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MYELOID NEOPLASIA
AML with mutated NPM1 carrying a normal or aberrant karyotype show
overlapping biologic, pathologic, immunophenotypic, and prognostic features
Claudia Haferlach,1 Cristina Mecucci,2 Susanne Schnittger,1 Alexander Kohlmann,1 Marco Mancini,3 Antonio Cuneo,4
Nicoletta Testoni,5 Giovanna Rege-Cambrin,6 Antonella Santucci,2 Marco Vignetti,7 Paola Fazi,7 Maria Paola Martelli,2
Torsten Haferlach,1 and Brunangelo Falini2
1Munich
Leukemia Laboratory GmbH, Munich, Germany; 2Institute of Hematology, University of Perugia, Perugia, Italy; 3Department of Cellular Biotechnologies
and Hematology, “La Sapienza” University, Rome, Italy; 4Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, Ferrara, Italy;
5Institute of Hematology “Ludovico & Ariosto Seragnoli,” University of Bologna, Bologna, Italy; 6Department of Clinical and Biological Sciences,
University of Turin, San Luigi Hospital, Orbassano, Turin, Italy; and 7GIMEMA Data Center, GIMEMA Foundation, Rome, Italy
Acute myeloid leukemia (AML) with mutated NPM1 usually carries normal karyotype (NK), but it may harbor chromosomal aberrations whose significance
remains unclear. We addressed this question in 631 AML patients with mutated/
cytoplasmic NPM1. An abnormal karyotype (AK) was present in 93 of 631 cases
(14.7%), the most frequent abnormalities
being ⴙ8, ⴙ4, ⴚY, del(9q), ⴙ21. Chromosome aberrations in NPM1-mutated AML
were similar to, but occurred less frequently than additional chromosome
changes found in other AML with recurrent cytogenetic abnormalities according
to WHO classification. Four of the 31
NPM1-mutated AML patients karyotyped
at different time points had NK at diagnosis but AK at relapse: del(9q) (n ⴝ 2),
t(2;11) (n ⴝ 1), inv(12) (n ⴝ 1). NPM1mutated AML with NK or AK showed
overlapping morphologic, immunophenotypic (CD34 negativity), and gene expression profile (down-regulation of CD34 and
up-regulation of HOX genes). No difference in survival was observed among
NPM1-mutated AML patients independently of whether they carried a NK or an
AK, the NPM1-mutated/FLT3-ITD negative
cases showing the better prognosis. Findings in our patients point to chromosomal aberrations as secondary events,
reinforce the concept that NPM1 mutation
is a founder genetic lesion, and indicate
that NPM1-mutated AML should be clinically handled as one entity, irrespective
of the karyotype. (Blood. 2009;114:
3024-3032)
Introduction
Acute myeloid leukemia (AML) carrying an NPM1 gene mutation
causing aberrant cytoplasmic expression of nucleophosmin1
(NPMc⫹ AML) accounts for approximately one-third of adult
AML. This large leukemia subgroup usually carries a normal
karyotype (NK)1 and shows distinctive biologic, pathologic, and
clinical features,2 such as mutual exclusion of recurrent genetic
abnormalities,3 high frequency of FLT3-ITD mutations,1 frequent
FAB M4 or M5 morphology,2 multilineage involvement,4 distinctive gene expression signature,5 and microRNA profile.6,7 Moreover, AML with mutated NPM1 is characterized by good response
to induction chemotherapy1 and favorable prognosis (in the absence of a concomitant FLT3-ITD mutation).8-13 Therefore, AML
with mutated NPM1 is now included as a provisional entity in the
4th Edition of the World Health Organization (WHO) classification
of tumors of hematopoietic and lymphoid tissues.14
In our original study describing AML with cytoplasmic/mutated
NPM1,1 we found that approximately 85% of patients carried a
NK, the remaining cohort harboring chromosomal aberrations.
However, the type, frequency, biologic and clinical significance of
these aberrations still remained poorly understood. Although the
patients with abnormal karyotype (AK) represent only a minority
of all AML with mutated NPM1, their definition with respect to
biologic, pathologic, and clinical terms is very important. Indeed,
during preparation of the WHO-2008 classification, one of the
points that was raised for designating AML with mutated NPM1 as
provisional rather than a definitive entity was that the biologic,
clinical, and prognostic significance of chromosomal aberrations in
AML with mutated NPM1 was still under debate. This is because
most studies on NPM1 mutations in AML mainly focused on
patients with NK and, in the few studies reporting on NPM1mutated patients carrying other chromosomal aberrations,10,11 the
frequency of this association and its biologic and clinical significance was not deeply investigated.
To address this issue in more depth, we analyzed the frequency and
type of chromosomal aberrations in 631AML patients with cytoplasmic/
mutated NPM1 and compared the results with the frequency and type of
additional aberrations occurring in the most common AML with
recurrent genetic abnormalities according to the WHO-2008 classification,14 ie, AML with t(8;21)(q22;q22), inv(16)(p13q22)/t(16;16)(p13;
q22), t(15;17)(q22;q12), and t(9;11) or other 11q23-abnormalities leading to an MLL-rearrangement. Other important goals of the study were
(1) to investigate variations in the karyotype of AML with mutated
NPM1 during the course of the disease and (2) to compare the biologic,
pathologic, clinical, and prognostic features of NPM1-mutated AML
carrying chromosomal aberrations with those of typical cases of
NPM1-mutated AML harboring a NK.
