Cytoplasmic Nucleophosmin Research Articles

Frequency of Nucleophosmin 1 Expression by Immunohistochemistry in Acute Myeloid Leukemia.

Nucleophosmin (NPM1) mutation is one of the most common recurring genetic abnormalities seen in acute myeloid leukemia (AML). Immunohistochemistry serves as a cost effective and simple surrogate testing method for detection of NPM1 mutation. This study was conducted to evaluate the frequency of aberrant cytoplasmic nucleophosmin 1 expression in leukemic blast cells on formalin fixed bone marrow trephine biopsy (BMB) sections and also to correlate this data with the reference molecular method (reverse transcriptase-polymerase chain reaction; RT-PCR and gene sequencing), where available. Immunostains were performed using mouse anti-NPM1 monoclonal antibody on 71 paraffin embedded bone marrow biopsies (BMB) of patients with AML of any French-American-British (FAB) subtype. Results of immunohistochemistry (IHC) were then compared with the reference molecular method. The proportion of NPM1 expression by immunostaining in AML cases was found to be 17%. Twelve of the total 71 cases demonstrated cytoplasmic nucleophosmin (NPMc+) on immunostaining. Eleven of the positive cases that were correlated with the molecular standard demonstrated mutation in exon 12 of NPM1 gene. Cytoplasmic nucleophosmin expression by immunostaining was found to be in complete agreement with the standard molecular method. In a resource restricted setup, the information from this study might help in providing an inexpensive and accurate detection method to facilitate introduction of this marker in diagnostic and prognostic workup of AML especially in patients showing normal karyotype and no common recurrent translocations.

Novel NPM1 exon 5 mutations and gene fusions leading to aberrant cytoplasmic nucleophosmin in AML

AbstractNucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but also sporadically affect exons 9 and 11, causing changes at the protein C-terminal end (tryptophan loss, nuclear export signal [NES] motif creation) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 patients with AML, we found non–exon 12 NPM1 mutations in 5 (1.3%) of 387 NPM1c+ cases. Besides mutations in exons 9 (n = 1) and 11 (n = 1), novel exon 5 mutations were discovered (n = 3). Another exon 5 mutation was identified in an additional 141 patients with AML selected for wild-type NPM1 exon 12. Three NPM1 rearrangements (NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13 979 AML samples screened by cytogenetic/fluorescence in situ hybridization and RNA sequencing. Functional studies demonstrated that in AML cases, new NPM1 proteins harbored an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting AML-associated NPM1 genetic lesions. This study highlights the need to develop new assays for molecular diagnosis and monitoring of NPM1-mutated AML.

DNA Damaging Agents Cause a Sub-Cellular Re-Localisation of Nucleophosmin in an npm1 Mutated Acute Myeloid Leukemia (AML) Cell Line and in Primary AML Cells Carrying an npm1 Mutation

