Acute myeloid leukemia with mutated nucleophosmin ( NPM1) : is it a distinct entity?

Following the discovery of NPM1 -mutated AML in 2005 and its subsequent inclusion as a provisional entity in the 2008 World Health Organization (WHO) classification of myeloid neoplasms, several controversial issues remained to be clarified. It was unclear whether the NPM1 mutation was a primary genetic lesion and whether additional chromosomal aberrations and multilineage dysplasia had any impact on the biological and prognostic features of NPM1 -mutated AML. Moreover, it was uncertain how to classify AML patients that were double-mutated for NPM1 and CEBPA . Recent studies have shown that: i) the NPM1 mutant perturbs hemopoiesis in experimental models; ii) leukemic stem cells from NPM1 -mutated AML patients carry the mutation; and iii) the NPM1 mutation is usually mutually exclusive of biallelic CEPBA mutations. Moreover, the biological and clinical features of NPM1 -mutated AML do not seem to be significantly influenced by concomitant chromosomal aberrations or multilineage dysplasia. All together, these pieces of evidence point to NPM1 -mutated AML as a founder genetic event that defines a distinct leukemia entity accounting for about one-third of all AML. engraftment of the CD34-negative in NPM1 -mutated AML In no or limited engraftment observed in In Taussig et al. reported a more consistent engraftment of the CD34-negative leukemic cells in immunocompromised mice. These may reflect some degree of heterogeneity in the leukemic stem cell compartment of NPM1 -mutated AML. N.B. studied the mechanisms of transport of NPM1 mutant protein and the zebrafish model; P.S. described the transgenic mouse model; A.L. produced the specific antibody for NPM1 mutant protein and analyzed multilineage involvement in NPM1 -mutated AML; E.T. performed gene expression profiling studies and immunohistochemical analysis; T.H. contributed to the clinical studies on the role of aberrant karyotype and myelodisplasia-related changes in NPM1 -mutated AML. All the authors contributed to write the manuscript.


Introduction
The remarkable molecular heterogeneity of acute myeloid leukemia (AML) 1 has made a genetic-based classification essential for accurate diagnosis, prognostic stratification, monitoring minimal residual disease and developing targeted therapies. The category of "AML with recurrent genetic abnormalities", which includes the genetically best defined myeloid neoplasms, underwent major changes in the 2008 World Health Organization (WHO) classification 2 . The four molecularly distinct entities that had been described in the 2001 WHO classification were expanded to comprise AML with t(6;9), AML with inv(3) or t(3;3), AML (megakaryoblastic) with (1;22) and two provisional entities, i.e. AML with mutated CEBPA and AML with mutated nucleophosmin (NPM1) ( Table 1).
The latter accounts for about one-third of all AML 3 and has distinct genetic, pathological, immunophenotypic and clinical characteristics 4,5 . The WHO synonym for AML with mutated NPM1, that is NPMc+ AML (c+ stands for "cytoplasmic positive") 3 , focuses on its most distinguishing functional feature, i.e. aberrant expression of nucleophosmin in the cytoplasm of leukemic cells 6 . This unique immunohistochemical pattern, which led in 2005 to the discovery of NPM1 mutations in AML 3 , is an excellent surrogate marker for molecular studies since it is fully predictive of NPM1 mutations 7,8 .
The present review is an update of the distinct genetic and clinical features of AML with mutated NPM1.