Our results point to chromosomal aberrations occurring in AML
with mutated NPM1 as secondary events, thus reinforcing the
Submitted January 9, 2009; accepted April 20, 2009. Prepublished online as
Blood First Edition paper, May 8, 2009; DOI 10.1182/blood-2009-01-197871.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2009 by The American Society of Hematology
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BLOOD, 1 OCTOBER 2009 䡠 VOLUME 114, NUMBER 14
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BLOOD, 1 OCTOBER 2009 䡠 VOLUME 114, NUMBER 14
concept that NPM1 mutation is a founder genetic lesion. In addition, the
finding that NPM1-mutated AML carrying a NK or an AK show
overlapping pathologic, immunophenotypic, and prognostic features
has important diagnostic and clinical implications.
NPM1-MUTATED AML IS NOT INFLUENCED BY KARYOTYPE
3025
Mutational analysis of the FLT3 gene
Analysis for fms-related tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) was performed in 600 cases. Point mutations in the FLT3tyrosine kinase domain (TKD), FLT3-TKD, were assessed in 491 cases.
Mutational assays were carried out as previously described.19,20
Methods
Cytogenetic studies
Leukemia patients
Cytogenetic studies were performed after short-term culture. Karyotypes,
analyzed after G-banding, were described according to the International
System for Human Cytogenetic nomenclature.21 All NPM1-mutated patients were studied at initial diagnosis. In 31 cases from the MLL,
cytogenetic studies were available both at first presentation and at relapse.
We investigated samples from 631 AML patients with mutated NPM1 for
whom cytogenetic studies were available: 390 were from the Munich
Leukemia Laboratory (MLL); 241 were enrolled in the Italian Group for
Adult Hematologic Diseases (GIMEMA) Leucemia Acuta Mieloide Protocollo begun in 1999 (LAM99P) and GIMEMA/European Organization
for Research and Treatment of Cancer (EORTC) AML12 trials. The frequency and type of cytogenetic aberrations found in NPM1-mutated AML
were compared with those of additional chromosomal aberrations detected
in 266 cases representative of the most common AML with recurrent
genetic abnormalities according to the WHO-2008 classification.14 The
latter cases (all from the MLL) included: 63 AML with t(8;21), 37 AML
with inv(16)/t(16;16), 83 AML with t(15;17), and 83 AML showing an
11q23/MLL-rearrangement.
For prognostic analysis, we focused on 576 AML patients: 390 from the
MLL; 186 cases from the GIMEMA LAM99P and GIMEMA/EORTC
AML12 trials who received the same treatment. Therapy in the 390 cases
from MLL was as follows: 141 patients (36%) were treated within the AML
Cooperative Group (AMLCG) trials,15 35 patients received treatment
according to AMLCG protocols but were not enrolled in the AMLCG trial,
and another 214 patients received other intensive AML therapy protocols.
All 186 GIMEMA/EORTC patients, both from the LAM99P and the
AML12 (registration phase only) protocols, received the same treatment,
consisting in a 3-drug induction cycle with daunorubicine (DNR), etoposide, and Ara-C followed, in case of complete remission (CR), by a single
course of consolidation therapy with intermediate-dose Ara-C (500 mg/m2
every 12 hours in a 2-hour infusion on days 1-6) plus DNR, given on days
4 to 6. After consolidation, younger patients with a sibling donor were then
assigned to undergo an allogeneic stem cell transplantation. Those without
such a donor, as well as older patients, were to receive an unpurged
autologous stem cell transplantation. Approval for the above studies was
provided by the Institutional Review Board of each participating center.
Informed consent was provided by patients at each participating center in
accordance with the Declaration of Helsinki.
Defining criteria for AML with mutated NPM1
All 390 cases from MLL were defined by molecular criteria (ie, mutational
analysis of the NPM1 gene). Nucleic acid isolation from leukemic cells,
cDNA synthesis, and screening for NPM1 gene mutations was performed
using a melting curve-based LightCycler assay (Roche Applied Science) as
previously described.8 AML samples with an aberrant melting curve
underwent subsequent nucleotide sequence analysis.
All 241 AML cases from GIMEMA LAM99P and GIMEMA/EORTC
AML12 trials were defined by immunohistochemical criteria, ie, presence
of aberrant cytoplasmic expression of nucleophosmin (NPMc⫹), which is
known to be fully predictive of NPM1 mutations.16,17 Nucleophosmin was
detected by immunostaining paraffin sections from B5-fixed/ethylenediaminetetraacetic acid (EDTA) decalcified bone marrow biopsies with a specific
monoclonal antibody directed against a fixative-resistant epitope of human
nucleophosmin (clone 376), as previously described.16 The antibodyantigen reaction was revealed using a highly sensitive alkaline phosphatase
anti-alkaline phosphatase (APAAP) technique.16 As expected,1,16 all cases
showed a nucleus-restricted positivity when stained with a monoclonal
antibody directed against the nucleolar protein nucleolin/C23 (Santa Cruz
Biotechnology). In 138 of the 241 NPMc⫹ AML cases, material was also
available for mutational studies or Western blot analysis with antibodies
specifically directed against NPM1 mutants. In all cases, these techniques
were performed as previously described1,18 and confirmed the presence of
NPM1 gene mutations or a mutated NPM1 protein.