Nucleophosmin (NPM) is a ubiquitously expressed phosphoprotein involved in many cellular functions including ribosome biogenesis, p53 activation, cell proliferation and genetic stability and is considered essential for cellular activity and survival. NPM contains a number of motifs which mediate interactions with a range of binding partners as well as influencing its cellular localisation. Primarily, NPM is located to the nucleolus due to a C-terminal nucleolar localisation signal (NoLS), however it is able to translocate through the nucleoplasm into the cytoplasm due to the presence of a bipartite nuclear localisation signal (NLS) and two nuclear export signals (NES).The most commonly mutated gene in Acute Myeloid Leukemia (AML) is npm1, occurring in approximately 35% of all AML patients. All npm1 mutations characterised to date occur within the C-terminal domain and alter the normal nucleo-cytoplasmic shuttling of NPM resulting in an abnormal cytoplasmic localisation of the protein. The mutations are always heterozygous, however mutant NPM oligomerises with wild type NPM resulting in an accumulation of both forms of the protein within the cytoplasm, leading to abnormal cellular functioning.Utilising an AML cell line containing a heterozygous npm1 mutation (OCI-AML3), we have shown that nucleolar localisation of NPM can be restored following short term exposure (4 - 24 hours) of the cells to the DNA damaging agent etoposide. Western blot analysis of the levels of NPM within cytoplasmic and nuclear fractions isolated from OCI-AML3 cells showed a significant decrease in cytoplasmic NPM following treatment relative to untreated cells (p=0.0045; N=3). Further analysis of OCI-AML3 cells using confocal microscopy identified an increase in nucleolar NPM following treatment relative to untreated controls (Figure 1). This post treatment increase of nucleolar NPM was also observed in primary leukemic cells isolated from patients with npm1 mutated AML.Similar results were observed with the DNA damaging agent cytarabine, however the potent anti-leukemic multi-kinase inhibitor, SB1317 had no effect on the sub-cellular localisation of NPM suggesting that the response is specific to DNA damage.In order to identify whether the nucleolar re-localisation of NPM following DNA damage could be abrogated by inhibiting the cellular response to DNA damage, OCI-AML3 cells were co-treated with etoposide and the PARP-1 DNA repair inhibitor, olaparib for 24 hours. Confocal microscope analysis of co-treated cells revealed that the sub-cellular distribution of NPM was predominantly cytoplasmic, with very little NPM-nucleolus co-localisation, i.e. resembling that of untreated cells.In summary, our data show that DNA damage caused by chemotherapeutic drugs results in a sub-cellular re-localisation of NPM in npm1 mutated AML cells, with the post-treatment NPM localisation resembling that of npm1 wildtype cells. In a chemotherapeutic context utilising DNA-damaging agents however, this re-localisation of NPM may not be desirable considering the involvement of NPM in DNA repair pathways. Re-localisation of NPM can be prevented however, by inhibiting the activity of other molecules involved in DNA damage response pathways, specifically PARP-1. Therefore these results suggest that the treatment of npm1 mutated AML with DNA damaging drugs should be considered within this context. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Arsenic trioxide inhibits anaplastic lymphoma kinase (ALK)‐positive diffuse large B‐cell lymphoma through targeting ALK‐fusion oncoprotein

Arsenic trioxide inhibits anaplastic lymphoma kinase (ALK)‐positive diffuse large B‐cell lymphoma through targeting ALK‐fusion oncoprotein

Relocation of NPM Affects the Malignant Phenotypes of Hepatoma SMMC-7721 Cells.

Nucleophosmin(NPM), heavily implicated in diverse solid tumors, is an important multifunctional protein mainly located in the nucleolus. Our previous study confirmed that NPM can also localize and accumulate in the cytoplasm of liver cancer cells. However, the role of cytoplasmic NPM (NPMc +) is unclear. Here, we showed that both nucleolar NPM and NPMc+ could promote cell proliferation, although the effect of NPMc+ was weaker than that of NPM. Cell adhesion ability of hepatoma cells was significantly reduced to a greater extent by NPMc+ expression. Nucleolar NPM enhanced cell migration and invasion, whereas NPMc+ impeded cell migration and invasion. The investigation of NPM interactional proteins by proteomic method demonstrated that the NPM was involved in multiple biological processes. By contrast, the interactional proteins of NPMc+ were mainly implicated in tRNA amino acylation regulation. The interactional network of NPMc+ was significantly small and simple. These results suggested that relocation of NPM altered its interactional network and consequently disturbed the primary functions, including cell proliferation, adhesion, migration, and invasion. NPM plays a promotional role in cancer and the reducing relocation may be a potential therapeutic target for hepatocellular carcinoma. J. Cell. Biochem. 118: 3225-3236, 2017. © 2017 Wiley Periodicals, Inc.

Low-Dose Actinomycin-D Induces Redistribution of Wild-Type and Mutated Nucleophosmin Followed by Cell Death in Leukemic Cells.

