Translocations and mutations involving the nucleophosmin (NPM1) gene in lymphomas and leukemias

Abstract

Nucleophosmin (NPM) is a ubiquitously expressed nucleolar phoshoprotein which shuttles continuously between the nucleus and cytoplasm. Many findings have revealed a complex scenario of NPM functions and interactions, pointing to proliferative and growth-suppressive roles of this molecule. The gene NPM1 that encodes for nucleophosmin (NPMI) is translocated or mutated in various lymphomas and leukemias, forming fusion proteins (NPM-ALK, NPM-RARa, NPM-MLF1) or NPM mutant products. Here, we review the structure and functions of NPM, as well as the biological, clinical and pathological features of human hematologic malignancies with NPM1 gene alterations. NPM-ALK indentifies a new category of T/Null lymphomas with distinctive molecular and clinicopathological features, that is going to be included as a novel disease entity (ALK+ anaplastic large cell lymphoma) in the new WHO classification of lymphoid neoplasms. NPM1 mutations occur specifically in about 30% of adult de novo AML and cause aberrant cytoplasmic expression of NPM (hence the term NPMc+ AML). NPMc+ AML associates with normal karyotpe, and shows wide morphological spectrum, multilineage involvement, a unique gene expression signature, a high frequency of FLT3-internal tandem duplications, and distinctive clinical and prognostic features. The availability of specific antibodies and molecular techniques for the detection of NPM1 gene alterations has an enormous impact in the biological study diagnosis, prognostic stratification, and monitoring of minimal residual disease of various lymphomas and leukemias. The discovery of NPM1 gene alterations also represents the rationale basis for development of molecular targeted drugs.

Nucleophosmin (NPM), also known as B23,1 NO38, and numatrin, is an abundant, highly conserved, ubiquitously expressed nucleolar phosphoprotein which belongs to the nucleoplasmin/nucleophosmin family of nuclear chaperones.2,3 Many findings have revealed a complex scenario of NPM functions and interactions, pointing to proliferative and growth-suppressive roles of this molecule.4 Nucleophosmin is an essential protein, since inactivation of the gene encoding for nucleophosmin (NPM1) in the mouse germ line leads to developmental defects that cause embryonic death in mid-gestation.5 In humans, accumulating evidence suggests that NPM is directly implicated in the pathogenesis of cancer. NPM is over-expressed in solid tumors of diverse histological origin6 or is involved in tumor progression 7–9 In several hematologic malignancies, the NPM1 locus is lost10 or translocated leading to the formation of oncogenic fusion proteins.11 Moreover, we recently discovered that NPM1 is mutated in about one-third of adult patients with acute myeloid leukemia (AML),12 which makes NPM1 mutations the most frequent genetic lesions so far identified in de novo AML. Here we review the structure and biological functions of the NPM molecule and discuss the role that NPM alterations play in the pathogenesis and clinicopathological behavior of human lymphomas and leukemias, focusing on AML carrying cytoplasmic/mutated NPM (NPMc AML).12,13

The NPM1 gene and the structure of the encoded protein

The NPM1 gene contains 12 exons14 and in humans maps to chromosome 5q35. The NPM protein exists in two alternatively spliced:14 B23.1, the prevalent isoform in all tissues,15 contains 294 amino acids,16 whereas B23.2, a truncated protein, lacks the last 35 C-terminal amino acids of B23.1 and is expressed at very low levels. The NPM molecule contains distinct domains17 that account for its multiple biochemical functions (Figure 1A). The N-terminal hydrophobic portion of NPM contains the regions that are responsible for the self-oligomerization and chaperone activities of the molecule18 (Figure 1A). Under native conditions, both in resting and proliferating cells, over 95% of cellular NPM protein exists as an oligomer.19 The oligomerization domain of NPM also contains a highly conserved motif that appears critical for mediating ADP-ribosylation factor (ARF) binding in vivo.20 Through its N-terminal hydrophobic domain, NPM also exerts a chaperone activity, preventing protein aggregation in the nucleolus, favoring histone and nucleosome assembly,21–23 and increasing acetylation-dependent transcriptional activity.24 Recently, NPM has also been found to act as a chaperone for Bax; that is, it induces the conformational changes of Bax that precede this molecule’s translocation into mitochondria,25 a key event in the control of the apoptotic pathway. The chaperone activity of NPM also depends upon the first of the two acidic domains17 (stretches of aspartic and glutamic acids) that are located in the middle portion of the protein.16 In fact, the negatively charged acidic cluster on the outside of the NPM oligomer may serve as a binding site for ribosomal basic proteins, possibly minimizing non-specific interactions between ribosomal proteins and rRNA, during ribosome assembly in the nucleoli.19 The first acidic segment also contains a major protein kinase casein kinase II (CK2) site (Ser 125). Phosphorylation by the abundant CK2 in the nucleolus is involved in NPM chaperoning26 since it reduces the affinity of NPM for substrate, promoting dissociation. Occurring during interphase, phosphorylation may help to regulate the function of NPM in the nucleolus.26,27 The segment between the two acidic stretches and part of the C-terminus of NPM accounts for the ribonuclease activity of the protein. The C-terminus also contains a short aromatic stretch with two tryptophans at positions 288 and 290, which are crucial for NPM binding to the nucleolus28 (the so-called nucleolar localization signal). NPM binding to the nucleolus and other subnuclear compartments27,29 is also conditioned by several potential cyclin-dependent kinase 1 (CDK1) phosphorylation sites in the C-terminal region. Phosphorylation induces NPM to shuttle between the nucleolus and nucleoplasm (a common feature of nucleolar components involved in ribosome biogenesis), and appears to reduce the affinity of NPM for other nucleolar components.27,30 Finally, the bipartite nuclear localization signal (NLS)17 and nuclear export signal (NES) motifs31,32 of NPM play major roles in regulating the nucleocytoplasmic traffic of the protein (see below).

