Nucleophosmin (NPM) is a nucleolar phosphoprotein that plays multiple roles in ribosome assembly and transport, cytoplasmic–nuclear trafficking, centrosome duplication and regulation of p53. In hematological malignancies, the NPM1 gene is frequently involved in chromosomal translocation, mutation and deletion. The NPM1 gene on 5q35 is translocated with the anaplastic lymphoma kinase (ALK) gene in anaplastic large cell lymphoma with t(2;5). The MLF1 and RARA genes are fused with NPM1 in myelodysplastic syndrome and acute myeloid leukemia (AML) with t(3;5) and acute promyelocytic leukemia with t(5;17), respectively. In each fused protein, the N-terminal NPM portion is associated with oligomerization of a partner protein leading to altered signal transduction or transcription. Recently, mutations of exon 12 have been found in a significant proportion of de novo AML, especially in those with a normal karyotype. Mutant NPM is localized aberrantly in the cytoplasm, but the molecular mechanisms for leukemia remain to be studied. Studies of knock-out mice have revealed new aspects regarding NPM1 as a tumor-suppressor gene. This review focuses on the clinical significance of the NPM1 gene in hematological malignancies and newly discovered roles of NPM associated with oncogenesis. (Cancer Sci 2006; 97: 963–969)
Nucleophosmin (NPM), also called B23, numatrin or NO38, was isolated as an abundant nucleolar phosphoprotein, whose expression level is increased significantly by several kinds of stimulation and transformation.(1,2) In 1989, a human NPM cDNA encoding a 294-amino-acid protein was cloned.(3) In 2002, a shorter isoform encoding a 259-amino acid protein that differs at the C-terminus was also isolated.(4) The NPM1 gene spans 25 kb, contains 12 exons and maps to chromosome 5q35.(4,5) Exon 8 is frequently skipped, and exon 10 is used only for the short isoform. Although the biological significance of the short isoform remains unclear, its expression is increased in radiation-insensitive cell lines and the product is localized in the cytoplasm as well as in the nucleus.(4,6) The structural features of NPM consist of an oligomerization domain, a metal-binding motif, a bipartite nuclear localization signal, two Asp/Glu-rich domains, phosphorylation sites for CDK2 and a nucleor localization signal.(6,7)
NPM1 has been recognized by oncologists as a partner gene for various chromosomal translocations: NPM–anaplastic lymphoma kinase (ALK) in anaplastic large cell lymphoma (ALCL) with t(3;5), NPM–RARA in acute promyelocytic leukemia (APL) with t(5;17), and NPM–MLF1 in acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) with t(3;5).(8–10) (Fig. 1, 2) In each chimeric gene product, the N-terminal NPM portion is thought to act as the interface for oligomerization and oncogenic conversion of the C-terminal functional domain such as a kinase or transcription factor. Recently, NPM was associated with centrosome duplication and the regulation of p53,(11,12) and might have a role as a tumor suppressor.
Expression and function of NPM
Nucleophosmin is an abundant and ubiquitously expressed phosphoprotein. It is located mainly in the nucleolus and shuttles between the nucleus and cytoplasm.(13,14) NPM has been proposed to be associated with the synthesis and processing of ribosomal RNA (rRNA), regulation of chromatin structure and transport of rRNA and ribosomal proteins.(15,16) However, a recent study using knock-out mice suggests that NPM is not indispensable for these biological processes, as shown below.
