Ijms 19 03492

International Journal of
Molecular Sciences
Review
Minimal/Measurable Residual Disease Monitoring in
NPM1-Mutated Acute Myeloid Leukemia: A Clinical
Viewpoint and Perspectives
Fabio Forghieri 1, *, Patrizia Comoli 2,3 , Roberto Marasca 1 , Leonardo Potenza 1 and Mario Luppi 1
1 Department of Medical and Surgical Sciences, Section of Hematology, University of Modena and Reggio
Emilia, Azienda Ospedaliero-Universitaria di Modena, Via del Pozzo 71, 41124 Modena, Italy;
[email protected] (R.M.); [email protected] (L.P.); [email protected] (M.L.)
2 Pediatric Hematology/Oncology Unit, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico
San Matteo, Viale Golgi 19, 27100 Pavia, Italy; [email protected]
3 Cell Factory, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico San Matteo,
Viale Golgi 19, 27100 Pavia, Italy
* Correspondence: [email protected]; Tel.: +39-059-4222447; Fax: +39-059-4222386

Received: 15 October 2018; Accepted: 3 November 2018; Published: 6 November 2018
Abstract: Acute myeloid leukemia (AML) with NPM1 gene mutations is currently recognized
as a distinct entity, due to its unique biological and clinical features. We summarize here the
results of published studies investigating the clinical application of minimal/measurable residual
disease (MRD) in patients with NPM1-mutated AML, receiving either intensive chemotherapy
or hematopoietic stem cell transplantation. Several clinical trials have so far demonstrated a
significant independent prognostic impact of molecular MRD monitoring in NPM1-mutated AML
and, accordingly, the Consensus Document from the European Leukemia Net MRD Working
Party has recently recommended that NPM1-mutated AML patients have MRD assessment at
informative clinical timepoints during treatment and follow-up. However, several controversies
remain, mainly with regard to the most clinically significant timepoints and the MRD thresholds
to be considered, but also with respect to the optimal source to be analyzed, namely bone marrow
or peripheral blood samples, and the correlation of MRD with other known prognostic indicators.
Moreover, we discuss potential advantages, as well as drawbacks, of newer molecular technologies
such as digital droplet PCR and next-generation sequencing in comparison to conventional RQ-PCR
to quantify NPM1-mutated MRD. In conclusion, further prospective clinical trials are warranted to
standardize MRD monitoring strategies and to optimize MRD-guided therapeutic interventions in
NPM1-mutated AML patients.
Keywords: NPM1-mutated acute myeloid leukemia; molecular minimal/measurable residual disease

monitoring; prognostic thresholds and timepoints; intensive chemotherapy; allogeneic hematopoietic
stem cell transplantation; clinical outcome
1. Introduction
The results of cytogenetic and molecular examinations at diagnosis of non-promyelocytic acute
myeloid leukemia (AML), combined with the achievement of morphologic complete remission (CR)
after remission induction chemotherapy, have significant prognostic impact on clinical outcome and
usually serve to guide post-remission therapeutic strategies in younger adult patients [1–3]. However,
the morphologic assessment of bone marrow (BM) blast percentage by light microscopy is significantly
hampered by limited sensitivity and inter-observer variability, so that it is generally recognized that,
when 5% myeloblasts is considered to be the cut-off to define morphologic CR, about 1010 leukemic
Int. J. Mol. Sci. 2018, 19, 3492; doi:10.3390/ijms19113492 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2018, 19, 3492 2 of 33
cells may still persist in the patient [3,4]. The risk stratification in AML patients actually remains
inadequate, and there is growing interest in the use of more sensitive laboratory tools, such as
multiparametric flow cytometry (MFC) or molecular techniques, to detect low levels of residual
disease in either BM or peripheral blood (PB) at different treatment timepoints [1]. The evaluation of
minimal residual disease, also referred to as measurable residual disease (MRD), is considered useful to
more precisely define AML response to intensive chemotherapy, thereby refining risk stratification [1].