Specific mutations involving C-terminal part of the nucleolar protein nucleophosmin (NPM) are associated with better outcome of acute myeloid leukemia (AML) therapy, possibly due to aberrant cytoplasmic NPM localization facilitating induction of anti-NPM immune response. Actinomycin D (actD) is known to induce nucleolar stress leading to redistribution of many nucleolar proteins, including NPM. We analyzed the distribution of both wild-type and mutated NPM (NPMmut) in human cell lines, before and after low-dose actD treatment, in living cells expressing exogenous fluorescently labeled proteins as well as using immunofluorescence staining of endogenous proteins in fixed cells. The wild-type NPM form is prevalently nucleolar in intact cells and relocalizes mainly to the nucleoplasm following actD addition. The mutated NPM form is found both in the nucleoli and in the cytoplasm of untreated cells. ActD treatment leads to a marked increase in NPMmut amount in the nucleoplasm while a mild decrease is observed in the cytoplasm. Cell death was induced by low-dose actD in all the studied leukemic cell lines with different p53 and NPM status. In cells expressing the tumor suppresor p53 (CML-T1, OCI-AML3), cell cycle arrest in G1/G0 phase was followed by p53-dependent apoptosis while in p53-null HL60 cells, transient G2/M-phase arrest was followed by cell necrosis. We conclude that although actD does not increase NPM concentration in the cytoplasm, it could improve the effect of standard chemotherapy in leukemias through more general mechanisms.

Immunohistochemical detection of cytoplasmic nucleophosmin in formalin-fixed paraffin-embedded marrow trephine biopsies in acute myeloid leukaemia

AimsNucleophosmin (NPM1) gene mutations resulting in cytoplasmic delocalisation of nucleophosmin (NPMc+) are the most common genetic abnormality in acute myeloid leukaemia (AML). In this study, we tested whether immunohistochemical (IHC)...

Cytoplasmic nucleophosmin has elevated T199 phosphorylation upon which G2/M phase progression is dependent.

The cytoplasmic mutant of nucleophosmin (NPMc) is found approximately in one-third of acute myeloid leukemia (AML) cases and is highly associated with normal karyotype. Whereas previous studies have focused on wtNPM in centrosome duplication, we further elucidate the role of NPM in the cell cycle by utilizing the increased cytoplasmic load of NPMc. Overexpression of NPMc causes increased phosphorylation of NPM on T199 and, to a lesser degree, S4. T199 phosphorylation is dependent on cdk2 but activators of cdk2 were not elevated. Upon inhibition of cdk2, NPMc-overexpressing cells demonstrate a greater G2/M phase arrest than wtNPM or GFP counterparts. However, the number of cells with 2 centrosomes did not increase concordantly. This suggests that the arrest was caused by a delay in centrosome duplication, most likely due to the inhibition of centrosome duplication caused by unphosphorylated NPMc. Overall, these results suggest that the phosphorylation of T199 is important in the mitotic progression of NPMc-expressing cells. This further supports the hypothesis that NPMc is associated with normal karyotypes in AML because the higher cytoplasmic load of NPM can better suppress centrosome overduplication which would otherwise result in unequal segregation of chromosomes during mitosis, leading to aneuploidy and other genomic instabilities.

NPMc+ cooperates with Flt3/ITD mutations to cause acute leukemia recapitulating human disease

Cytoplasmic nucleophosmin (NPMc(+)) mutations and FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations are two of the most common known molecular alterations in acute myeloid leukemia (AML); they frequently occur together, suggesting cooperative leukemogenesis. To explore the specific relationship between NPMc+ and FLT3/ITD in vivo, we crossed Flt3/ITD knock-in mice with transgenic NPMc+ mice. Mice with both mutations develop a transplantable leukemia of either myeloid or lymphoid lineage, definitively demonstrating cooperation between Flt3/ITD and NPMc+. In mice with myeloid leukemia, functionally significant loss of heterozygosity of the wild-type Flt3 allele is common, similar to what is observed in human FLT3/ITD+ AML, providing further in vivo evidence of the importance of loss of wild-type FLT3 in leukemic initiation and progression. Additionally, in vitro clonogenic assays reveal that the combination of Flt3/ITD and NPMc+ mutations causes a profound monocytic expansion, in excess of that seen with either mutation alone consistent with the predominance of myelomonocytic phenotype in human FLT3/ITD+/NPMc+ AML. This in vivo model of Flt3/ITD+/NPMc+ leukemia closely recapitulates human disease and will therefore serve as a tool for the investigation of the biology of this common disease entity.