AML with mutated NPM1 shows distinct genetic features
Several pieces of evidence suggest the NPM1 mutation is a founder genetic alteration (Table 2) in AML.
With the exception of rare cases of myelodysplastic (MDS)/myeloproliferative neoplasms 9 that require further confirmation, the NPM1 mutation or its immunohistochemical surrogate (cytoplasmic nucleophosmin) appears to be restricted to For personal use only. on August 28, 2017. by guest www.bloodjournal.org From AML 3,10 and is usually expressed in the whole leukemic population. It has a recurrence rate of about 30% in AML and is mutually exclusive of other AML recurrent genetic abnormalities 3,11 (see below). As expected for a founder genetic lesion, the NPM1 mutation is stable over the course of disease 12,13 . Notably, it has been detected in AML at relapse, even many years after the initial diagnosis 14 , in patients experiencing more than one relapse and in relapses occurring in extramedullary sites 15 . Although loss of NPM1 mutation has been sporadically observed in NPM1-mutated AML 16 , no extensive investigations were performed to exclude secondary, clonally unrelated, AML 17 . Since many groups currently employ NPM1 mutation as a tool to evaluate minimal residual disease, further data on the stability of NPM1 mutations should be soon available. Finally, when AML with mutated NPM1 carries a concomitant FLT3-ITD (about 40% of cases) 3 , the NPM1 mutation appears to precede FLT3-ITD 18,19 .
As expected for a founder genetic lesion, the NPM1 mutation defines a subgroup of AML with a distinct gene expression profile (including down-regulation of CD34 and upregulation of HOX genes) 20-22 and microRNA signature 22-24 (including up-regulation of miR-10a and miR-10b). Sequencing of the whole genome from two cases of AML with normal karyotype (AML-NK) at 91% 25 and 98% resolution 26 , respectively, did not reveal any recurrent lesion, other than the NPM1 mutation, which showed features of a primary genetic hit. In fact, in one case 25 , the NPM1 and FLT3 genes were involved, while the other patient 26 harboured a mutated NPM1 gene and concomitant NRAS and IDH1 gene mutations. Mutations of FLT3 and NRAS in AML are widely recognized as secondary genetic events which are associated with tumour progression. The impact of IDH1 mutation 26,27 on the molecular pathogenesis of AML remains to be elucidated.
Interestingly, one NPM1-mutated/IDH1-mutated AML patient was recently reported to have lost IDH1 mutation at relapse while retaining the NPM1 mutation, suggesting that at least in this case IDH1 mutation was probably a secondary event 28 . Studies of additional For personal use only. on August 28, 2017. by guest www.bloodjournal.org From genomes from AML patients with normal karyotype are warranted to clarify the pathogenetic role of NPM1 mutation and its relationship with other mutations.
Overall, the features of NPM1-mutated AML appear to overlap with those of wellrecognized primary AML genetic lesions, such as the AML1-ETO fusion gene (Table 3).
Similar characteristics are also shown by AML carrying double CEBPA mutations, but not by AML-NK associated with other mutations (Table 3) since the latter are probably secondary genetic events. As an example, FLT3-ITD and FLT3-TKD are less stable, being lost at relapse in about 9% and 50% of cases, respectively 29, 30 . Instability has been also reported for NRAS 31 and WT1 32 mutations. Consequently, if recurrence and the other distinctive features shown in Tables 2 and 3 are to be considered, the main criteria for judging the relevance of an individual genetic alteration for pathogenesis, the NPM1 mutation emerges as the most likely candidate as the primary, driving genetic lesion in about 60% of AML-NK. This view is further supported by recent evidence showing the NPM1 mutant perturbs hemopoiesis in experimental models and is expressed in the leukemic stem cells from patients with NPM1-mutated AML (see below).
Besides the primary genetic event, secondary cooperating mutations are thought to play a major role in leukemogenesis 33 . Recurrent genetic lesions that probably cooperate with the NPM1 mutation include chromosomal aberrations (in about 15% of cases) 3 and mutations such as those affecting the FLT3-ITD, FLT3-D835, NRAS, IDH1, and TET2 genes (in about 60% of cases). Hypothetical steps of leukemic transformation in NPM1mutated AML are shown in Figure 1.