Figure 1.A. Structure and functional domains of wild-type NPM1. Starting from the N-terminus, the protein displays two nuclear export signal (NES) motifs (residues 42–49 and 94–102), a metal binding domain, two acidic regions (residues 120–132 and 160–188), a bipartite nuclear localization signal (NLS) motif (residues 152–157 and 190–197), a basic cluster inside a moderately basic region, and an aromatic region at the C-terminus unique to NPM isoform 1 containing the nucleolar localization signal (NLS) with tryptophan residues 288 and 290. B. Structure and functional domains of NPM altered proteins in hematologic malignancies. The NPM/ALK fusion protein is constituted by the amino-terminal portion of NPM (amino acids 1–116) and the cytoplasmic tail of ALK protein which contains the tyrosine kinase (TK) domain. The NPM/RARα fusion protein is constituted by the amino-terminal portion of NPM (amino acids 1-147) and the DNA binding, dimerization and ligand binding domain of RARα protein. A variant fusion protein of 563 amino acids has been described. The NPM/MLF1 fusion protein is constituted by the amino-terminal portion of NPM (amino acids 1-175) and almost the entire MLF1 protein, excluding only the first 16 amino acids at the N-terminus. The 14-3-3 binding domain of MLF1 is indicated. Mutated NPM protein in AML: (NPMmut) mutations at the NPM C-terminus introduce a new NES motif and disrupt tryptophan residues 288 and 290 (or 290 only) at the nucleolar binding domain (asterix). See, for comparison, Figure 1A.

NPM expression

NPM is more highly expressed in proliferating cells than in quiescent ones33 and increases in response to mitogenic stimuli.34 NPM over-expression promotes survival and recovery of hematopoietic stem cells under conditions of stress 35 and decreases the sensitivity of human HL-60 leukemic cells to retinoic acid-induced differentiation and apoptosis.36 Conversely, NPM levels drop after retinoic acid-induced differentiation of HL-60 leukemic cells37 and TPA-induced megakaryocytic differentiation of K562 cells.38 Loss of NPM, or inhibition of NPM shuttling, blocks protein translation and arrests the cell-cycle.32 NPM control of cell growth and proliferation is probably the result of several activities which include modulation of ribosome biogenesis as well as interactions with histones, p53 and ARF oncosuppressor proteins (see below). The close association of NPM over-expression with increased proliferation concurs with the finding that NPM is a transcriptional target of the Myc oncogene.39

The NPM isoforms have different patterns of subcellular distribution: B23.1 is located in the granular component of the nucleolus,15 which contains maturing pre-ribosomal particles;40 retention is probably due to its nucleic acid-binding domain interacting with other nucleolar components, possibly RNA.23 The rare B23.2 isoform mostly localizes in the nucleoplasm.30,41

Although most NPM resides in the nucleolus,42,43 this molecule shuttles from the nucleus to cytoplasm.44,45 The NLS signal drives NPM from the cytoplasm to the nucleoplasm, where it is translocated to the nucleolus through its nucleolar binding domain, particularly tryptophans 288 and 290.28 NPM remains in nucleoli, even though it contains highly conserved hydrophobic leucine-rich NES motifs within residues 94–10231 and 42–49,32 which drive it out of the nucleus. Therefore, in physiological conditions, nuclear import of wild-type NPM predominates over export, thus explaining why most of the wild-type NPM is found in the nucleolus (Figure 2A).

Figure 2.Mechanism of altered nucleocytoplasmic traffic of wild-type and mutant NPM. A. Export from the nucleus to the cytoplasm of wild-type NPM is minimal since the wild-type protein contains only weak physiological NES motifs (purple circles). Moreover, the two tryptophans at the C-terminus (red circles) drive wild-type NPM to the nucleolus, where the bulk of protein accumulates. Immunofluorescence analysis (lower panel) clearly shows nucleolar localization of NPM in NIH3T3 mouse fibroblasts transfected with eGFP/wild-type NPM. B. NPM mutants accumulate in the cytoplasm (immunofluorescence image in lower panel) since the mutation creates an additional NES motif at the C-terminus (gray circles) which is available for Crm1-mediated nuclear export. Moreover, mutations of the two tryptophans (cian stars) impair nucleolar localization. Dimerization between mutated and wild-type NPM partially delocalize the latter into cytoplasm. NPC is the Nuclear Pore Complex. The image in the lower panel is a confocal micrograph of NIH3T3 cells transfected with eGFP-NPM mutant A. Nuclei are counterstained red with propidium iodide. Cells were analysed with a Zeiss LSM 510 confocal microscope and reconstructed with Imaris software (Bitplane, Zurich, CH, Switzerland).