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.

Detection of cytoplasmic nucleophosmin expression by imaging flow cytometry

Mutations within the nucleophosmin NPM1 gene occur in approximately one-third of cases of acute myeloid leukemia (AML). These mutations result in cytoplasmic accumulation of the mutant NPM protein. NPM1 mutations are currently detected by molecular methods. Using samples from 37 AML patients, we investigated whether imaging flow cytometry could be a viable alternative to this current technique. Bone marrow/peripheral blood cells were stained with anti-NPM antibody and DRAQ5 nuclear stain, and data were acquired on an ImageStream imaging flow cytometer (Amnis Corp., Seattle, USA). Using the similarity feature for data analysis, we demonstrated that this technique could successfully identify cases of AML with a NPM1 mutation based on cytoplasmic NPM protein staining (at similarity threshold of 1.1 sensitivity 88% and specificity 90%). Combining data of mean fluorescence intensity and % dissimilar staining in a 0-2 scoring system further improved the sensitivity (100%). Imaging flow cytometry has the potential to be included as part of a standard flow cytometry antibody panel to identify potential NPM1 mutations as part of diagnosis and minimal residual disease monitoring. Imaging flow cytometry is an exciting technology that has many possible applications in the diagnosis of hematological malignancies, including the potential to integrate modalities.

Cytoplasmic nucleophosmin (cNPM) in acute myeloid leukaemia: Relation to disease characteristics

BackgroundNucleophosmin/B23 (NPM) is a 38 kDa molecular phosphoprotein involved in ribosome assembly and transport. Findings have revealed a complex scenario of NPM functions and interactions, pointing to proliferative and growth-suppressive roles. NPM appears to be more abundant in tumour cells than in normal cells. ObjectiveThe aim of the work was to identify cytoplasmic localization of NPM using bone marrow clot biopsy and correlate it with disease and patient characteristics and the known prognostic factors, induction chemotherapy response and survival after 12 months of follow up. MethodsThe present work was undertaken on 50 cases of normal karyotype acute myeloid leukaemia classified according to modified FAB system. Bone marrow aspirates were obtained, clotted and a formalin-fixed, paraffin embedded cell block was prepared. Sections were immunostained for NPM. ResultsTwenty-six cases (52%) had cytoplasmic positivity (cNPM+) while the remaining 24 cases (48%) had nuclear restricted NPM (cNPM−). cNPM positivity was significantly variable among the different FAB subtypes being the most prevalent among M5 and M2 cases, was associated with high blast and white blood cell count at diagnosis. It was significantly correlated with CD34 negativity, CD14 positivity but not with FLT3 mutations. Clinically no relation between cNPM positivity and age, sex nor extramedullary involvement of the studied patients was proven. The cytoplasmic positivity for NPM was significantly correlated with increased survival and better outcome after cycles of chemotherapy. So it can be regarded as a good prognostic marker. FLT3 positivity affected the survival of the cNPM negative group of patients remarkably, denoting the importance of combining both their statuses to predict outcome of therapy and survival. ConclusionOur data confirm that cytoplasmic NPM1 immunoreactivity is predictive of NPM1 mutations and can be included in the routine diagnostic and prognostic workup of AML.

CD34+ cells from AML with mutated NPM1 harbor cytoplasmic mutated nucleophosmin and generate leukemia in immunocompromised mice

AbstractAcute myeloid leukemia (AML) with mutated NPM1 shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34+ cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34+ cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34+ leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34+ subfraction exhibiting the phenotype (CD34+/CD38−/CD123+/CD33+/CD90−) of leukemic stem cells. When transplanted into immunocompromised mice, CD34+ cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patient's disease. These results indicate that the CD34+ fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34− cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1 mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.