The NPM level is increased in proliferating cells as well as tumor cells, perhaps due to an increased requirement for ribosomal synthesis. Overexpression or downregulation of NPM reportedly alters the cellular status with respect to proliferation, differentiation and apoptosis, although some contradictory results have been reported.(17,18) NPM function appears to be different depending on whether or not wild-type p53 is present.(12,19)
It has been reported that NPM has an important role in the cell cycle. NPM interacts with the centrosome and protects it from duplication in G1 phase.(20) It is expressed highly during S and G2 phases and is duplicated concomitantly with the initiation of DNA synthesis. Okuda et al. reported that the temporal activation of cyclin-dependent kinase (CDK)/cyclin E, which is associated with DNA replication–initiation factors, occurs simultaneously with phosphorylation of NPM, which occurs initially in centrosome duplication.(21,22) Phosphorylated NPM leaves the centrosomes during their duplication. Dissociation of NPM from the centrosome allows the centrosome duplication process during S phase. NPM was reported to reassociate with the centrosome in mitosis. Cha et al. reported that CDK1/cyclin B, a key regulator of M phase, phosphorylates another site of NPM and allows NPM to target to the centrosome during mitosis.(23)
Regarding the anti-oncogenic role of NPM, it is notable that NPM binds to p53 and its associated proteins. NPM has been implicated in the acute response of mammalian cells to various DNA-damaging stresses, such as radiation and ultraviolet (UV) light.(24) The stability of p53 is regulated primarily by MDM2, which is a ubiquitin E3 ligase.(25) MDM2 is regulated negatively by Arf, which binds to MDM2 and promotes its rapid degradation. NPM has been reported to form a molecular complex consisting of p53, MDM2 and Arf.(26,27)
Colombo et al. reported that NPM interacts directly with p53, increases its stability and activates the transcriptional function of p53 after DNA-damaging stress, and induces p53-dependent premature senescence in mouse embryonic fibroblasts.(12) Kurki et al. demonstrated that NPM activated by UV or viral stress is redistributed to the nucleoplasm, binds to MDM2, and leads to the stabilization of p53.(19) However, although Arf is a nucleolar protein that binds and inactivates MDM2 in the nucleoplasm, Korgaonkar et al. showed that Arf functions primarily outside the nucleolus, and it is sequestered and held inactive in the compartment by NPM.(28) Most likely, NPM inhibits Arf's p53-dependent activity by targeting it to nucleoli and impairing ARF–MDM2 association.
In accordance with these findings, knock-out (KO) mice of the NPM1 gene show mid-stage embryonic lethality due to the accumulation of DNA damage, activation of p53, and widespread apoptosis.(29,30) Double KO mice of TP53 and NPM1 rescue apoptosis in vivo and fibroblast proliferation in vitro.(29) In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. It has been suggested that NPM regulates DNA integrity through Arf–MDM2–p53.
Conversely, it was also shown that Arf inhibits the production of rRNA, retarding the processing of 47/45S and 32S precursors.(31) Itahana et al. further reported that Arf promotes polyubiquitination and degradation of NPM.(32) Accordingly, there is a molecular association between NPM and Arf, which implies a new role for the nucleolus in oncogenesis.
NPM–ALK chimeric kinase in ALCL
Anaplastic large cell lymphoma is a T-cell lymphoma that is characterized by abundant cytoplasm, pleomorphic nuclei and CD30 expression.(33) ALCL accounts for approximately 3% of adult non-Hodgkin's lymphoma and 10–30% of childhood lymphoma. Cytogenetically, the 2p23 locus is translocated frequently with 5q35, and less frequently with 1p25, 3q21 or 2q35.(34) In 1994, Morris et al. showed that the t(2;5) translocation fused the NPM1 gene on 5q35 to a previously unidentified protein tyrosine kinase gene, ALK, on 2p23.(35)
In the NPM–ALK chimeric kinase, the N-terminus of NPM (amino acids 1–117) is fused to the catalytic domain of ALK (amino acids 1058–1620). Because its expression is regulated by the NPM1 promoter, the fusion protein is expressed ectopically in lymphoid tissue. Wild-type ALK is a receptor tyrosine kinase that is expressed in the brain, spinal cord, small intestine and testis, but not in lymphoid cells. ALK belongs to the insulin receptor subfamily, although the natural ligand remains unclear.(36) In NPM–ALK, the N-terminal NPM domain is associated with oligomerization (Fig. 3) and causes constitutive activation of the ALK kinase. This mechanism is similar to that of BCR–ABL. NPM–ALK can associate with several adaptor proteins such as SHC, Grb2 and IRS-1, and is associated with the activated RAS–MAPK, PLCg, PI3K–Akt and JAK–STAT pathways.(34,37–39) Experiments using Ba/F3 cells revealed that NPM–ALK abrogates the interleukin-3 dependency of the cells and mediates signals for proliferation and survival.