Although the levels of the NPM pools in the nucleoplasm and the cytoplasm are very low, they are crucial for proper execution of the diverse cellular activities of NPM, such as centrosome duplication and ribosome biogenesis.31,32 The size of these NPM pools closely depends upon regulation of NPM traffic across the different subcellular compartments. The exchange of NPM between the nucleolus and the nucleoplasm is strictly regulated by the capacity of this molecule to bind more or less efficiently to nucleolar components, which seems in turn to depend upon several variables: the oligomerization state of the molecule; the interaction of NPM with ARF or other nucleolar proteins; the phosphorylation state of CDK1 sites (mutants carrying changes of all four CDK1 sites delocalize in the nucleoplasm);27 and critical tryptophans at position 288 and 290. Interactions between NPM and other proteins may be involved in nucleoli assembly and disassembly.46 Finally, regulation of NPM traffic between the nucleus and cytoplasm mostly occurs through the NLS and NES motifs.31,32

Alterations in the regulation of NPM cellular traffic may contribute to tumorigenesis. Indeed, an abnormal subcellular distribution of fusion and wild-type NPM proteins (see below) is a general property of lymphomas and leukemias carrying NPM1 gene alterations.

The functions of NPM

NPM is a multifunctional protein. Here is a brief description of the main functions of NPM that have been observed to date. NPM is a key player in ribosome biogenesis, supporting cell growth and proliferation. It mediates 5S rRNA nuclear export, interacting with ribosomal protein L5.32 NPM intervenes in processing and/or assembly of ribosomes through its nucleocytoplasmic shuttling properties and intrinsic RNAse activity,47 and its ability to bind nucleic acids,48 to process pre-RNA molecules,49 and to act as a chaperone,21 impeding protein aggregation in the nucleolus during ribosome assembly.50 Recently, Pelletier et al.51 demonstrated a mechanistic link between TSC1/mTOR signaling, nucleophosmin-mediated nuclear export of ribosome subunits, protein synthesis levels, and cell growth.

NPM maintains genomic stability5,52 by controlling DNA repair mechanisms53,54 and centrosome duplication55 during mitosis. In resting cells, NPM associates with the unduplicated centrosome and after CDK2-cyclinE-mediated phosphorylation on threonine 95, it dissociates from it,31,56 thereby enabling proper chromosome duplication. Following phosphorylation on serine 4 by PLK1 and NEK2A,57 NPM re-associates with centrosomes at the mitotic spindle during mitosis.58 A recent study points to ROCK II kinase (an effector of Rho small GTPase) being the effector of the CDK2/cyclinE-NPM pathway in the regulation of centrosome duplication.59 As NPM inactivation leads to unrestricted centrosome duplication and genomic instability,60 NPM appears to protect from centrosome hyper-amplification and from the consequent heightened risk of cellular transformation. This view is supported by the observation that NPM1 mice develop a myelodysplastic-like syndrome with signs of dyserythropoiesis which is caused by unrestricted centrosome duplication.5

NPM is involved in the apoptotic response to stress and oncogenic stimuli,61 and can modulate the activity and stability of the oncosuppressor protein p53.62,63 Notably, nucleolar integrity, NPM and p53 stability are functionally linked.62,64,65 Cellular stress-inducing stimuli disrupt nucleolar integrity and relocate NPM from the nucleolus to the nucleoplasm, leading to p53 activation,64,66 and growth arrest. Moreover, nucleoplasmic NPM reinforces p53 stability by binding to, and inhibiting, Mdm2,65 a p53 E3-ubiquitin ligase,67,68 even though nucleolar stress may activate p53 independently of Mdm2.69

NPM stabilizes the oncosuppressor ARF and determines its subcellular localization,70 thus contributing to modulating growth-suppressive pathways.4 NPM co-localizes with ARF in the nucleolus,71 forming high molecular weight complexes.72 NPM protects ARF from degradation,52,73 stabilizing and maintaining a basal ARF level in the nucleolus, probably because ARF assumes a stable structure only when bound to NPM,70 thus escaping the rapid proteasome-mediated destruction which many misfolded proteins are subjected to.74 Increased ARF levels after oncogenic stress affect NPM polyubiquitination,75 promoting NPM degradation and interfering with the NPM nucleocytoplasmic shuttling which regulates several functions of NPM. Subnuclear compartimentalization of ARF is important for its regulation.76 In response to cellular stresses, NPM and ARF are displaced to the nucleoplasm where competitive binding between Mdm2 and NPM for ARF leads to formation of NPM-Mdm2 and ARF-Mdm2 binary complexes where Mdm2 sequestration strongly activates the p53 pathway.77 Thus, the ARF-NPM interaction site is bi-compartimental. In the nucleolus, ARF directly accesses the rRNA processing machinery through its association with NPM, thus preventing export of processed rRNA and inhibiting cell growth.78 In the nucleoplasm ARF may regulate the p53 cell cycle pathway through multiple interactions with NPM, Mdm2 and other proteins such as ARF-BP1,79 leading to a block of cell-cycle progression. It has been proposed that ARF acts as a parasitic peptide on the NPM molecule, since in exploiting this chaperone for its own survival it antagonizes normal NPM activities.80 However, the biological consequences of the NPM-ARF interaction still remain poorly defined. NPM seems to fall into a newly discovered category of genes that function both as oncogenes and oncosuppressors.4 When NPM expression is aberrantly increased, NPM may function as an oncogene by promoting aberrant cell growth through enhancement of ribosome machinery81 and, possibly, by inhibiting programmed cell death.4 Paradoxically, when NPM expression is reduced and its subcellular distribution altered (as in tumors carrying alterations of the NPM1 gene), NPM behaves as an oncosuppressor, possibly facilitating tumorigenesis through destabilization and functional impairment of the ARF-tumor suppressor pathway and increased genomic instability.4

Lymphomas and leukemias carrying NPM1 gene translocations

The NPM1 gene is translocated in CD30 anaplastic large-cell lymphoma and in rare variants of AML11 (Figure 1B). Cells from these tumors contain an oncogenic fusion protein (NPM-ALK, NPM-RARα or NPM-MLF1) (Figure 1B) and a reduced level of wild-type NPM due to the loss of one functional allele of the NPM1 gene (hemizygosity).