How does mutated NPM1 promote leukemia?
The NPM1 gene encodes for a protein which, although nucleolar at steady state 6 , shuttles between nucleus and cytoplasm 34 . Acting as a molecular chaperone to establish multiple protein-protein interactions, NPM1 is involved in critical cell functions 35 , such as For personal use only. on August 28, 2017. by guest www.bloodjournal.org From control of ribosome formation and export, stabilization of the oncosuppressor p14 Arf protein in the nucleolus, and regulation of centrosome duplication. Although the NPM1 gene was strongly implicated in cancer pathogenesis 35 , how the NPM1 mutant protein promotes leukemia remains elusive. Since the NPM1 mutation always results in aberrant cytoplasmic dislocation of the mutant protein 36,37 , this event appears critical for leukemogenesis 6,38 . Increased nucleophosmin export into cytoplasm probably perturbs multiple cellular pathways by "loss-of-function" (NPM1 nucleolar interactors are delocalized by the mutant into leukemic cell cytoplasm) and/or "gain-of-function" (the hyper-shuttling NPM1 mutant work in a deregulated fashion). Moreover, the NPM1 mutant could have neomorphic features, e.g. capability to interact with new protein partners in cytoplasm 4,6 .
NPM1 mutant-mediated cytoplasmic delocalization of nuclear proteins 6 was implicated in knocking-down the oncosuppressor Arf 39,40 and activating the c-MYC oncogene 41 . In addition, the function of wild-type nucleophosmin in NPM1-mutated AML cells is profoundly affected by its reduction at the nucleolar physiological site. Reduction of wild-type NPM1 in the nucleolus is due to both heterozygosity and dislocation into cytoplasm through forming heterodimers with NPM1 mutant 6 . In the Npm knock-out mouse, Npm inactivation led to genomic instability which, in turn, promoted in vitro and in vivo cancer susceptibility. Npm heterozygous cells were more susceptible to oncogenic transformation and Npm +/mice developed spontaneous tumours, especially myeloid malignancies 42 indicating how NPM1 acts as haploinsufficient tumor suppressor in vivo.
The NPM1 mutant may also exert its transforming properties through gain-of function in cytoplasm. Interestingly, the NPM1 mutant bound and inhibited caspase 6 and 8 signalling in leukemic cell cytoplasm 43 . In the future, functional alterations of other NPM1 interactors are expected to be identified in NPM1-mutated AML.
In vitro studies demonstrated the NPM1 mutant promoted oncogenic transformation of primary cells in cooperation with oncogenic E1A 44 . In vivo, the NPM1 mutant impacted For personal use only. on August 28, 2017. by guest www.bloodjournal.org From directly on myelopoiesis, favoring myeloid proliferation in transgenic mice 45 and in a zebrafish embryonic model 46 . In the transgenic mouse model, the most frequent human NPM1 mutation (type A) was driven by the myeloid-specific human MRP8 promoter.
NPMc+ transgenic mice developed a non-reactive myeloproliferation with mature GR-1+;Mac-1+ cells accumulating in bone marrow and spleen 45 . In zebrafish, ubiquitous mutant NPM1 not only caused expansion of primitive myeloid cells, but also resulted in increased numbers of definitive erythro-myeloid progenitors (gata1+/lmo2 bright ) and hematopoietic stem cells (c-myb+/cd41+) in the aorta ventral wall ( Figure 2).
However, in none of these models was the NPM1 mutant alone able to initiate AML.
In the mouse model inability of enhanced myeloproliferation to progress to spontaneous overt AML may have been determined by either the cell type expressing NPMc+ or by low level mutant expression in hemopoietic cell cytoplasm which does not reproduce the features of human NPM1-mutated AML exactly. In the zebrafish embryo, follow-up for AML development was not possible due to the transient nature of mutant NPM1 expression.
Consequently, to exert its oncogenic effect, NPM1 may need to act under different conditions, such as targeting a specific myeloid precursor and/or achieving a mutant to wild-type expression ratio that is appropriate for cytoplasmic delocalization of both nucleophosmin forms 6,38 and/or being accompanied by a secondary cooperating event 44 .
Knock-in mice models mimicking human NPM1-mutated AML more closely are needed to address these issues.

Origin of NPM1-mutated AML
Consistent CD34 negativity in the great majority of NPM1-mutated AML cases 3 raises the question of whether a minimal pool of CD34+/CD38-NPM1-mutated progenitors exists. In NPM1-mutated AML we and other investigators 47,48 found that the small fraction of CD34+ hemopoietic progenitors, including CD34+/CD38-cells, carried the NPM1 The engraftment potential of the CD34-negative fraction in NPM1-mutated AML appears more controversial. In one study 47 , no or limited engraftment was observed in NOG mice. In contrast, Taussig et al. 48 reported a more consistent engraftment of the CD34-negative leukemic cells in immunocompromised mice. These findings may reflect some degree of heterogeneity in the leukemic stem cell compartment of NPM1-mutated AML.
Despite CD34 negativity, HOX genes, which are involved in stem cell maintenance, are consistently upregulated in NPM1-mutated AML 20-22 . However, it remains to be elucidated whether leukemic stem cells in NPM1-mutated AML originate from very early progenitors or from committed myeloid precursors, with subsequent reactivation of stem cell self-renewal machinery through HOX gene reprogramming.

Relationships between AML with mutated NPM1 and other myeloid neoplasms
AML with mutated NPM1 shows distinctive genetic, pathological, immunophenotypic and clinical features 4,5 (

Diagnosis of NPM1 mutated AML: the strength of flexibility
One important prerequisite for a disease being included as an entity in the WHO classification is that it can be easily recognized worldwide, according to well-established and reproducible criteria. Fortunately, several molecular assays and surrogate methods are currently available for diagnosing AML with mutated NPM1 54 ( Figure 3).