Because NPM–ALK+ lymphomas express CD30, a transmembrane receptor for CD30L, it has been asked whether CD30 and NPM–ALK are connected functionally.(40) In Hodgkin's lymphoma, overexpressed CD30 molecules aggregate on the cell surface ligand-independently, and form a TRAF–IKK–IKBα complex, which leads to nuclear factor kappa-B (NF-κB) activation.(41) In contrast, Horie et al. clarified that NPM–ALK disrupts CD30 signaling and constitutive NF-κB activation in ALCL.(42) TRAF2/5 was shown to be bound with NPM–ALK on both the kinase domain of ALK and the N-terminal domain of NPM. In the same study, wild-type NPM was shown to be tyrosine-phosphorylated by NPM–ALK, although the significance remains unclear.
Studies of subcellular localization of NPM and its chimeric proteins reveal another aspect of the oncogenic role of NPM.(43) As shown in Fig. 1, NPM–ALK consists structurally of the oligomerization and metal-binding domains of NPM together with an almost full-sized intracytoplasmic domain of ALK. It lacks the two nuclear localization domains and Asp/Glu-rich domains of NPM. Accordingly, NPM–ALK is thought to be localized to the cytoplasm instead of nucleoli. Immunohistochemistry using anti-ALK stains both the nucleus and cytoplasm of ALCL cells(36,43,44) (Fig. 4). Anti-C-terminal NPM antibody is able to discriminate wild-type NPM from the fused protein. Interestingly, wild-type NPM is localized to the nucleus but not the cytoplasm, whereas NPM–ALK is distributed in both.
There are many in vivo studies on the oncogenic role of NPM–ALK.(39) Mice transplanted with bone marrow cells infected with retrovirus containing human NPM1–ALK cDNA developed B-cell lymphoma, not ALCL. However, in transgenic mice carrying NPM1–ALK cDNA under the control of the murine CD4 promoter or Moloney murine leukemia virus Long Terminal Repeat (LTR), T-cell tumors resembling human ALCL developed, suggesting that the phenotype depends on the promoter.
NPM–RARα chimeric transcription factor in APL
Acute promyelocytic leukemia is characterized by myeloid malignancies with a maturational block at the promyelocytic stage.(33) Treatment with all-trans retinoic acid (ATRA) overcomes this maturation arrest and induces differentiation of APL blasts.(45) APL is usually accompanied by t(15;17), which forms the PML–RARA fusion gene.(46) In addition, molecular variants of APL have been described in which RARA is fused to one of four other genes: PLZF, NUMA, STAT5b or NPM1.(46,47) Common features of all of these proteins are that the B through F regions of RARα, which contain its DNA and ligand-binding domains, have N-terminal non-RARα moieties that add dimerization ability to each fusion protein. Homodimerization or heterodimerization is thought to be associated with the repression of retinoid-responsive transcription and perhaps with other alterations in transcription.
The first APL case with a t(5;17) translocation was described in 1994 and the sequence of chromosomal joint was cloned in 1996.(48,49) This variant type of APL was clinically sensitive to ATRA, and the sensitivity was later confirmed in vitro.(50) The N-terminal NPM portion fuses with RARα in a similar manner to other APL fusion products. Like other APL fusion products, NPM–RARα forms a heterodimer with RXR or NPM. These dimers recruit corepressor complexes and repress retinoid-responsive transcription in a dominant-negative manner.(51) Homodimer formation of RARα may be a crucial event in APL pathogenesis in vivo.(52) In transgenic mice, NPM–RARα enhanced the proliferation of myeloid cells, mimicking myeloproliferative disease and, later, the transgenic mice developed an APL-like disease with blasts that were sensitive to ATRA.(53)
In the case of PML–RARα, immunostaining with anti-PML antibody revealed a microgranular pattern, which differs from the microspeckled one observed in the case of wild-type PML.(54) ATRA treatment restored the aberrant localization to a speckled pattern through degradation of PML–RARα.(55,56) This finding implies that NPM–RARα also acts in a dominant-negative manner relative to wild-type PML, and this effect is cancelled by ATRA. Anti-NPM (N-terminus) antibody stained the nucleus with a diffuse microgranular pattern, whereas PML localization was normal.(57) It is interesting but remains to be studied how NPM–RARα affects the function of wild-type NPM.
NPM–MLF1 chimeric protein in AML/MDS
Yoneda-Kato et al. reported that the t(3,5) (q25.1;q34) translocation associated with AML/MDS (most frequently with the M6 French–American–British [FAB]-type) produces a 5′-NPM-coding sequence fused in-frame to a new gene, which they named MLF1.(10) The MLF1 gene encodes a 268-amino-acid polypeptide that has no homology to any previously characterized protein and does not have known functional motifs. Expression of MLF1 mRNA is observed in testis, ovary, skeletal muscle, heart, kidney and colon, but not in normal hematopoietic cells.