ALCL expressing NPM-ALK

Anaplastic large cell lymphoma (ALCL) is characterized by a proliferation of tumor cells, usually with anaplastic morphology, which express the CD30 molecule, tend to grow cohesively and invade lymph node sinuses.82,83 Due to ALK gene translocations, about 60% of ALCL express the anaplastic lymphoma kinase (ALK) protein, hence the term ALK ALCL.11 About 85% of ALK ALCL carry the t(2;5)(p23;q35) chromosome translocation,11,83 in which the ALK gene on chromosome 2 is fused with the NPM1 gene on chromosome 5.84 The chimeric gene encodes for a fusion protein comprising the amino-terminal portion of NPM (containing the oligomerization domain) and the entire cytoplasmic region of ALK (containing the tyrosine kinase domain)85 (Figure 1B). The other 15% of ALK ALCL are molecularly heterogeneous, since the ALK gene fuses with many different partners.82,83 ALK ALCL exhibits distinctive molecular, pathological and clinical features83 and, unlike other peripheral T-cell lymphomas, has a good prognosis.86 Tumor cells of ALCL with t(2;5) contain the NPM-ALK fusion protein and the wild-type NPM protein. They characteristically express the ALK protein in the cytoplasm and ectopically in the nucleus because, through the NPM oligomerization domain, NPM-ALK forms heterodimers with wild-type NPM, which in turn, via shuttling, import NPM-ALK into nucleoli11,87 (Figure 3, top and middle). Due to the presence of the NPM-ALK fusion protein,11 ALCL cells also show aberrant NPM cytoplasmic expression. This can be detected by an antibody against the N-terminus of NPM which is retained in the fusion protein87 (Figure 3, bottom). In contrast, wild-type NPM, which is recognized by an antibody against the NPM C-terminus (not retained in NPM-ALK), maintains its nucleolar localization (not shown).11,87 Through the oligomerization domain of NPM, NPM-ALK can also form homodimers (Figure 3, top panel) that lead to constitutive activation of the ALK tyrosine kinase domain, a major trigger of lymphomagenesis.85 This appears a general mechanism that also underlies the formation of homodimers between ALK fusion proteins other than NPM-ALK.85 Thus, the NPM moiety of NPM-ALK would appear to play no other role in transformation beyond serving as an oligorimerization domain for the chimera.88 Since wild-type NPM in ALCL with t(2;5) retains its nucleolar localization,11,87 its functions may not be perturbed by the NPM-ALK fusion product. Whether the loss of one functional NPM1 allele contributes to tumourigenesis4 remains an open question.

Figure 3.Localization of wild-type NPM and NPM-ALK fusion protein in ALCL with t(2;5). (Top) Schematic representation of wild-type NPM and NPM-ALK fusion proteins in ALCL with t(2,5). Cytoplasmic NPM positivity is due to the presence in the cytoplasm of NPM-ALK. Wild-type NPM can form heterodimers with NPM-ALK causing its dislocation into the nucleus (arrow). Wild-type NPM is nucleus-restricted. (Middle) Immunostaining for ALK protein. ALK is expressed both in the cytoplasm and nucleoli of tumor cells (arrow). Normal residual lymphoid cells are ALK-negative. Alkaline phosphatase anti-alkaline phosphatase (APAAP) technique; x 800. (Bottom) Immunostaining for NPM N-terminus (retained in the fusion protein). NPM shows the expected nuclear localization in addition to aberrant expression in the cytoplasm (long arrow). Normal residual lymphoid cells display nucleus-restricted NPM positivity (short arrows) (APAAP technique; x 800).

AML carrying the NPM-RARα protein

This extremely rare genetic alteration has been so far reported in three children with acute promyelocytic leukemia (APL) carrying the t(5;17) translocation.89–91 The t(5;17) causes the NPM1 gene to fuse with the retinoic acid receptor-α gene (RARα) (Figure 1B). Morphologically these cases look like the typical APL cases carrying the t(15;17)/PML-RARα rearrangement.91

Leukemic cells express the NPM-RARα fusion protein and its reciprocal, as well as wild-type NPM and RARα from the uninvolved alleles.92 In two cases investigated by immunocytochemistry,90,91 NPM showed diffuse nuclear positivity which was very unlike the typical wild-type NPM nucleolar staining. Furthermore, after a long latency, NPM-RARα transgenic mice in which the fusion gene is expressed under the control of a human cathepsin G (hCG) minigene, develop monoblastic leukemia with the NPM/RARα oncoprotein localizing in leukemic cell nucleoli.93 These findings suggest that NPM-RARα physically interacts with wild-type NPM, either by delocalizing it from the nucleolus or by altering its function in the nucleolus. The NPM-RARα fusion protein does not appear to interact with PML, nor alter its localization.94

Like PML-RARα, the NPM-RARα fusion protein modulates retinoid-responsive gene expression,95 perturbing the retinoid acid signal pathway and arresting myeloid differentiation at the promyelocyte stage. APL carrying NPM-RARα shows a good response to differentiation therapy with all-transretinoic acid (ATRA).91