Gene expression profiling
The sample preparation assay was performed as previously reported
(HG-U133 Plus 2.0 microarrays; Affymetrix).22-24 Gene expression raw
data were processed according to the manufacturer’s recommendations.
After quality control, raw data were normalized using the robust multiarray
average normalization algorithm as implemented in the R-package affy
version 1.18.0.25 For supervised statistical analyses, samples were grouped
accordingly, and for each disease entity, differentially expressed genes were
calculated using t statistics. To visualize similarity of gene expression
patterns, hierarchical clustering and principal component analyses were
applied. Transformed gene expression data were analyzed using GeneMaths
XT version 2.1 (Applied Maths) and Partek Genomics Suite version
6.4 (Partek). Microarray data can be found at Gene Expression Omnibus,
GEO, under accession number GSE16015.
Statistical analysis
Statistical analysis was performed to investigate the different distribution of
quantitative variables, such as age, and qualitative variables, such as sex,
CD34 expression, and FLT3 status in the 2 different cytogenetic groups
(NK vs AK) of AML with mutated NPM1. In particular, the t test was used
to analyze mean differences, and the ␹2 test was applied in case of
contingency tables. In case of 2 ⫻ 2 contingency tables, the Fisher exact
test was applied.
Prognostic analysis was performed on 576 NPM1-mutated AML
patients treated as described above. Survival curves were calculated for
overall survival (OS) and event-free survival (EFS) according to KaplanMeier and compared using the log rank test. OS was calculated from time of
diagnosis to death, and EFS was calculated from time of diagnosis to death,
documentation of persistent leukemia, or relapse.
For all above analyses, results were regarded as significant at a P value
less than .05 at both sides. SPSS version 14.0.1 software (SPSS) was used
for statistical analysis of patients from the MLL. Statistical analyses of
patients enrolled in the GIMEMA LAM99P and GIMEMA//EORTC
AML12 studies were performed using the SAS 9.1 software (SAS
Institute).
Results
Chromosomal aberrations in AML with mutated NPM1 are
similar to, but occur at lower frequency than, those associated
with AML carrying other recurrent genetic abnormalities
Frequency and type of chromosomal aberrations were investigated
by chromosome banding analysis in 631 AML with mutated/
cytoplasmic NPM1. A NK was detected in 538 of 631 (85.3%) and
an AK was present in 93 of 631 cases (14.7%). This distribution in
a large number of cases is almost identical to what we previously
observed in a cohort of 166 patients with NPMc⫹ AML
(AK ⫽ 14.5%).1
A total of 142 chromosome abnormalities were observed in the
93 NPM1-mutated AML cases with AK (Table 1). The most
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BLOOD, 1 OCTOBER 2009 䡠 VOLUME 114, NUMBER 14
HAFERLACH et al
Table 1. Types and frequency of clonal chromosome abnormalities detected in 93 NPM1-mutated AML and 266 AML with recurrent
cytogenetic abnormalities
AML
Karyotype
Additional abnormalities
NPM1-mut* (n ⴝ 631)
t(8;21) (n ⴝ 63)
inv(16) (n ⴝ 37)
t(15;17) (n ⴝ 83)
11q23/MLL (n ⴝ 83)
93/631† (14.7%)
44/63 (69.8%)
13/37 (35.1%)
39/83 (47%)
28/83 (33.7%)
⫺X/⫺Y
11
32
⫹4
11
2
⫺7
3
⫹8
33
⫹13
2
2
1
3
2
5
12
2
⫹19
4
⫹21
5
⫹22
1
4
6
del(7q)
del(9q)
8
2
2
9
del(11q)
10
2
2
ider(17)(q10)t(15;17)
7
Other
67‡
11
8
20
30
Total
142†
59
22
44
52
*AML with mutated NPM1.
†More than one abnormality was present in a subset of cases with aberrant karyotype.
‡Includes 4 cases fulfilling the definition of a complex aberrant karyotype but without a typical pattern of chromosomal gains and losses:
(1) 90, XXXX, ⫺3, ⫹8, ⫹8, ⫺10, ⫺11, ⫺17; FLT3-ITD⫺ (74 days, alive);
(2) 49, XY, ⫹der(5)t(5;17)(q11;?), ⫹8, ⫹del(13)(q12); FLT3-ITD⫺ (30 days, alive);
(3) 53, XX, ⫹4, ⫹5, ⫹8, ⫹8, der(12)t(12;13)(q24;q?), ⫹16, ⫹18, ⫹20; FLT3-ITD⫹ (86 days, dead); and
(4) 55, XY, ⫹X, ⫹4, ⫹5, ⫹8, ⫹10, ⫹13, ⫹14, ⫹17, ⫹18; FLT3-ITD⫹ (5 days, dead).
frequent abnormalities were ⫹8, ⫹4, ⫺Y, del(9q), and ⫹21. Other
aberrations were nonrecurrent balanced rearrangements (n ⫽ 10)
and nonrecurrent unbalanced abnormalities (n ⫽ 57). In 4 cases,
the definition of a complex AK was fulfilled (3 or more clonal
chromosome aberrations according to the Southwest Oncology
Group [SWOG]). However, these karyotypes did not show the
typical pattern of chromosomal gains and losses observed in the
majority of cases with complex AK, such as loss of 5q, 7q 12p, 16q,
and 17p and gain of 8q, 11q, and 21q (Table 1). Two of these cases
were FLT3-ITD positive, and 2 were negative.