Some studies have demonstrated that MRD persistence in a condition of morphologic CR confers high
relapse risk and adverse prognosis, comparable to the one associated with persisting leukemic cells
at microscopic examinations [4–6]. Interestingly, the recently revised European Leukemia Net (ELN)
recommendations for AML management have introduced the category CR without MRD (CRMRD− ) [7].
Moreover, long-term MRD monitoring in patients in CR may serve to early detect leukemia relapse [1].
MRD assessment captures the diversities of the underlying cytogenetic/genetic AML characteristics
and also recapitulates patients’ heterogeneity regarding chemotherapy bioavailability, metabolism
and resistance, thus resulting in a unique in vivo tool to evaluate chemosensitivity of leukemic
cells [4,8,9]. Moreover, even within homogeneous genetic subgroups, the long-term outcome of AML
patients depends on the clearance of the molecular lesion, as clearly demonstrated in other myeloid
neoplasms, namely chronic myeloid leukemia and acute promyelocytic leukemia [1,4]. Beyond
MFC, several molecular techniques are currently available for MRD determination in AML patients,
including real-time quantitative polymerase chain reaction (RQ-PCR), which is highly sensitive, reliable,
rapid and reproducible between different laboratories, as well as newer but less standardized tools such
as next-generation sequencing (NGS) and digital droplet PCR (ddPCR) [2,4]. RQ-PCR allows MRD
detection in patients with documentation of chimeric fusion genes generated by balanced chromosomal
rearrangements, especially in cases of acute promyelocytic leukemia and core-binding factor (CBF)
leukemias, but also in AML cases with other genetic alterations, such as insertions/duplications (e.g.,
NPM1, FLT3-ITD, MLL-PTD), point mutations (CEBPA, IDH1/2, KIT, RAS, RUNX1, TP53) or gene
overexpression (WT1, EVI1, ERG) [1,2,4]. While MFC, despite its limitations, could enable MRD
assessment in the vast majority of AML patients, a major drawback of RQ-PCR is its applicability only
to those cases, accounting for approximately 50–60% of younger AML patients, who bear at least one
molecular lesion, specific and stable over the treatment course, which could reliably be monitored
using this molecular technique [2,4,10].
2. NPM1 Mutations in AML: Biological and Clinical Features

Nucleophosmin (NPM1) gene encodes for a protein which physiologically shuttles between
nucleus and cytoplasm, acting as a molecular chaperone to establish multiple protein-protein
interactions [11]. NPM1 protein is normally involved in critical cell functions, such as control of
ribosome formation and export, stabilization of the oncosuppressor p14Arf protein in the nucleolus,
and regulation of centrosome duplication [12–14]. NPM1 gene mutations, occurring in approximately
30% of adult AML cases, and in 50–60% of AML cases with normal karyotype, represent one of the
most frequent molecular lesions observed in AML [14,15]. Since the discovery of NPM1 mutations
in 2005 by Falini et al. [16], more than 55 different mutations, mainly occurring in the exon 12 of the
gene, have been described, but three mutation types (A, B and D) account for 95% of all cases [8,14,17].
NPM1 gene mutations result in structural changes of the C-terminus of NPM1 protein, with subsequent
aberrant cytoplasmic delocalization, leading to a unique immunohistochemical pattern detectable
on BM trephine biopsy [16,18]. This cytoplasmic accumulation of NPM1-mutated protein causes
perturbations of multiple cellular pathways through a combination of loss of functions and gain of
functions, critical for leukemogenesis [12–14,17]. Notably, it was recently reported that NPM1-mutated
protein dislocated PU.1 into cytoplasm with it, whereas CEBPA and RUNX1, the master transcription
factors that collaborate with PU.1 to activate granulo-monocytic lineage-fates, remained in the nucleus.
However, without nuclear PU.1, their coregulator interactions were toggled from coactivators to
corepressors, thus repressing >500 granulocyte and monocyte terminal differentiation genes [19].