Cytoplasmic FANCA-FANCC Complex Interacts and Stabilizes the Cytoplasm-dislocalized Leukemic Nucleophosmin Protein (NPMc)

Eight of the Fanconi anemia (FA) proteins form a core complex that activates the FA pathway. Some core complex components also form subcomplexes for yet-to-be-elucidated functions. Here, we have analyzed the interaction between a cytoplasmic FA subcomplex and the leukemic nucleophosmin (NPMc). Exogenous NPMc was degraded rapidly in FA acute myeloid leukemia bone marrow cells. Knockdown of FANCA or FANCC in leukemic OCI/AML3 cells induced rapid degradation of endogenous NPMc. NPMc degradation was mediated by the ubiquitin-proteasome pathway involving the IBR-type RING-finger E3 ubiquitin ligase IBRDC2, and genetic correction of FA-A or FA-C lymphoblasts prevented NPMc ubiquitination. Moreover, cytoplasmic FANCA and FANCC formed a cytoplasmic complex and interacted with NPMc. Using a patient-derived FANCC mutant and a nuclearized FANCC, we demonstrated that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability. Finally, depletion of FANCA and FANCC in NPMc-positive leukemic cells significantly increased inflammation and chemoresistance through NF-κB activation. Our findings not only reveal the molecular mechanism involving cytoplasmic retention of NPMc but also suggest cytoplasmic function of FANCA and FANCC in NPMc-related leukemogenesis.

Mutant nucleophosmin deregulates cell death and myeloid differentiation through excessive caspase-6 and -8 inhibition

AbstractIn up to one-third of patients with acute myeloid leukemia, a C-terminal frame-shift mutation results in abnormal and abundant cytoplasmic accumulation of the usually nucleoli-bound protein nucleophosmin (NPM), and this is thought to function in cancer pathogenesis. Here, we demonstrate a gain-of-function role for cytoplasmic NPM in the inhibition of caspase signaling. The NPM mutant specifically inhibits the activities of the cell-death proteases, caspase-6 and -8, through direct interaction with their cleaved, active forms, but not the immature procaspases. The cytoplasmic NPM mutant not only affords protection from death ligand-induced cell death but also suppresses caspase-6/-8–mediated myeloid differentiation. Our data hence provide a potential explanation for the myeloid-specific involvement of cytoplasmic NPM in the leukemogenesis of a large subset of acute myeloid leukemia.

Inhibition of CRM1-mediated Nuclear Export of Transcription Factors by Leukemogenic NUP98 Fusion Proteins

NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

Ribosome‐associated nucleophosmin 1: increased expression and shuttling activity distinguishes prognostic subtypes in chronic lymphocytic leukaemia

Two distinct groups of chronic lymphocytic leukaemia (CLL) are distinguished by the presence or absence of somatic hypermutation of the immunoglobulin heavy-chain gene. CLL without somatic hypermutation has an adverse outcome, but the precise biological differences that underlie this more aggressive clinical-course are unclear. Using a proteomic approach, we found that the two prognostic forms of CLL were consistently distinguished according to their protein expression pattern. The most important difference observed related to the different expression of nucleophosmin 1 between the two forms of CLL. This different expression was not related to apoptosis, proliferation or gene mutation. However, co-immunoprecipitation experiments identified an association between nucleophosmin 1 and ribosomal proteins. Using immunocytofluorescence, nucleophosmin 1 expression was identified in the nucleoli and nucleoplasm of all cells, but in a proportion of cells, nucleophosmin had been transferred from the nucleoplasm to the cytoplasm. Both the fluorescent intensity, and the frequency of cytoplasmic nucleophosmin 1 expression, was higher in CLL without somatic hypermutation. We propose therefore, that nucleophosmin 1, in association with ribosomal proteins, undergoes nucleo-cytoplasmic shuttling in CLL. This process is most prominent in un-mutated CLL and may signify altered protein biosynthesis.