The function of MLF1 protein remains unclear, but it inhibits erythropoietin-induced differentiation, cell-cycle exit and p27KIP1 accumulation.(58) Hanissian et al. identified an MLF1-interacting protein (MLF1IP) that associates specifically with MLF1. MLF1IP has nuclear localization signals, two nuclear receptor-binding motifs (LXXLL), two leucine zippers, two polypeptide enriched in proline, glutamine, serine and threonine (PEST) residues and several potential phosphorylation sites.(59) MLF1IP seems to have an important role in erythroid differentiation, and MLF1 seems to regulate MLF1IP function negatively.
Immunostaining studies have shown that MLF1 is localized mainly in the cytoplasm, whereas the NPM–MLF1 fusion protein is localized in the nucleus, especially the nucleolus(60) (Fig. 4). NPM is a nuclear protein and therefore the N-terminal portion that is fused to MLF1 may carry the nuclear targeting signal. Thus, both the ectopic expression and aberrant subcellular localization of MLF1 seem to be associated with the interruption of erythroid differentiation, which may be the reason that MPL–MLF1 is found entirely in M6 FAB-type AML/MDS.
Frameshift mutation of NPM1 in AML
During an extensive study of the subcellular localization of NPM, Falini et al. found a correlation between the presence of cytoplasmic NPM and clinical and biological features in AML samples.(61) Cytoplasmic NPM was detected in 35.2% of 591 bone marrow specimens from patients with primary AML but not in 135 secondary AML specimens or in 980 hematopoietic or non-hematopoietic neoplasms other than AML. It was associated with a wide spectrum of morphological subtypes of the disease from M0 to M7 except M3. A normal karyotype and responsiveness to induction chemotherapy were also related to cytoplasmic NPM. There was a high frequency of internal tandem duplications of FLT3 (FLT3/ITD) and lack of CD34 and CD133 expression. AML with cytoplasmic NPM carried novel mutations in exon 12 of the NPM1 gene. In the most common mutation, called type A, a 4-bp nucleotide (TCTG) is inserted at the position encoding the 288th amino acid residue, causing a frameshift of the downstream coding sequence (Table 1). As a result, the C-terminal amino acid residues, 286DLWQWRKSL-COOH, are changed to 286DLCLAVEEVSLRK-COOH. So far, a total of 29 variant sequence mutations have been reported in the NPM1 genes of 1557 patients. All of the NPM mutant proteins lose at least one of W288 and W290, and share the same last five amino acid residues (VSLRK). Thus, despite the genetic heterogeneity, all of these NPM1 gene mutations have the common feature of a frameshift mutation at the C-terminal region. Nakagawa et al. noticed that this frameshift not only loses the nucleolar localization signal (WXW) but also gains a nuclear export signal (NES) consisting of LXXXVXXVXL.(62) Falini et al. further confirmed that both alterations are crucial for NPM mutant export from the nucleus to the cytoplasm.(63)
Table 1. Representative mutations of NPM1 in AML
The four inserted nucleotides are underlined. Stop codon is in bold. The nucleolar localization signal WXW in the wild-type sequence is substituted by LXXXVXXVXL, which corresponds to a nuclear export signal, shown in the predicted amino-acid column.
Wild type (NPM1.1)
GAT CTC TGG CAG TGG AGG AAG TCT CTT TAA GAAAATAG
GAT CTC TGT CTG GCA GTG GAG GAA GTC TCT TTA AGA AAA TAG
GAT CTC TGC ATG GCA GTG GAG GAA GTC TCT TTA AGA AAA TAG
GAT CTC TGC CTG GCA GTG GAG GAA GTC TCT TTA AGA AAA TAG
Falini et al. found that cytoplasmic staining of NPM could define the NPM1 mutation-positive AML cases that had a normal karyotype, NPM1 gene mutations, and responsiveness to induction chemotherapy.(61) Grisendi and Pandolfi noted that NPM staining in cases of AML with aberrant cytoplasmic localization of the protein is mostly cytoplasmic, which suggests that the mutant NPM acts predominantly on the product of the remaining wild-type allele, causing its retention in the cytoplasm by heterodimerization(64) (Fig. 4).