Molecular analysis
Since AML with mutated NPM1 was first identified in 2005, highly specific and sensitive molecular assays have been developed for detecting NPM1 mutations 55 . One of the most frequently used at diagnosis is fragment analysis (genescan analysis) 18 which has the advantage of multiplexing with FLT3-specific or CEBPA-specific assays 56 . It does not, however, discriminate type A NPM1 mutation from rare variants and all samples that are positive at fragment analysis have to be sequenced for detailed characterisation. On the other hand, melting curve assays which include mutation specific probes are not only useful in screening but also discriminate between type A, B, and D mutations 57 and sequencing is required only for 5% of patients with rare mutation types. These methods at diagnosis show a sensitivity of approximately 5%.
More sensitive methods have to be applied to detect minimal residual disease and the mutation sequence at diagnosis needs to be known. Although gene expression 20-22 , microRNA 23,24 and methylation 67 profiles identified distinct signatures associated with NPM1-mutated AML, these procedures are currently not used for diagnostic or prognostic purposes in the every day clinical practice.

Detection of cytoplasmic nucleophosmin: a surrogate for molecular analysis
For personal use only. on August 28, 2017. by guest www.bloodjournal.org From One of the WHO's primary goals is the widespread use of the genetic-based AML classification. As molecular techniques are not always available for diagnosis, especially in developing countries, there is great interest in suitable substitutes. Morphology and immunophenotype (frequent CD34 negativity) cannot be used since NPM1-mutated AML encompasses various FAB categories and absence of CD34 is also observed in other AML genetic subtypes. Appearing to fill the gap for AML with mutated NPM1 is a simple, low-cost and highly specific immunohistochemical assay which predicts NPM1 mutations Which antibodies should be used to visualise subcellular expression of nucleophosmin ? Some anti-NPM antibodies recognize both wild-type and mutated NPM1 3 while others identify only the NPM1 mutant 68,74 . Immunohistochemistry as first line screening for NPM1-mutated AML is best achieved using the former, since they detect all NPM1 mutated proteins, including those generated by the very rare NPM1 mutations occurring in exons other than 12. In contrast, reagents that are specific for NPM1 mutant A 74 fail to identify some mutants and may be more suitable for flow cytometry monitoring of minimal residual disease.

Prognostic features of NPM1-mutated AML
AML with mutated NPM1 is highly responsive to induction chemotherapy 3,4 . About 80% of patients achieve complete remission with clearance of leukemic cells as early as 16 days after starting treatment 75 . The exquisite chemosensitivity of NPM1-mutated AML is probably related to the aberrant dislocation of nucleophosmin from nucleolus to cytoplasm, but the underlying mechanism through which this occurs remains unknown.
The prognostic significance of NPM1 mutations was mainly investigated in AML with normal karyotype. In patients < 60 years old, the outcome is similar to the "good-risk" AML categories carrying t(8;21) or inv (16)  As for any type of AML that has attained complete remission the question is whether the patient should undergo an allogeneic stem cell transplantation, which is so far the most effective treatment modality for AML. Because of its intrinsic risk of morbidity and mortality, this procedure is generally reserved for young AML patients carrying high-risk genetic abnormalities. In contrast, AML patients with relatively good prognosis, such as those carrying t(15;17), t(8;21) or inv(16), are usually not transplanted in first complete remission 1 . This policy was also proposed for AML with mutated NPM1 in the absence of concurrent FLT3-ITD, since no apparent benefit seems to derive from allogeneic transplantation in these patients 76 who account for about 16% of all newly diagnosed denovo AML under 60 years of age 1 . These cases are currently treated with conventional therapy, with or without autologous stem cell transplantation. Further prospective studies are warranted to confirm these findings.

AML with mutated NPM1: new insights into controversial issues of the 2008 WHO classification
In the 2008 WHO classification, NPM1-mutated AML was listed as a provisional entity since uncertainties persisted about the biological significance and prognostic impact of additional chromosomal aberrations and multilineage dysplasia in AML with mutated NPM1 and how AML patients who were double-mutated for NPM1 and CEBPA should be classified. Recent studies provided insights into these areas.