As expected,3 none of the 93 cases of NPM1-mutated AML with
AK harbored any of the AML-associated recurrent genetic abnormalities, as defined by the WHO-2008 classification.14 No differences in the type and frequency of chromosomal aberrations were
observed in AML carrying NPM1 mutation A, B, D, or other rare
mutation types.
Our results in a large series of leukemia patients clearly
demonstrate that chromosomal aberrations are rather uncommon in
AML with mutated/cytoplasmic NPM1.
We then compared the frequency and types of clonal chromosomal aberrations detected in 93 of 631 AML with mutated NPM1
with those found in 266 AML with recurrent genetic abnormalities
of WHO classification. The results are summarized in Table 1 and
clearly indicate that the type of chromosome aberrations detected
in AML with mutated NPM1 are similar to, but occur at lower
frequency than, additional chromosome changes found in AML
with recurrent genetic abnormalities,26-28 according to the WHO2008 classification.14
Chromosomal aberrations in AML with mutated NPM1 are
secondary events
To assess whether chromosomal aberrations in AML with mutated
NPM1 were primary or secondary events, we applied chromosome
banding at diagnosis and relapse in 31 patients with NPM1-mutated
AML (all from MLL). All 31 cases showed the same NPM1
mutation at diagnosis and relapse. Therefore, in no case a loss of
the NPM1 mutation was observed. Median time from diagnosis to
relapse was 301 days (range, 71-986). Most cases (22/31; 71%) had
a normal karyotype both at diagnosis and relapse. However,
4 patients showing a normal karyotype at diagnosis were found to
have an AK at relapse: del(9q) (n ⫽ 2), t(2;11) (n ⫽ 1), and inv(12)
(n ⫽ 1). One case with ⫹8 at diagnosis showed ⫹8 also at relapse.
One case with ⫹4 at diagnosis showed ⫹4 and additional
aberrations at relapse. In one case, clonal regression was observed
(⫹21 to NK). One case with an unbalanced 1;3-translocation at
diagnosis showed a der(17;18)(q10;q10) at relapse; and one case
with ⫺Y at diagnosis showed a del(3p) at relapse.
We next investigated the occurrence of acquired NPM1 mutations at relapse in AML cases that, at first presentation, showed a
germ line NPM1 gene and a chromosomal aberration, such as ⫹8
or del(9q). Here, we focused on cases with ⫹8 or del(9q), because
these aberrations were commonly detected in the small subgroup of
NPM1-mutated AML with AK (Table 1). Notably, all 7 cases of
AML with germ line NPM1 that, at the time of initial diagnosis, had
presented with ⫹8 (n ⫽ 5) or del(9q) (n ⫽ 2), as sole abnormality,
maintained the same karyotype and did not acquire an NPM1
mutation at relapse.
Taken together, these results point to chromosomal aberrations occurring in AML with mutated NPM1 as acquired
secondary events.
AML with mutated NPM1 carrying a NK or chromosomal
aberrations show a similar gene expression profile
To further investigate the biologic nature of the 2 subset of
NPM1-mutated AML (NK vs AK), gene expression microarray
studies were carried out. First, restricting analysis to cases of AML
with NK, a group of 42 NPM1-unmutated (NPMc⫺) cases was
compared against a group of 55 NPM1-mutated cases (NPMc⫹).
The resulting signature of the top 500 differentially expressed
probe sets clearly separated the 2 AML groups both in a hierarchical clustering approach and a principal component analysis. The
gene list is provided in the supplementary materials (available on
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BLOOD, 1 OCTOBER 2009 䡠 VOLUME 114, NUMBER 14
Figure 1. Gene expression profiling analysis of NPM1mutated AML (NK vs AK). Analysis based on the
top-500 differentially expressed genes between AML
cases NPM1c⫺ (unmutated NPM1) and NPM1c⫹ (mutated NPM1). The gene discovery process was restricted
to AML with NK (ie, a supervised analysis was performed). (A) Hierarchical clustering heatmap also including 10 NPM1 mutated AML cases with other chromosomal aberrations (other). The similarity of the genes
was computed by Euclidean distance, and then Ward
method was used to cluster the gene expression profiles
based on these measures. The normalized expression
value for each probe set is coded by color (standard
deviation from mean). Red cells indicate high expression
and green cells indicate low expression. (B) Principal
component analysis (PCA). The AML samples were
plotted in a 3-dimensional space using the 3 principal
components (PC) capturing most of the variance in the
original
dataset
(PC1 ⫽ 34.0%,
PC2 ⫽ 7.0%,
PC3 ⫽ 5.2%). Each patient sample is represented by a
single color-coded sphere.