Based on the above report, Suzuki et al. identified similar NPM1 mutations, including four novel sequence variants, in 64 of 257 (24.9%) Japanese patients with de novo AML.(65) NPM1 mutations were associated with normal karyotypes and with FLT3 mutations, but not with other mutations. In 190 patients without the M3 FAB subtype who were treated using the protocol of the Japan Adult Leukemia Study Group, multivariate analyses showed that the NPM1 mutation was a favorable factor for achieving complete remission but was associated with a high relapse rate. Importantly, sequential analysis using 39 paired samples obtained at diagnosis and relapse showed that NPM1 mutations were lost at relapse in two of the 17 patients who had NPM1 mutations at diagnosis. The loss of NPM1 mutation at relapse suggests that it is not necessarily needed for maintenance of the disease.
Schnittger et al. screened 401 AML patients with normal karyotypes treated using the German AML Cooperative Group Protocol 99 for NPM1 mutations.(66) NPM1 mutations were detected in 212 (52.9%) of the 401 patients. Fourteen mutations, including eight new variants, were identified. NPM1-mutated cases were frequently associated with FLT3 mutations but rarely associated with MLL tandem duplication, NRAS, KIT and CEBPA mutations. The NPM1-mutated group had a higher complete remission (CR) rate, a tendency of longer overall survival (OS), and significantly longer event-free survival (EFS).
Roel et al. examined NPM1 mutation status in a cohort of 275 patients with AML by denaturing high-performance liquid chromatography.(67) NPM1 mutations are less frequent in younger patients than in those aged over 35 years. NPM1 mutations are positively correlated with AML with high white blood cell counts, normal karyotypes, and FLT3/ITD. NPM1 mutations are correlated inversely with the occurrence of CEBPA and NRAS mutations. AML patients with NPM1 mutations have a significantly better OS and EFS than those without NPM1 mutations. Finally, in multivariate analysis, NPM1 mutations have an independent favorable prognostic value with regard to OS, EFS and DFS.
In 300 patients entered into the AML Study Group trials, NPM1 mutations were identified in 48% of the patients, including 12 novel sequence variants, all leading to a frameshift in the C-terminus of NPM.(68) AML patients with NPM1 mutations in the absence of FLT3/ITD define a distinct molecular and prognostic subclass of young adult AML patients with normal cytogenetics.
In a larger study, the clinical significance of NPM1 mutation was further suggested. One thousand four hundred and eighty-five patients with AML were examined for NPM1 exon 12 mutations using fragment analysis.(69) A 4-bp insert was detected in 408/1485 patients (27.5%). Sequence analysis revealed known mutations (type A, B and D) as well as 13 novel alterations in 229 of the cases analyzed. NPM1 mutations were more prevalent in patients with normal karyotype (324/709; 45.7%) than in those with karyotype abnormalities (58/686, 8.5%; P < 0.0001), and were significantly associated with several clinical parameters (high bone marrow blasts, high white blood cell and platelet counts, women). NPM1 alterations were associated with FLT3/ITD mutations, even if the cases analyzed were restricted to patients with normal karyotype. Analysis of the clinical impact in four groups (NPM1 and FLT3/ITD single mutants, double mutants, and wild-type for both) revealed that patients having only an NPM1 mutation had significantly better overall and disease-free survival and a lower cumulative incidence of relapse. In conclusion, NPM1-mutations represent a common genetic abnormality in adult AML. If not associated with FLT3/ITD mutations, mutant NPM1 appears to identify patients with favorable response to treatment.