AML carrying the NPM-MLF1 fusion protein

The rare chromosomal translocation t(3;5)(q25;q35) occurs in <1% of cases of AML and generates a chimeric gene named NPM-MLF1(myelodysplasia/myeloid leukemia factor 1).96 In the encoded NPM-MLF1 fusion protein, the N-terminal portion of NPM is fused to almost the entire MLF1 protein, excluding only the first 16 amino acids at the N-terminus96 (Figure 1B). AML with t(3;5) embraces all French-American-British (FAB) categories but is most frequently M6.97

Immunohistochemical hallmarks of leukemic cells carrying NPM-MLF1 are aberrant nuclear MLF1 expression, with a mechanism similar to the t(2;5), and aberrant cytoplasmic NPM positivity.98 Cytoplasmic NPM positivity is due to the NPM-MLF1 fusion protein in the cytoplasm and to wild-type NPM which is dislocated, through an unclear mechanism, from the nucleus to the cytoplasm.98 As these aberrant staining patterns are frequently observed in myeloid and erythroid cell precursors, at least one myeloid-committed cell appears to be involved.98 Interestingly, HLS7 (the murine gene counterpart to human MLF1) influences erythroid/myeloid lineage switching and development of normal hematopoietic cells.99 The mechanism underlying the NPM-MLF1 fusion protein induction of malignant transformation remains unknown. Since MLF1 is not usually expressed in normal hematopoietic tissues, NPM-MLF1 may promote ectopic MLF1 expression in hematopoietic cells, thus contributing to myelodysplasia/AML.100 Interestingly, MLF1 prevents erythroleukemic cells from undergoing biological and morphological maturation in response to erythropoietin,101 possibly in part through an interaction with the recently identified MLF1IP protein.102 This finding concurs with the frequent association of t(3;5) AML with the FAB M6 subtype.97

The contribution of NPM to NPM-MLF1-induced leukemogenesis remains to be determined. The interaction between the NPM moiety of the fusion protein and wild-type NPM causes formation of heterodimers (wild-type NPM/NPM-MLF1)96,98 and transport of the chimeric protein into the nucleus, which may result in functional alterations.103 Reduced levels of wild-type NPM (due to the loss of one NPM1 functional allele) and its cytoplasmic dislocation may also contribute to leukemogenesis. In fact, NPM heterozygous mice develop a hematologic disorder with features resembling those of human myelodysplastic syndromes.4 Despite sharing some features with NPMc AML (see below), AML patients carrying t(3;5) are younger at presentation, may have suffered from previous myelodysplasia, show no NPM1 mutations and have a poorer prognosis (Table 1).

Table 1.AML with t(3;5) vs NPMc⁺ AML: common and distinctive features.

AML carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML)

In early 2005, we reported that NPM1 mutations closely associate with AML carrying a normal karyotype12 (AML-NK). Molecular alterations in AML-NK have always been difficult to study because, although this leukemia subgroup accounts for 40–50% of adult AML,104–106 it lacks chromosomal rearrangements that direct researchers towards cloning and characterization of oncogenic fusion genes, such as those encoded by the translocations t(15;17), t(8;21) or Inv(16).107 Attempts to stratify AML-NK using gene microarrays108,109 succeeded in associating gene expression patterns with differences in response to treatment, but no specific genetic subgroups emerged. In the difficult setting of AML-NK, our discovery of NPM1 mutations is yet another example of how simple observations at the microscope110 sometimes provide the key. The clue to the identification of NPM1 mutations in AML-NK came from our immunohistochemical studies on ALCL with t(2;5).11 Having observed that NPM, instead of being restricted to nucleoli as in normal tissues,111 is aberrantly expressed in the cytoplasm87 (due to the presence of the NPM-ALK protein) (Figure 3, bottom) we reasoned that immunohistochemical detection of cytoplasmic NPM in routine biopsy samples might serve as a quick and sensitive method to screen tumors for the presence of NPM1 gene alterations. Consequently, we extended our immunohistochemical studies to a wide range of human neoplasms, including leukemias, and found the first cases of AML with cytoplasmic NPM expression (NPMc AML) but no known NPM fusion proteins. Subsequent analysis of a large cohort of AML patients established that cytoplasmic NPM was associated with normal karyotype and other distinctive biological and clinical features.12 These findings led us to hypothesize that yet unidentified genetic lesion underlies NPMc AML. As the only distinguishing feature was NPM cytoplasmic dislocation, the NPM1 gene emerged as the most likely candidate and indeed, gene sequencing revealed mutations at exon 12.12

General characteristics of NPM1 mutations

NPM1 mutations appear to be specific for de novo AML.12,13 Although mutational studies of NPM1 in tumors other than AML are scarce, immunohistochemical analysis of NPM in thousands of hematopoietic and extrahematopoietic neoplasms has consistently revealed nucleus-restricted NPM expression,12 which indicates no NPM1 mutations are present.112 Rare reports of NPM1 mutations in chronic myelomonocytic leukemia113,114 are questionable, since most such cases developed AML within 1 year and probably represented early stage M4 or M5 AML with marked monocytic differentiation. Finding NPM1 mutations in 2/38 (5.2%) myelodysplastic patients115 does not concur with our immunohistochemical studies in 50 patients with various myelodysplastic syndromes who showed nucleus-restricted NPM expression, indicating an unmutated NPM1 gene (Falini, unpublished results). Thus, whether NPM1 mutations occur occasionally in myelodysplastic syndromes remains to be clarified in larger series of patients. Although NPM1 mutations are found in about one-third of adult AML patients, they were detected in only 1/79 human myeloid leukemic cell lines.116 This cell line, called OCI-AML3 was originally generated at the Ontario Cancer Institute, Ontario, Canada,117 from a 57-year old male patient with AML-M4 and an unknown karyotype at diagnosis. The OCI-AML3 cell line exhibits the distinctive features of NPMc AML, since it carries NPM1 mutation A and expresses cytoplasmic NPM.116