NPM1-MUTATED AML IS NOT INFLUENCED BY KARYOTYPE
A
3027
B
normal karyotype
NPM1c-
normal karyotype
NPM1c+
other
42 AML normal karyotype, NPM1c55 AML normal karyotype, NPM1c+
10 AML other aberrations, NPM1c+
the Blood website; see the Supplemental Materials link at the top of
the online article). Subsequently, the analysis was expanded to
include 10 additional cases of NPM1-mutated AML carrying
chromosomal aberrations: 45,X,⫺Y (n ⫽ 4); 47,XX,⫹21 (n ⫽ 3);
47,XX,⫹8 (n ⫽ 2); and 46,XX,del(9)(q22q34) (n ⫽ 1). Interestingly, we observed that, when using the gene list from AML with
NK, the NPM1-mutated AML cases carrying chromosomal aberrations had an expression profile similar to that of the group of
NPM1-mutated AML with NK (Figure 1).
AML with mutated NPM1 carrying a NK or chromosomal
aberrations show overlapping pathologic, immunophenotypic,
and clinical features
NPM1-mutated AML carrying a NK or an AK were then compared
in terms of age distribution, gender, morphologic appearance
according to French-American-British (FAB)/WHO, CD34 expression, FLT3-ITD, and FLT3-TKD status.
In the full cohort of 631 NPM1-mutated AML patients, mean
age (⫾ SD) was 54.39 (⫾ 14.53) years in the NK group and 55.78
(⫾ 15.78) in the AK group (P ⫽ .39). No significant difference in
age distribution of the patients emerged when they were stratified
by decades of age (ages ⬍ 21 years, 21-30 years, 31-40 years,
41-50 years, and 51-60 years). Males were 252 of 538 (46.8%) in
the NK group versus 47 of 93 (50.5%) in the AK group (P ⫽ .57).
FAB categories were available in 507 cases with a distribution in
NK and AK groups not statistically significant (P ⫽ .076).
Down-regulation of CD34 is a distinguishing immunophenotypic feature of AML with cytoplasmic/mutated NPM1.1 Therefore,
we were interested to assess this parameter in NPM1-mutated AML
with NK and AK. Material for analysis of CD34 expression by
immunocytochemical labeling of cell suspensions or bone marrow
tissue sections was available for 422 AML with mutated NPM1
(357 with NK; 65 with AK). Cases were regarded as negative if the
percentage of CD34⫹ cells was less than 10%. Absence of CD34
expression was found in 284 of 357 (79.6%) and 47 of 65 (72.3%)
of the NK and AK group, respectively (P ⫽ .19; Figure 2).
Internal tandem duplication (ITD) analysis of the FLT3 gene
was available in 600 NPM1-mutated AML cases (515 with NK; 85
with AK). FLT3-ITD was detected in 190 of 515 (36.9%) of NK
group and 23 of 85 (27.1%) of AK group (P ⫽ .087). Analysis of
FLT3-TKD mutation was available in 491 cases (422 NK; 69 AK).
FLT3-TKD mutations were detected in 38 of 422 of the NK group
(9.0%) and 14 of 69 of the AK group (20.3%), respectively
(P ⫽ .01; Figure 3).
Figure 2. Morphology and immunophenotype of NPMcⴙ AML with AK. The figure
shows the morphologic and immunohistologic features of a representative example
of NPM1-mutated AML with AK 47,XX,⫹8,der(19)t(13;19)(q12;q13) (top panel).
These features are identical to those observed in the typical NPM1-mutated AML
cases with NK. (Middle left) Diffuse marrow infiltration by leukemic cells with
myelomonocytic appearance. The arrow points to a dysplastic megakaryocyte
(paraffin section from bone marrow biopsy; hematoxylin-eosin; ⫻1000). (Middle right)
Leukemic cells are CD34⫺. The single arrow indicates a dysplastic megakaryocyte,
while the double arrows indicate a CD34⫹ vessel. (Bottom left) Myelomonocytic
leukemic cells and a dysplastic megakaryocyte (arrow) show aberrant cytoplasmic
expression of nucleophosmin (NPM1), indicating they belong to the same clone; cells
showing nucleus-restricted expression of nucleophosmin represent normal residual
hemopoietic precursors. (Bottom right) Leukemic cells and a dysplastic megakaryocyte (arrow) show nucleus-restricted positivity for C23/nucleolin. (Middle right and
bottom panels) Immunostaining of paraffin sections from bone marrow biopsy using
the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) technique;
hematoxylin counterstaining; ⫻1000. All images were collected using an Olympus
B61 microscope and a UPlan FL 100⫻/1.3 NA oil objective; Camedia 4040, Dp_soft
Version 3.2; and Adobe Photoshop 7.0.
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HAFERLACH et al
400
FLT3/ITD +
360
FLT3/ITD -
No. of Cases
320
280
(P = .08)
400
FLT3/TKD +
Figure 3. Distribution of FLT3-ITD and FLT3-TKD mutations in NPM1-mutated AML with NK and AK.
FLT3/TKD -
360
320
(P = .01)
280
240
200
160
120
240
200
160
120
80
40
0
NPMc+AML NPMc+AML
(NK=515)
(AK=85)
80
40
0
NPMc+AML NPMc+AML
(NK=422)
(AK=69)
The above findings indicate that, with the exception of a
difference in the distribution of FLT3-TKD mutations, AML with
mutated NPM1 carrying a NK or an AK show overlapping
morphologic, immunophenotypic, and clinical features.