Can cytoplasmic nucleophosmin be detected by immunocytochemical staining of cell smears in acute myeloid leukemia?

Mutations in the C-terminal region of nucleophosmin in acute myeloid leukemia (AML) result in aberrant cytoplasmic nucleophosmin (cNPM) in leukemic blast cells which is detectable by immunocytochemistry in bone marrow trephine (BMT) biopsy sections. We tested whether cNPM is detectable by immunocytochemistry in air-dried smears of AML with nucleophosmin1 (NPM1) mutations. An immunoalkaline phosphatase method was developed using the OCI-AML3 cell line, known to have mutated NPM1, and assessed on blood and marrow smears of 60 AML cases. NPM was detectable in all blast cell nucleoli and cNPM in 21 of 31 of NPM1 mutated and 15 of 29 wild-type cases. Paired air-dried smears and BMT biopsies from the same case (mutated and wild-type) gave discrepancies in cNPM expression and there was no correlation in 10 of 22 cases. Due to the high false positive and negative rates for cNPM in cell smears, this method should not be used as a surrogate for NPM1 mutations in AML.

Dissecting the Hierarchical Level of Hematopoietic Progenitors' Involvement in AML with NPM1 Gene Mutation and Their Engraftment Potential in Immunocompromised Mice.

Abstract 480Acute myeloid leukemia with mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM 2005;352:254-266] is a new entity of WHO classification that shows distinctive biological and clinical features [Falini B et al, Blood 2007;109:874-885] which include negativity for CD34 antigen expression at both immunohistochemistry and gene expression profiling. Flow cytometric analysis shows that, in most NPM1-mutated AML, percentages of CD34+ cells are in the low range (< 5-10%). Detection of NPM1 mutations by molecular techniques and/or immunohistochemistry and Western Blot analysis with specific antibodies provides an important tool for tracking the genetic lesion in leukemic cells at different hierarchical stage. We previously reported involvement by NPM1 gene mutation of the CD34+ cell fraction isolated from patients with NPM1-mutated AML, and, in one case, the involvement, in particular, of the early progenitor CD34+/CD38- [Martelli MP et al, Blood (ASH Annual Meeting Abstracts) 2008;112:307]. Here we expand and confirm our previous observation in 5 cases of CD34-negative NPM1-mutated AML. CD34+/CD38- cells were isolated by either FACS (3 cases, purity >98%) or MACS-sorting (2 cases, purity >92%) and analyzed by molecular analysis and Western Blot with a specific anti-NPM1 mutant antibody, respectively. The presence of either NPM1 gene mutation or mutant protein was demonstrated in all samples analyzed proving the CD34+/CD38- cells belong to the leukemic clone. This cell subpopulation displayed also immunophenotypic features classically associated to leukemic stem cells (LSCs) (CD123+/CD33+/CD90-) in all (16/16) samples analyzed, suggesting they might actually represent the LSCs in NPM1-mutated AML. Indeed, CD34+ cell fraction isolated from NPM1-mutated AML was able to generate leukemia in immunocompromised mice resembling the original patient's disease. However, there is experimental evidence that, at least in some CD34-negative AML, also the CD34- population may contain LSCs. Whether the CD34- cell compartment in NPM1-mutated AML is also able to engraft and outgrow into leukemia in mice remains to be clarified. For this purpose, we assessed the engraftment ability of CD34- cells from 5 NPM1-mutated AML patients. No engraftment was observed in one case. Interestingly, in three patients with myelomonocytic (M4, 2 cases) and myelocytic (M2, 1 case) AML, the CD34- fraction resulted into marrow engraftment by human CD45+/CD33+ myeloid cells that, at morphological and immunohistological grounds, consisted of a mixed population of macrophage cells expressing the CD68 (PG-M1) antigen and mature looking myeloperoxidase (MPO)-positive cells. This pattern possibly reflects short-term engraftment by leukemic cells devoid of self-renewal potential that differentiated into mature elements. However, the neoplastic nature of engrafted cells could be established with certainty only in one case by western blotting detection of NPM1 mutant protein. Immunohistochemistry could not help in these cases to establish the leukemic nature of human cells since terminally differentiated leukemic cells in NPM1-mutated AML show nucleus-restricted NPM1 positivity. In contrast, the pure CD34+ fraction (availabel for comparison in one of these three cases) engrafted as AML with clear blastic morphology and cytoplasmic dislocation of nucleophosmin. In a fourth patient, the highly purified CD34- fraction from relapsed NPM1-mutated AML engrafted in mice with a typical AML picture. These preliminary findings suggest that in general the CD34- fraction from NPM1-mutated AML may have more limited engraftment potential than the CD34+ fraction. Further studies are ongoing to address this issue. Disclosures:Falini:Xenomics: Patents & Royalties.