NPM1 mutations are consistently heterozygous and a wild-type allele is retained. With very few exceptions, i.e. involvement of exon 11118 and exon 9,119 NPM1 mutations are restricted to exon 12.13 About 40 molecular variants of NPM1 mutations have been described to date in AML patients,13 with >95% occurring at nucleotide position 960. The most common mutation (so-called mutation A12) duplicates a TCTG tetranucleotide at positions 956 to 959 of the reference sequence (GenBank accession number NM_002520), and accounts for 75%–80% of adult NPMc AML cases. Mutations B and D are observed in about 10% and 5% of cases, respectively; other mutations are very rare. Independently of their type, all mutation variants generate common alterations at the C-terminus of the NPM leukemic mutants which are responsible for their dislocation into cytoplasm (see below). NPM1 mutations tend to be stable.120,121 Loss of mutations at relapse is extremely rare and sometimes associated with a change from a normal to an abnormal karyotype.120,121

Cytoplasmic/mutated NPM closely associates with AML-NK, and it is mutually exclusive with major recurrent genetic abnormalities.120,122–127 About 86% of patients with NPMc AML have a normal karyotype: the other 14% show chromosomal abnormalities that are likely to be secondary.12,13 The interaction of NPM1 mutations with other genetic alterations in AML-NK is interesting. FLT3-ITD targets NPMc AML twice as often as AML-NK carrying nuclear/unmutated NPM.12,120,124–127 NPM1 mutations are likely primary events, preceding the acquisition of FLT3-ITD127 or other mutations. In contrast, partial tandem duplications within the MLL gene (MLL-PTD) and cytoplasmic/mutated NPM are usually mutually exclusive.12,124,127 In two large studies, CEBPA, KIT and NRAS mutations did not appear to differ between NPM1-mutated and NPM1-unmutated AML-NK.124,125 Mutations of p53 are rare in NPMc AML (about 3% of cases),120 concurring with their being mainly found in AML with karyotype abnormalities.128,129

Properties of NPM leukemic mutant proteins

One of the most distinctive features of NPM mutants is their aberrant localization in the cytoplasm of leukemic cells12 (Figure 2B). This is causally related to two alterations at the leukemic mutant C-terminus: (i) generation of an additional leucine-rich NES motif;130 and (ii) loss of tryptophan residues 288 and 290 (or residue 290 alone) which are crucial for NPM nucleolar localization.28 We recently demonstrated that both alterations are needed to ensure NPM cytoplasmic dislocation131 (Figure 2B). Observing that rare NPM mutants, despite retaining tryptophan 288, still delocalize in cytoplasm, led us to discover a close correlation between the type of NES motif and C-terminal mutated tryptophans.131 Mutations of both tryptophans are always found with the very common NES motif, L-xxx-V-xx-V-x-L; retention of tryptophan 288 is always associated with rare NES variants in which valine at the second position is replaced by leucine, phenylalanine, cysteine or methionine.131 One explanation might be that, in order to counterbalance tryptophan 288, which drives mutants to the nucleolus,28 the rare variant NES motifs need to be more efficient than the L-xxx-V-xx-V-x-L sequence in binding Crm1 and dislocating NPM to the cytoplasm.

All NPM leukemic mutants interfere with wild-type NPM and oncosuppressor ARF functions by binding and recruiting them into the nucleoplasm and cytoplasm.131–133

Diagnosis of NPMc+ AML

NPMc AML is easily diagnosed by molecular biology techniques134–137 or immunohistochemistry, since detection of aberrant cytoplasmic NPM expression is predictive of NPM1 mutations.112 Immunohistochemistry is the first choice technique for diagnosing NPMc AML in the case of a dry-tap or biopsy from extramedullary sites,138 and for the analysis of hematopoietic lineage involvement.139 Immunohistochemistry must be applied on paraffin sections since fresh material (smears or cytospins) is not suitable for reliably detecting cytoplasmic dislocation of NPM. Molecular analysis is the first choice assay for monitoring minimal residual disease.140

Three different types of antibodies are available for the immunohistochemical detection of NPM in tissue sections. Monoclonal antibodies directed against fixative-resistant epitopes of NPM12,111,131 do not distinguish between mutated and wild-type NPM and stain NPMc AML cells in the nucleus (mostly containing wild-type NPM) and in the cytoplasm (Figure 4A) which contains the NPM mutant protein and wild-type NPM (recruited by the mutant, as shown in Figure 2B). Despite lack of specificity for NPM mutants, these antibodies are the most widely used, reliable reagents for the immunohistochemical diagnosis of NPMc AML.112 Polyclonal antibodies recognizing NPM mutants but not wild-type NPM116,131,139 stain only the cytoplasm of NPMc AML cells, indicating that mutants are mostly cytoplasmic (Figure 4, middle). Due to their high specificity for NPM mutants, these antibodies are useful when studying lineage involvement in NPMc AML,139 but because of antigenic denaturation, are suitable for diagnostic purposes in only 50–60% of samples.139 Future efforts should be devoted to generating antibodies against NPM mutants that can be more widely applied. Finally, a monoclonal antibody recognizing wild-type NPM but not NPM mutants131,139 stains NPMc AML cells in the nucleus and cytoplasm (not shown), providing evidence that wild-type NPM is dislocated into the cytoplasm. This antibody is of little diagnostic relevance. The best control for assessing specificity of NPM cytoplasmic positivity is immunostaining with an antibody against C23/nucleolin, another abundant shuttling nucleolar protein,44 which in NPMc AML must have nucleus-restricted localization12 (Figure 4C).