AML with mutated NPM1 carrying NK or chromosomal
aberrations show a similar outcome
The impact of chromosomal aberrations on prognosis was assessed
in a total of 576 AML patients with mutated NPM1, including
390 cases from the MLL and 186 cases from the GIMEMA LAM99P
and GIMEMA/EORTC AML12 trials. In patients from the MLL,
the CR rate was comparable in cases with NPM1 mutation and a
NK to those with an AK (80.9% vs 85.7%). OS and EFS of patients
from MLL did not significantly differ between 328 NPM1-mutated
AML with NK and 62 NPM1-mutated AML with AK (median OS:
not reached vs not reached; percentage alive at 2 years 63.5% vs
59.0%, P ⫽ .804; median EFS: 16.0 months vs 14.0 months,
P ⫽ .849; Figure 4A-B). OS was significantly shorter in NPM1mutated cases with additional FLT3-ITD (n ⫽ 138) compared with
NPM1-mutated cases without FLT3-ITD (n ⫽ 241; median OS: not
reached vs not reached; percentage alive at 2 years 50.9% vs
70.5%, P ⫽ .015; supplemental Figure 1). Also EFS was significantly shorter in the NPM1-mutated/FLT3-ITD⫹ subgroup versus
NPM1-mutated/FLT3-ITD⫺ (11.6 months vs 17.0 months; P ⫽ .045;
supplemental Figure 1).
Considering only FLT3-ITD negative cases, no statistically
significant difference emerged in OS and EFS of NPM1-mutated
AML with NK versus AK (median OS: not reached vs 21.0 months;
A
B
normal karyotype, n=328
aberrant karyotype, n=62
normal karyotype, n=328
aberrant karyotype, n=62
P = .804
P = .849
10
20
30
40
50
10
Overall Survival in months
C
Figure 4. Survival curves of NPM1-mutated AML patients (NK vs AK) from the MLL. (A) No significant
differences in OS between NPM1-mutated AML with NK
(n ⫽ 328) and NPM1-mutated cases with AK (n ⫽ 62) are
observed (P ⫽ .804). (B) No significant differences in
EFS between NPM1-mutated AML with NK (n ⫽ 328) and
NPM1-mutated cases with AK (n ⫽ 62) are observed
(P ⫽ .849). (C) No significant differences in OS between
NPM1-mutated/FLT3-ITD⫺ AML with NK (n ⫽ 197) and
NPM1-mutated cases with AK (n ⫽ 44) are observed
(P ⫽ .075). (D) No significant differences in EFS between
NPM1-mutated/FLT3-ITD⫺ AML with NK (n ⫽ 197) and
NPM1-mutated cases with AK (n ⫽ 44) are observed
(P ⫽ .176).
20
30
40
50
Event Free Survival in months
D
FLT3-ITD negative
FLT3-ITD negative
normal karyotype, n=197
normal karyotype, n=197
aberrant karyotype, n=44
aberrant karyotype, n=44
P = .075
P = .176
10
20
30
40
Overall Survival in months
50
10
20
30
40
Event Free Survival in months
50
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BLOOD, 1 OCTOBER 2009 䡠 VOLUME 114, NUMBER 14
NPM1-MUTATED AML IS NOT INFLUENCED BY KARYOTYPE
3029
Figure 5. Survival curves of NPM1-mutated AML patients (NK vs AK) from the GIMEMA LAM99P and GIMEMA/EORTC AML12 trials. (A) No significant differences in
OS are observed between NPM1-mutated AML with NK and AK (P ⫽ .877). (B) No significant differences in EFS are observed between NPM1-mutated AML with NK and AK
(P ⫽ .827). (C) No significant differences in OS are observed between NPM1-mutated/FLT3-ITD⫺ AML with NK and AK (P ⫽ .814). (D) No significant differences in EFS are
observed between NPM1-mutated/FLT3-ITD⫺ AML with NK and AK (P ⫽ .970).
percentage alive at 2 years 74.5% vs 48.5%, P ⫽ .075; and median
EFS was 18.1 months vs 8.6 months, P ⫽ .176; Figure 4C-D).
Also in the subgroups of FLT3-ITD positive cases, no difference in
OS and EFS was observed between NPM1-mutated AML with NK
or AK (median OS 21.1 months vs not reached; percentage alive at
2 years 47.5% vs 83.3%, P ⫽ .245; and median EFS was 9.5 months
versus 14.9 months, P ⫽ .354; supplemental Figure 2).
The same analysis was done for the 186 GIMEMA LAM99P
and GIMEMA/EORTC AML12 patients who received the same
treatment (although enrolled in 2 consecutive studies). The CR rate
difference between NPM1-mutated AML with NK or AK was not
statistically significant (79.4 vs 76.0, respectively, P ⫽ .7). No
differences were observed both for OS and for EFS in the whole
population according to karyotype (Figure 5A-B). In addition,
considering separately the FLT3-ITD⫺ (Figure 5C-D) and the
FLT3-ITD⫹ cases (supplemental Figure 3), again, the outcome of
NPM1-mutated AML in terms of OS and EFS was not influenced
by the karyotype.
Discussion
This study clearly shows that chromosomal aberrations occur
infrequently in AML with mutated NPM1 (⬃ 15% of cases), are
different from the typical AML-associated recurrent genetic abnormalities (as defined by the 2008 WHO Classification),14 and are
likely represent secondary genetic events. Our results also indicate
that AML with mutated NPM1 carrying a NK or concomitant
chromosomal aberrations has overlapping biologic, pathologic,
immunophenotypic, and clinical features. Taken together, these
findings strongly suggest that AML with mutated NPM1, irrespective of concomitant chromosome abnormalities, represents a single
disease entity whose molecularly defining feature is the presence of
a mutated NPM1 gene. These results have also important diagnostic and clinical implications.