i) What is the biological and clinical significance of chromosomal aberrations in AML with mutated NPM1?
About 15% of AML with mutated NPM1 harbour chromosomal aberrations other than typical recurrent cytogenetic abnormalities 3 . The significance of these chromosomal abnormalities was addressed in 631 AML patients with mutated/cytoplasmic NPM1 84 .
Chromosomal aberrations were found in 14.7%, with the most frequent anomalies being For personal use only. on August 28, 2017. by guest www.bloodjournal.org From +8, +4, -Y, del(9q) and +21 84 (Table 4). Several findings suggested these chromosomal aberrations were secondary events 84 . Although less frequent, they were mostly similar to additional chromosome aberrations that are widely regarded as secondary events in AML with t(8;21), inv(16), t(15;17) or 11q23/MLL-rearrangements 84 . They were often subclones within the leukemic population with normal karyotype 3 (mosaicism). More importantly, 4/31 NPM1-mutated AML patients with NK at diagnosis remained NPM1-mutated while switching to the following abnormal karyotype at relapse: del(9q) (n=2), t(2;11) (n=1), inv(12) (n=1) 84 . In addition, few NPM1-mutated AML with abnormal karyotype at diagnosis NPM1-mutated AML also differs from AML with MD-related changes as it does not usually evolve from previous MDS or MDS/MPN 3 and shows distinctive features that seem to be independent of whether the karyotype is normal or abnormal 84 , further supporting the view that these two leukemias are distinct entities (Table 5).

iii) What is the significance of rare AML cases carrying both NPM1 and CEBPA mutations?
A minority (about 4%) of NPM1-mutated AML also carry a CEBPA mutation 90 . At the time of preparation of 2008 WHO classification, this fact was thought to be difficult to reconcile with the claim that NPM1 and CEBPA mutations defined distinct AML entities. In

Future perspectives
Recent findings point to "AML with mutated NPM1" and "AML with biallelic CEBPA mutations" as distinct leukemia entities. Additional information is expected to accumulate over the next few years that will help to assess whether they should be incorporated as such in the next revision of the WHO classification. Since NPM1-mutated/FLT3-ITD negative AML patients seem to have good prognosis, independently of normal or abnormal karyotype 84 , one critical issue requiring clarification will be how to best risk-stratify AML patients according to molecular criteria. The current assessment of the prognostic values of NPM1, CEBPA and FLT3-ITD mutations in the framework of normal karyotype 18,21,57 . has two major limitations: i) it excludes AML patients in whom cytogenetic analysis fails; and ii) it prevents AML patients from being assigned to the group with favourable genotype (e.g., NPM1 mutated/FLT3-ITD negative), if a chromosomal aberration is present. Use of "normal karyotype" as initial framework for risk-stratification, may be more appropriate for AML patients without NPM1 or biallelic CEBPA mutations. In this subgroup, which comprises about 40% of AML with normal karyotype, increasing application of whole genome sequencing is expected to unravel novel causal mutations that may serve as new diagnostic and prognostic markers.

Genetic features
-NPM1 mutation° is specific for AML, usually "de novo" -Usually all leukemic cells carry the NPM1 mutation -Mutually exclusive of other "AML with recurrent genetic abnormalities" -NPM1 mutation is stable (consistently retained at relapse) -NPM1 mutation usually preceeds other associated mutations (e.g. ° Or its immunohistological surrogate (cytoplasmic NPM, NPMc+); GEP:gene expression profiling; * Lower incidence in Chinese children; ** In most but not all studies; ^Less than 10% CD34+ cells.   is depicted in the central column while its more differentiated CD34-negative progeny is depicted in the right and left columns. The primary, driving NPM1 mutation (red dot) in a HSC causes transformation that leads to the "leukemic phenotype". Other mutations (light-blue dots) such as FLT3-ITD occur later in clonal evolution. Leukemic cells in about 15% of NPM1-mutated AML can also acquire a chromosomal abnormality (X) whilst in 85% of cases they maintain a normal karyotype.
Both later mutations and chromosomal abnormalities are usually expressed in a leukemic cell subclone whose size may vary from a patient to another. For simplicity, occurrence of the second mutation and a chromosomal abnormality in the same cells is not depicted. According to the two-hit hypothesis only two mutations are indicated but additional mutations may be involved.
Light grey circles indicate normal HSC and multipotent progenitors. Green circles indicate the normal hemopoietic progenitor compartment where primary NPM1 mutation (red dot) and secondary mutations (blue dot) and/or chromosomal aberrations (X) occur, giving raise to the leukemic bulk population.

Figure 2. NPM1 mutant in zebrafish model
In zebrafish, where mutant NPM1 was expressed ubiquitously, not only it caused expansion of primitive myeloid cells, but also resulted in increased numbers of both definitive erythro-myeloid progenitors (gata1+/lmo2 bright ) and hematopoietic stem cells (c-myb+/cd41+) in the ventral wall of the aorta.