eIF4E levels in MNPM+/− cells likely reflect transcriptional activation by the oncoprotein c-Myc, protein levels of which are also elevated in MNPM1+/− cells. We propose that increased eIF4E then induces increased C/EBPαp30 translation. C/EBPαp30 is a dominant negative inhibitor of full length C/EBPαp42 activity and disrupts normal neutrophil development. Furthermore, we demonstrate that C/EBPαp30 but not C/EBPαp42, activates the eIF4E promoter. We propose a positive feedback loop, wherein increased C/EBPαp30 induced by eIF4E further increases the expression of eIF4E. Our data suggest that NPM1 deficiency modulates neutrophil-specific gene expression by altering C/EBPα. We propose an aberrant feed-forward mechanism that increases levels of both eIF4E and C/EBPαp30 and likely contributes to MDS associated with NPM1 deficiency. Disclosures:No relevant conflicts of interest to declare.