Several findings in this study point to chromosomal aberrations
detected in a minority of our patients, as secondary genetic events.
The type of chromosome aberrations found in NPM1-mutated
AML are mostly similar to the additional chromosome aberrations
detectable in AML with t(8;21), inv(16), t(15;17), or 11q23/MLLrearrangements, which are thought to be secondary alterations.26-28
In AML with cytoplasmic/mutated NPM1, cytogenetic studies
frequently showed mosaicism (ie, the presence of cells with an
abnormal karyotype as subclones within the population with a
NK).1 More importantly, a proportion of NPM1-mutated AML
cases carrying a NK at initial diagnosis acquired a chromosomal
aberration during the course of the disease, while retaining the
original NPM1 mutation. It should be added that in this study, a few
NPM1-mutated AML patients with AK at diagnosis showed either
clonal regression (AK to NK) or changing to a different AK at
relapse, while maintaining the original NPM1-mutated gene status.
Finally, we found that NPM1-mutated AML with NK or chromosomal aberrations have a very similar gene expression profile
characterized by the expected down-regulation of the CD34 and
CD133 genes and overexpression of most HOX genes.5,10,29
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3030
HAFERLACH et al
Secondariness of chromosomal aberrations in NPM1-mutated
AML is in line with the growing body of evidence that NPM1
mutation is a founder genetic lesion in AML, as supported by the
following observations: (1) cytoplasmic mutated nucleophosmin is
specific for AML1,30,31 and clinically shows close association with
AML of de novo origin1,32-34; (2) NPM1 mutations are mutually
exclusive of other recurrent genetic abnormalities in AML3 (with
the exception of rare cases in which both NPM1 and CEPBA
mutations coexist); (3) AML with mutated NPM1 shows distinctive
gene expression signatures5,10,29 and microRNA profiles6,7; (4) all
NPM1 mutations generate common changes at the C terminus of
nucleophosmin protein that appear to maximize nuclear export of
NPM1 leukemic mutants,1,35-37 pointing to their cytoplasmic dislocation as the central event for leukemogenesis36,38; (5) NPM1
mutations are stable during the course of the disease,39-41 as the
same type of NPM1 mutation is consistently detected at relapse in
medullary and extramedullary sites. Loss of NPM1 mutation has
been rarely observed in NPM1-mutated AML.42 These cases,
however, could represent secondary treatment-related AML rather
than relapse of the de novo AML43; and (6) quantitative real-time
polymerase chain reaction (PCR) shows that NPM1 mutations
disappear at CR.44,45
Interestingly, we found that the incidence of associated chromosomal aberrations was lower in AML with mutated NPM1 (14.7%)
than in AML with 11q23/MLL rearrangement, inv(16), t(15;17),
and t(8;21) (frequency range, 34%-70%). These findings and the
fact that approximately 85% of AML with mutated NPM1 carry a
NK suggest that this type of leukemia may be characterized by a
higher genomic stability than other leukemias listed in the group of
“AML with recurrent genetic abnormalities” of the WHO classification,14 such as AML with 11q23/MLL rearrangement, inv(16),
t(15;17), and t(8;21).
High genomic stability was recently observed in an NPM1mutated/FLT3-ITD positive AML patient who, at whole-genome
sequencing,46 showed no deletions or loss of heterozygosity (LOH)
and mutations of yet unknown significance of only 8 genes (in
addition to those affecting NPM1 and FLT3). The 8 mutated genes
were: CDH24 and PCLK (encoding protocadherin/caderin family
members), GPR123 and EBI2 (encoding G protein–coupled receptors), PTPRT (encoding a protein phosphatase), KNDC1 (encoding
a potential guanine nucleotide exchange factor), SLC15A1 (encoding a peptide/drug transporter), and GRINL1B (encoding a glutamate receptor gene). Notably, when the nonsense and missense
mutations affecting these 8 genes were searched in an additional
187 AML cases (including 43 carrying an NPM1 mutation), none of
them were detected.46 In our opinion, these results further support
the concept that NPM1 is a primary genetic lesion, because it is
specific for AML,1,31 is recurrent in approximately 30% of adult
AML,2 and defines a type of leukemia with distinctive biologic and
clinical features2 as well as unique gene expression5,10,29 and
microRNA signatures.6,7 On the contrary, the 8 additional mutated
genes are likely to represent secondary events that, in this particular
whole-sequenced patient,46 may signify either “passenger” or
cooperating genetic alterations.
One possible explanation for the high genomic stability of AML
with mutated NPM1 is that the NPM1 mutant alone induces
leukemic transformation. Notably, in transfected cells, NPM1
leukemic mutants decreased the activity of tumor oncosuppressor
alternative reading frame (ARF)47,48 and caused activation of the
MYC oncogene.49 When combined with haploinsufficiency for
wild-type NPM1,50 this double-edged sword could result into full
potential to induce leukemic transformation. This would be also in
BLOOD, 1 OCTOBER 2009 䡠 VOLUME 114, NUMBER 14
accordance with a recently developed one-mutation mathematical
model51 for NPM1-mutated AML. However, clarification of the
leukemogenic role of NPM1 mutants awaits the development of
appropriate animal models.