Figure 4.Aberrant cytoplasmic expression of NPM in NPMc⁺ AML. (Top) Nuclear plus cytoplasmic expression of NPM in leukemic blasts (arrows). Normal residual hematopoietic cells show nucleus-restricted NPM (arrowhead). Bone marrow biopsy paraffin section stained with monoclonal antibody 376 that recognizes wild-type and mutated NPM (APAAP technique; hematoxylin counter-staining; x 1,000). (Middle) Cytoplasmic-restricted expression of mutant NPM protein in NPMc⁺ AML (arrow). Bone marrow biopsy paraffin section stained with a polyclonal antibody (Sil-A) that recognizes mutated but not wild-type NPM (APAAP technique; hematoxylin counterstaining; ×1,000) (Bottom) Nucleus-restricted expression of C23/nucleolin in NPMc⁺ AML (arrows). Double arrows indicated the expected NPM cytoplasmic positivity in a mitotic figure. Bone marrow biopsy paraffin section stained with a specific anti-C23 monoclonal antibody (APAAP technique; hematoxylin counterstaining; ×1,000).

Since cytoplasmic NPM is fully predicitve of NPM1 mutations,112 immunohistochemistry could be used as a first-line screening to rationalize cytogenetic and molecular studies in AML. A potential approach is shown in Figure 5.

Figure 5.NPM-immunostaining-based approach to molecular and cytogenetic analysis of AML. AML patients can be subdivided by the results of immunohistochemistry on paraffin sections into two subgroups, i.e. those expressing nucleus-restricted NPM (NPMc⁻) (left) or cytoplasmic NPM (NPMc⁺) (right). The arrow in the NPMc⁻sample indicates the expected cytoplasmic positivity of a mitotic figure. The arrows in the NPMc⁺ sample indicate leukemic cells with aberrant cytoplasmic expression of NPM. NPM mutational analysis can be avoided in NPMc⁻AML but should be performed in NPMc⁺ cases, since molecular information about NPM1 status in NPMc⁺ AML can serve as a prognostic factor (in association with FLT3-ITD) and is critical for monitoring minimal residual disease. NK: normal karyotype; AK: abnormal karyotype (other than recurrent cytogenetic abnormalities); MRD: minimal residual disease.

Pathological, phenotypic and genotypic features of NPMc+ AML

Although associated with a wide morphological spectrum, NPMc AML is mostly of M4 and M5 categories.12,124,125 Interestingly, up to 90% of AML-M5b carry cytoplasmic/mutated NPM;12,124 gene expression profiling of AML41 also revealed a cluster dominated by monocytic leukemias and a high frequency of NPM1 mutations. A high frequency of NPM1 mutations has been reported in AML with prominent nuclear invaginations (cup-like nuclei).142 NPMc AML shows frequent multilineage involvement.139 At immunohistochemistry, over 95% of NPMc AML cases are negative for CD34,12 as was subsequently confirmed by gene expression profiling143 which also showed CD133/PROM1 gene down-regulation. Another striking feature of the NPMc AML gene-expression signature is activation of numerous members of the homeodomain-containing family of transcription factors, including HOX and TALE genes,126,143 some of which are oncogenes implicated in hematopoietic development and leukemogenesis.144,145 Interestingly, NPMc AML cases with a normal karyotype and those carrying minor chromosomal abnormalities show the same molecular signature,143 providing evidence that NPMc AML is a distinct subgroup of leukemia regardless of karyotype, and that the minor chromosomal abnormalities which occasionally accompany NPM1 mutations are secondary events (see above).

Clinical features of NPMc+ AML and response to therapy

The incidence of NPM1 mutations increases with age,12,120,124,127 accounting for 2.1%–6.5% of childhood AML121,122,146 (9%–26.9% of pediatric AML-NK)121,122,146 but 25%–35%120,121,123–127 of adult AML (45.7%–63.8% of adult AML-NK).120,121,123–127 These findings suggest that the molecular pathogenesis of AML-NK may be different in adults and children. Notably, there is also evidence for a variability of NPM1 mutation types according to patient age, with uncommon non-typical (i.e. non-type A) mutations being most prevalent in children and younger adults.147 Three large clinical studies124,125,127 found that NPM1 mutations are significantly more frequent in females.

Cases of NPMc AML-NK are associated with higher blast counts and more extramedullary involvement (mainly gingival hyperplasia and lymphadenopathy) than cases of AML-NK without NPM1 mutations.125,127 Platelet counts are significantly higher in NPMc than in NPMc AML-NK.125,127 Concurring with this clinical observation is the enhanced ability of K562 cells for megakaryocytic differentiation after transfection with a C-terminal NPM mutant.38 Most investigators have found that cytoplasmic/mutated NPM predicts a good response to induction therapy.12,120,124,127 In contrast, Döhner et al.125 observed the best complete remission rate in the NPM1-mutated/FLT3-ITD negative group, whilst AML patients carrying both mutations showed poor responses.