In summary, the NPM1 mutation(61,65–70) is observed in a high percentage of de novo AML, and is associated with normal karyotype, FLT3/ITD and better response to chemotherapy (Table 2). It remains controversial whether the presence of NPM1 mutation indicates a good prognosis, but it is clear that it indicates a good prognosis in patients without FLT3/ITD. Structurally, a newly generated NES sequence at the C-terminus is a common feature, although more than 20 different variant NPM1 mutations have been identified.(63) How the acquired NES changes the function of NPM in addition to altering the subcellular localization should be studied. The second question is why only FLT3 mutations frequently accompany the NPM1 mutation. According to a Japanese study, D835 of FLT3 is also frequently mutated in NPM1-mutated AML. One possibility is that mutated NPM may sequester some transcription factors associated with differentiation. Activated FLT3 might tyrosine-phosphorylate cytoplasmic NPM, which has been observed in NPM–ALK. Another possibility is that mutated NPM lowers the replication or repair fidelity of DNA. Umekawa et al. reported that the C-terminal sequence of NPM is important for its elevated DNA polymerase alpha activity compared with the short isoform NPM1.2.(71) As described below, NPM1 haploinsufficiency increases the number of centrosomes to more than two and causes karyotype abnormalities in mice.(30) However, NPM1 mutations are observed exclusively in AML with normal karyotype, and FLT3 is the sole target for mutation. Recently Colombo et al. showed that mutant NPM forms a direct complex with Arf but is unable to protect it from degradation.(29) AML cells and cell lines harboring mutant NPM have low levels of cytoplasmic Arf. Colombo et al. suggested that inactivation of Arf, a key regulator of the p53-dependent cellular response to oncogene expression, might contribute to leukemogenesis in AML with mutated NPM. Further study is needed to clarify the underlying molecular mechanisms.
Table 2. Clinical relevance of NPM1 mutation in acute myeloid leukemia
Total patients (%) (normal karyotype)
Nucleophosmin mt (%) (normal ka ryotype)
CR, complete remission; DFS, disease-free survival; EFS, event-free survival; F, favorable; NS, not significant; OS, overall survival; RFS, relapse-free survival; U, unfavorable; NA, not analyzed.
Haploinsufficiency and gene deletion in hematological malignancies
To study the function of NPM in vivo, Grisendi et al. generated NPM1 heterozygous-null, hypomorphic-mutant and homozygous-null mice.(30) They observed that NPM1 homozygous-null and hypomorphic mutants had aberrant organogenesis and died between embryonic days 11.5 and 16.5 owing to severe anemia resulting from defects in primitive hematopoiesis. They showed that NPM1 inactivation leads to unrestricted centrosome duplication and genomic instability. Notably, NPM1 heterozygous mice developed a hematological syndrome with features of human MDS. They concluded that their data uncovered an essential developmental role for NPM and implicated its functional loss in tumorigenesis and MDS pathogenesis.
According to the KO mice study, mutation or downregulation of NPM1 as a tumor-suppressor gene may also be associated with human MDS. The evidence that NPM1 is located on 5q35, which is deleted or rearranged in AML/MDS, suggests the loss of the NPM1 gene in myeloid malignancies. Using fluorescence in situ hybridization and reverse transcription–polymerase chain reaction methods, Berger et al. analyzed eight AML/MDS cases with chromosomal breakpoints at 5q31–5q34.(72) Two bacterial artificial chromosome signals spanning the NPM1 and MLF1 genes were colocalized in three of the eight cases. However, the breakpoints were outside the NPM1 gene in the remaining five cases, and one copy of the NPM1 gene was deleted in three of the five. Further study is needed to determine the frequency and significance of NPM1 deletion in the unbalanced translocation and chromosomal deletion.
Notably, the NPM1 gene has a CpG island in its promoter region, which is potentially hypermethylated. However, no study has been reported about the methylation of the NPM1 gene. The significance of promoter hypermethylation should also be investigated.
Conclusions and future directions
The NPM1 gene is one of the most frequent targets for deletion and insertion mutations in addition to acting as a component of fusion genes. In fusion genes, translocation of the NPM1 gene on the 5′ side is associated with ectopic expression, aberrant subcellular localization and oligomerization of the partner gene products. However, the biological significance of the NPM1 gene mutation at exon 12 remains unclear. It may be associated with a mutator phenotype, dysregulated transcription or apoptosis. One interesting issue is altered nuclear–cytoplasmic trafficking of mutant NPM. The export system of mutant NPM might be a candidate for targeted therapy for NPM-mutated AML. Clarification of the expression and subcellular localization of NPM-interacting proteins such as p53, MDM2 and Arf in NPM-mutated AML would help to further our understanding of the pathogenesis of this disease. It would also yield insights into the masked function of NPM in myeloid hematopoiesis. The interaction between FLT3 and NPM1 mutations is another important issue for future studies. The evidence that FLT3 and NPM1 mutations are observed in a variety of types of AML from M0 to M7 (except M3) raises the question of what determines the phenotype of AML from M0 to M7. Thus, one discovery generates 10 more questions.