Acute Myeloid Leukemia with Mutated NPM1 Presenting with Life-Threatening, Either Arterial or Venous, Thromboembolism: a Report of 4 Cases.

Abstract 4135Acute myeloid leukemia (AML) expressing mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM 2005;352:254-266] is a new entity of WHO classification that shows distinctive biological and clinical features. AML with mutated NPM1 usually presents with a high white blood cell count; the bone marrow biopsy is usually markedly hypercellular and leukemic cells frequently show myelomonocytic or monocytic features, with dysplasia and involvement of two or more cell lineages in about 25% of cases. Lack, or low expression, of CD34 in over 90% of cases is the most distinctive immunophenotypic feature of NPM1-mutated AML and is independent of leukemic cell maturation. NPM1 gene mutation without concomitant FLT3-ITD identify a subgroup of AML patients with a favorable prognosis and has been associated with an approximately 50-60% probability of survival at 5 years in younger patients. Here we report 4 out of 41 (10%) patients, admitted at our Hospital in the last year, with new-diagnosed AML with mutated NPM1 presenting with life-threatening thromboembolic (either arterial or venous) events. The main characteristics of these patients are summarized in Table 1. The patients had neither personal nor family history concerning thromboembolism. Hyperleukocytosis was a common feature of the vast majority of NPM1-mutated AML patients at diagnosis. Immunophenotypic analysis did not show a peculiar phenotype in these patients.Table 1Characteristics of patients with NPM1-mutated AML and thrombosis.Case report noAgeSex (M/F)FAB subtypeWBC/mmcType of thrombosisSite of thrombosis141FM114970arterialAnterior interventricular branch of left coronary artery256MM493990arterialexternal iliac and femoral (right limb)363MM2113000deep venousgreat saphenous veins (bilateral)473FM4190000deep venousiliac and femoralIn two patients (cases 1 and 2), the arterial thromboembolic event (acute myocardial infarction and acute ischemia of right lower limb, respectively) presented about one month before diagnosis of leukemia. In the other 2 patients (cases 3 and 4), deep venous thromboembolism was concomitant with the diagnosis of leukemia. One patient (case 4), who could not initiate chemotherapy for severe concomitant renal failure, died few days after diagnosis. The other patients recovered from the acute event and upon diagnosis of leukemia were promptly treated with standard polychemotherapy which allowed to obtain complete hematological remission associated with complete resolution of the thromboembolic event. The clinical course after chemotherapeutic treatment of the patients outlines the importance and life saving role of early chemotherapy even under adverse circumstances. The pathogenesis of thromboembolic disease in hematological malignancies is complex and multifactorial: tumor cell-derived procoagulant, fibrinolytic or proteolytic factors and inflammatory cytokines affect clotting activation. Other important factors include infectious complications and hyperleukocytosis. However, large vessel thrombosis is a very rare clinical presentation. Our report of severe thromboembolic events at presentation in AML with mutated NPM1 suggests some still unidentified biological features of this leukemia which we are currently investigating. Disclosures:No relevant conflicts of interest to declare.