NPM1 mutations without concomitant FLT3-ITD identify a subgroup of AML-NK patients with a favorable prognosis124–127,148 and has been associated with an approximately 60% probability of survival at 5 years in younger patients.125 In a donor versus no-donor comparison, the good prognostic group of NPM1-mutated/FLT3 ITD–negative patients did not benefit from allogeneic stem cell transplantation,125 suggesting that such treatment should not be recommended for NPMc AML in first complete remission. These results underline the value of molecular genetic screening in improving risk stratification of patients with AML-NK.149 Screening is facilitated by recently developed assays that simultaneously detect NPM1, FLT3-ITD and CEBPA mutations,150,151 all of which have a prognostic impact in AML-NK.124–127,152

Unsolved issues

Several questions remain unanswered on the role of NPM in the development of hematopoietic malignancies and on the clinical significance of NPM1 gene alterations. Although the ALK, RARα and MLF1 moieties are believed to dominate in promoting tumorigenesis in lymphomas and leukemias carrying NPM fusion proteins, there is evidence that NPM moieties are not only innocent bystanders. This view is supported by the finding that leukemias developing in transgenic mice expressing either PML/RARα, PLZF/RARα, or NPM/RARα show different phenotypes.93 Since NPM behaves as an oncosuppressor,4 a reduction in wild-type NPM may also contribute to tumorigenesis, due to ineffective ARF stabilization with consequent inactivation of the ARF growth-inhibitory function. However, dose reduction of NPM at the nucleolar level may differ among tumor types, e.g. lower levels in AML carrying t(3;5) in which one allele is altered and the wild-type NPM is dislocated into the cytoplasm98 than in ALCL with t(2;5), in which one allele is altered but the location of wild-type NPM is apparently unaffected by the translocation.87

The molecular mechanism underlying the development of NPMc AML is unclear. The ability of NPM leukemic mutants to recruit the oncosuppressor p19 in the cytoplasm and interfere with its stability and activities132,133 may contribute to the development of AML. However, p19 is only one potential target of NPM mutants. In NPMc AML, the perturbed cellular traffic of the wild-type and mutant NPM proteins could influence the nuclear/nucleolar import and functions of other, as yet unidentified, critical molecules.153–155 The close associations of FLT3-ITD with NPM1 mutations in clinical practice12,124–127 suggests that these genetic lesions may co-operate to promote leukemic transformation. There is also experimental evidence that the NPM leukemic mutants (but not wild-type NPM) co-operate with adenovirus E1A to transform primary MEF in soft agar.156 NPMc/E1A MEF cells also show high efficiency at forming foci in low-density seeding assays, comparable to that of RasV12/E1A MEF cells.156

Interestingly, the gene expression profile of NPMc AML is characterized by up-regulation of genes involved in stem cell maintenance (such as HOX genes).126,143 NPMc AML also shows Notch1-ligand JAG1 up-regulation and CDKN2C/p18-INK4C repression,143 both of which are associated with hematopoietic stem cell expansion.157,158 Accordingly, NPM leukemic mutants may confer self-renewal properties on hematopoietic targets.

Studies on NPM abnormalities in leukemias may lead to updating of the World Health Organization (WHO) classification of AML.159 The term primary AML not otherwise characterized,160 which is defined according to slightly modified FAB criteria, encompasses about 60% of AML cases, including all AML-NK and, therefore, NPMc AML. We propose that, because of its distinctive genetic, biological and clinical features, NPMc AML and NPMc myeloid sarcoma161 should be included as a separate AML entity in the upcoming version of WHO classification.

Multicenter studies are warranted to assess the favorable prognostic value of NPM1 mutations (in the absence of FLT3-ITD) in various clinical settings and to provide answers to a series of questions. Does the prognostic value of mutated NPM1 also apply to the poor prognostic category of AML patients over 60 years old? Should NPM1-mutated/FLT3-ITD negative AML-NK patients be exempted from allogeneic stem cell transplantation as first-line therapy?162,163 Is the NPM1 mutation a better molecular marker than FLT3-ITD164 or Wilms’ tumor gene (WTI)165 for monitoring minimal residual disease in AML-NK? Will quantification of NPM1-mutated copies140 predict early relapse and patients with long-term survival? Recent retrospective studies indicate that this may be the case,166 but large prospective clinical trials are required to confirm this exciting finding. Finally, better understanding of the mechanism underlying leukemogenesis in NPMc AML may eventually lead to the development of more specific anti-leukemic therapies.

Acknowledgments

we are indebted to Laura Pasqualucci, Barbara Bigerna, Alessandra Pucciarini, Roberta, Pacini, Alessia Tabarrini, Manola Carini, Roberta Mannucci, Roberto Rosati, and Giovanni Roti for generating some of the data described in the paper.

We would also like to thank Claudia Tibido for her excellent secretarial assistance and Dr. Geraldine Anne Boyd for her help in editing this paper. We apologize to those whose papers could not be cited owing to space limitations.

Footnotes

• Authors’ Contributions BF had the original idea which, inspired by his immunohistochemical studies, led first to the identification of ALK⁺ anaplastic large cell lymphomas as a distinct clinicopathologic entity, and subsequently to the discovery of AML carrying cytoplasmic mutated NPM (NPMc⁺ AML); IN and NB clarified the molecular mechanism of altered NPM transport in NPMc⁺ AML; MPM, AL performed the biochemical analysis of NPMc⁺ AML; CM, PG: involved in the study of NPM1 mutations; FM, MFM: carried out the clinical analysis of NPMc⁺ AML patients.
• Conflict of Interest BF and CM applied for a patent on clinical use of NPM mutants. All other authors declare no conflict of interest.
• Funding: supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) and the Fondazione Monte dei Paschi di Siena, Perugia. NB is supported by the Federazione Italiana per la Ricerca sul Cancro (FIRC).

• Received November 7, 2006.
• Accepted February 20, 2007.