Int. J. Mol. Sci. 2018, 19, 3492 3 of 33
As expected for founder genetic lesions, NPM1 mutations are specific, being almost
exclusively restricted to AML, usually de novo, and generally expressed in the whole leukemic
population [13,14,20]. Notably, NPM1-mutated AML, showing distinctive genetic, pathologic,
immunophenotypic and clinical features, has now been recognized as a full distinct entity among
AML with recurrent genetic abnormalities in the 2016 revision of World Health Organization (WHO)
classification of myeloid neoplasms and acute leukemia [21]. While the infrequent presence of
coexisting chromosomal abnormalities, observed in only 15% of patients, does not appear to modify
the prognostic effects of NPM1 mutations [7,22,23], prognosis may be significantly influenced by
accompanying molecular lesions, mainly FLT3 and DNMT3A gene mutations, documented in about
40% and 50% of NPM1-mutated AML cases, respectively [7,15,17]. In detail, the better risk outcomes
observed in NPM1-mutated AML patients were generally considered limited to cases without
concurrent FLT3-ITD mutations [17,24]. Furthermore, the deleterious prognostic effects of FLT3-ITD
have previously been found to be most clinically relevant when co-occurring with NPM1 and DNMT3A
mutations, as opposed with either mutation alone [25]. However, recent studies have suggested that
patients with NPM1 mutation and FLT3-ITD with a low (<0.5) allelic ratio have a similar favorable
outcome as patients with NPM1-mutated AML without FLT3-ITD [26–29]. Thus, both of these latter
groups are currently considered favorable according to the 2017 ELN risk stratification, in contrast
to NPM1-mutated AML associated with FLT3-ITD with high allelic ratio, which is characterized by
higher relapse rate and poorer overall survival (OS) [7].
3. ELN Recommendations for MRD Assessment

Based upon the above-mentioned biological and clinical characteristics, especially their
homogeneous mutation pattern in AML patients, NPM1 mutations may be considered an ideal
leukemia-specific target for MRD detection [2]. Since the first application by Gorello et al. of
sensitive and specific RQ-PCR assays as a reliable system to quantitatively assess NPM1-mutated
gene copies [30], several studies have investigated the clinical implications of MRD monitoring in
NPM1-mutated AML undergoing intensive therapeutic approaches (Table 1) [30–73].
Int. J. Mol. Sci. 2018, 19, 3492 4 of 33
Table 1. MRD monitoring in NPM1-mutated AML series: review of the literature.
Reference/Type of Number of Patients/Median Number of Samples Number of Samples per Molecular Sensitivity of the
NPM1 Mutation Type
Study Age (Years, Range) (PB/BM) Patient (Median, Range) Method/Material Assay
NA (13 patients analyzed at
NA (PB and/or BM diagnosis and
Gorello et al., samples at diagnosis post-induction). MRD RQ-PCR/cDNA (5 cases), A, B (cDNA); A, B, D, E,
20/NA 10−3 –10−6
2006 [30]/retrospective and/or at different kinetics during follow-up of DNA (15 cases) G, H (DNA)
timepoints) 3 representative patients
is reported
Chou et al.,
38/47 (17–87) 194 BM 5 RQ-PCR/DNA 7 different mutations 10−5
2007 [31]/retrospective
NA (26 patients analyzed at
diagnosis and at least at
Papadaki et al., 2 timepoints during therapy;
51/58 (22–78) 154 (18 PB/136 BM) RQ-PCR/cDNA A 10−5
2008 [32]/retrospective 27 patients analyzed at
diagnosis and after
induction therapy)
Barragan et al.,
24/17 cases (71%) <60 yeas 97 (5 PB/92 BM) NA RQ-PCR/cDNA A 10−5
2008 [33]/-
Bacher et al.,
13/47 (20–66) 139 (PB/BM) 7 (2–25) RQ-PCR/DNA A, B 10−4 –10−6
Schnittger et al., 1227 (28 PB at
252/59 (20–79) 4 (2–16) RQ-PCR/cDNA 17 different mutations 10−4 –10−6
2009 [35]/retrospective diagnosis/1199 BM)
Stahl et al.,
25/53 (21–73) 76 (38 PB/38 BM) 1–2 RQ-PCR/DNA A 10−4 –10−6
Dvorakova et al.,
25/51 (43–75) 1026 (339 PB/687 BM) 28 (11–68) RQ-PCR/DNA 9 different mutations 10−4 –10−6
Ommen et al., 193 CCR and 70 relapse
180 (54 in HR)/NA NA RQ-PCR/cDNA NA 10−4 –10−6
2010 [38]/retrospective samples
Kristensen et al.,
20/61 (41–76) 204 NA RQ-PCR/DNA A 2.4 × 10−5
Kronke et al.,
245/49 (19–61) 1682 (410 PB/1272 BM) NA RQ-PCR/cDNA 6 different mutations 10−5 –10−6
3 different mutations
Thol et al., NGS, RQ-PCR/DNA,
10/NA (adult patients) 45 NA (8 cases A, 1 case D, 10−4
2012 [41]/retrospective cDNA
1 case atypical)
Int. J. Mol. Sci. 2018, 19, 3492 5 of 33
Table 1. Cont.