Our results have also important diagnostic and clinical implications. The finding that NPM1-mutated AML with NK or AK have
overlapping biologic, pathologic, immunophenotypic, and clinical
features justifies their inclusion in the new WHO classification, as a
single entity under the term of “AML with mutated NPM1.” 14
According to our results, cytogenetic status (NK vs AK)
seems to have no significant impact in the prognosis of patients
with NPM1-mutated AML. The most clinically relevant finding
was that FLT3-ITD negative NPM1-mutated AML patients
carrying a NK or an AK showed the same relatively favorable
prognosis. These results were based upon the comparison
between NPM1-mutated AML patients with NK or AK group, as
a whole. Thus, we cannot exclude that single sporadic chromosomal aberrations within the NPM1-mutated AK group may
have an impact on prognosis. Despite this potential limitation,
our findings raise the question of whether assessment of the
prognostic value of NPM1 and FLT3-ITD mutations in the
framework of NK8-13 represents the most rationale way for
risk-stratification of AML patients. In fact, this approach has
2 major limitations: (1) it excludes a significant number of AML
patients because of failure of cytogenetic analysis (up to 25%
in large multicenter studies, including GIMEMA/EORTC), and
(2) it prevents assignment of AML patients to the group with
favorable genotype (NPM1 mutated/FLT3-ITD negative) if a
chromosomal aberration is present. Thus, in line with the new
WHO classification,14 a patient should simply be classified as
“AML with mutated NPM1” (when carrying an NPM1 mutation
or aberrant cytoplasmic expression of nucleophosmin), independently of whether the karyotype is normal or not. In the absence
of FLT3-ITD, this patient should then be regarded as potentially
belonging to the good favorable prognostic group. This approach would also offer the opportunity to investigate in future
large multicenter clinical trials whether single, rare chromosomal abnormalities may affect the prognosis of NPM1-mutated
AML patients. Use of NK as initial framework for analysis of
other mutations or prognostic factors is likely to be more
important in the group of AML patients that does not carry
NPM1 mutations (⬃ 40% of all AML with NK), whose molecular nature still remains uncertain.
In conclusion, our studies further support the concept that AML
with mutated NPM1 represents a distinct entity and have important
diagnostic and clinical implications.
Acknowledgments
We are indebted to Prof. Martin Dugas, University of Münster,
Germany, for support in microarray analyses and to Profs.
Giuseppe Saglio, Francesco Lo Coco, Daniela Diverio, and
Fabrizio Pane for performing molecular analyses of the FLT3
gene. The authors from the Munich Leukemia Laboratory
(MLL) thank all physicians and especially participants of the
AML Cooperative Group (AMLCG) for sending bone marrow
or blood samples to the laboratory for reference diagnosis and
also for submitting clinical data.
This work was supported by the Associazione Italiana per la
Ricerca sul Cancro (AIRC). We thank Mrs Claudia Tibidò for
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BLOOD, 1 OCTOBER 2009 䡠 VOLUME 114, NUMBER 14
NPM1-MUTATED AML IS NOT INFLUENCED BY KARYOTYPE
secretarial assistance and Dr Geraldine Boyd for assistance in
editing the manuscript.
Authorship
Contribution: C.H. studied the cases from MLL cytogenetically and
contributed to the writing of the paper; C.M., M.M., A.C., N.T., and
G.R.-C. performed the cytogenetic studies on patients from the
GIMEMA/EORTC studies and contributed to the writing of the
paper; S.S. carried out the molecular studies on patients from MLL
and contributed to the writing of the paper; A.K. investigated the
gene expression profile of cases from MLL and contributed to the
writing of the paper; A.S. performed the statistical analyses on
patients enrolled in the GIMEMA/EORTC trials; M.P.M. character-
3031
ized the samples from the GIMEMA/EORTC patients by immunohistochemistry/Western blot analysis; M.V. and P.F. carried out the
prognostic analysis of the GIMEMA/EORTC patients; T.H. carried
out clinical and statistical correlations among AML patients from
the MLL and contributed to the writing of the paper; and B.F. had
the original idea for the study and wrote the paper.
Conflict-of-interest disclosure: B.F. and C.M. applied for a
patent on clinical use of NPM1 mutants. All other authors declare
no competing financial interests.
For a complete list of the Italian Group for Adult Hematologic
Diseases (GIMEMA) study group participants and AMLCG participant centers, see the supplemental appendices.
Correspondence: Brunangelo Falini, Institute of Hematology, University of Perugia, Ospedale S. Maria della Misericordia, S. Andrea delle
Fratte, 06132 Perugia, Italy; e-mail: [email protected].
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2009 114: 3024-3032
doi:10.1182/blood-2009-01-197871 originally published
online May 8, 2009
AML with mutated NPM1 carrying a normal or aberrant karyotype show
overlapping biologic, pathologic, immunophenotypic, and prognostic
features
Claudia Haferlach, Cristina Mecucci, Susanne Schnittger, Alexander Kohlmann, Marco Mancini,
Antonio Cuneo, Nicoletta Testoni, Giovanna Rege-Cambrin, Antonella Santucci, Marco Vignetti,
Paola Fazi, Maria Paola Martelli, Torsten Haferlach and Brunangelo Falini
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