NPM1 Mutation Type
116 (20 at diagnosis,
Abdelhamid et al., 96 follow-up samples, 3 different mutations
20/55 (27–69) 4.5 (2–11) RQ-PCR/DNA 10−4 –10−5
2012 [42]/retrospective namely 55 PB and (A, B, insCACG)
41 BM)
Schiller et al., 30 (among 54 FLT3-ITD+
NA NA RQ-PCR/cDNA NA 10−4 –10−6
2012 [43]/retrospective patients)/62
Shayegi et al.,
155/51 (20–79) 1750 (817 PB/933 BM) NA RQ-PCR/DNA A, B, D 10−5
Jeziskova et al., 6 (among 8 patients with
60 (17 PB/43 BM) 3–14 RQ-PCR/DNA A, B 10−4 –10−6
2013 [45]/retrospective IDH2 mutations)/57 (53–72)
Salipante et al., No need for
6/NA 22 BM 2–6 NGS/DNA 10−5
2014 [46]/retrospective mutation-specific probes
Hubmann et al.,
158/57 (18–80) 588 BM NA RQ-PCR/cDNA A, B, D 10−6
Bacher et al.,
99/NA 498 4 (1–28) digital PCR/cDNA 37 different mutations 10−4 –10−5
Lambert et al., 77 patients with NPM1
250 (125 PB/125 BM) NA RQ-PCR/cDNA A, B, D 10−5
2014 [49]/prospective mutation/61 (57–65)
RQ-PCR for NPM1; NGS
Debarri et al.,
31/60 (23–70) 94 NA for IDH1/2 and A, B, D 10−5
DNMT3A/cDNA
Pettersson et al.,
19/64 (28–78) 63 (2 PB/61 BM) 1–9 RQ-PCR/DNA A 10−5
2016 [51]/-
Karas et al.,
60/54 (30–66) 60 BM 1 (pre-HSCT) RQ-PCR/cDNA A, B, D NA
Alizad Ghandforoush
et al., 11/42 (28–63) 71 (PB/BM) NA RQ-PCR/DNA A 10−5
346 in preliminary
Ivey et al., 2569 in first phase
development phase, 91 in 6 RQ-PCR/cDNA 27 different mutations 10−5
2016 [54]/prospective (1667 PB/902 BM)
validation cohort/50 (6–68)
Int. J. Mol. Sci. 2018, 19, 3492 6 of 33
Table 1. Cont.
NPM1 Mutation Type
pre-transplant MRD was
Kayser et al., 406 (BM) at different available for 39/51 (76.4%)
67/55 (21–70) RQ-PCR/cDNA A, B, D 10−5 –10−6
2016 [55]/retrospective timepoints patients in CR at HSCT
(22 cases, 56% MRD-positive)
Malmberg et al., 17 (3 NPM1-mutated
NA NA RQ-PCR, NGS/DNA A 10−5
2017 [56]/retrospective patients)/39 (2–71)
304 (PB/BM) at
samples obtained at
Balsat et al., diagnosis and 270
152/49 (21–61) diagnosis and after RQ-PCR/cDNA A, B, D 10−5
2017 [57]/retrospective post-induction
induction CHT
(135 PB/135 BM)
4 PB/BM samples (at
Schieppati et al., diagnosis, TP1 at CR, TP2
68/56 (27–74) NA RQ-PCR/cDNA NA 10−4 –10−5
2017 [58]/- post-consolidation, TP3 post
1st cycle of Ara-C)
multiplex assay effective
Mencia-Trinchant et al., Sequential determination of in a range of diverse
3/- NA ddPCR/cDNA 10−4 –10−5
2017 [59]/- MRD levels common and rare NPM1
mutations (14)
58 BM at diagnosis,
Getta et al., 104 (10 with NPM1
83 BM before HSCT NA NGS, MFC/DNA 2 different mutations 10−4
2017 [60]/retrospective mutation)/58 (21–78)
for NGS
Bill et al., samples collected directly
51/62 (33–74) 51 (40 PB/11 BM) ddPCR/cDNA A, D 10−4
2018 [61]/retrospective before HSCT
482 PB/BM samples at
Jongen-Lavrencic et al., 430 (168 with NPM1
diagnosis, 430 BM 2 (at diagnosis and in CR) NGS, MFC/DNA NA 10−4
samples after treatment
Zhou et al.,
59/57(21–79) 104 BM pre-HSCT and post-HSCT NGS, MFC/DNA - 10−4
Zappasodi et al., Availability of samples
201 (116 with NPM1
2018 [64]/retrospective, NA during treatment and RQ-PCR/cDNA NA 10−4 –10−5
mutation)/58
real-life study follow-up was variable.
Int. J. Mol. Sci. 2018, 19, 3492 7 of 33
Table 1. Cont.
NPM1 Mutation Type
all recurrent insertion
Delsing Malmberg et al., 3 (at diagnosis, before and mutations in NPM1 exon
29/49 (18–66) 83 (6 PB/77 BM) NGS/DNA 10−4
2018 [65]/retrospective after HSCT) 12 (mutation A in
25 cases)
NA (samples analyzed at
Kapp-Schwoerer et al., 6339 (2812 PB/
611/18–60 diagnosis, during treatment RQ-PCR/cDNA NA 10−5 –10−6
2018 [66]/retrospective 3527 BM)
and follow-up)
Caprioli et al.,
27/57 (23–65) 27 BM 1 (pre alloHSCT) RQ-PCR/cDNA NA 10−4
Patkar et al.,
83/NA NA NA NGS/DNA 12 different mutations 10−5
106 BM (51 after
Onecha et al., 63 (57 with NPM1
induction, 55 post 2 NGS/DNA A 10−5
consolidation CHT)
90 (22 positive for IDH1/2 NA (90 at diagnosis, 22 after
Petrova et al., RQ-PCR for NPM1
mutations, 11 with NPM1 149 BM induction, 37 during A NA
2018 [70]/retrospective mutations/cDNA
mutation)/61 (22–82) follow-up)
34 with newly diagnosed MRD assessment
Prata et al.,
NPM1-mutated AML/77 available on BM NA RQ-PCR/NA A, B, D NA
(55–85) samples in 6 patients
NA (BM samples at NA (NPM1-mutated
Ottone et al., 556 de novo AML (177 with
diagnosis, during transcripts monitoring in RQ-PCR/cDNA A 10−5
2018 [72]/retrospective NPM1 mutation)/49 (16–89)
follow-up, at relapse) 51 cases)
Multiplex analysis of
34 cytogenetically normal 34 BM or PB at 2 (at diagnosis and after at
Gaksch et al., NGS/DNA extracted 19 genes, including
AML (16 cases with NPM1 diagnosis, 27 BM least one 10−2
2018 [73]/retrospective from BM slides NPM1 mutational
mutation)/47 (22–79) samples in remission consolidation therapy)
hotspots
MRD, minimal/measurable residual disease; AML, acute myeloid leukemia; PB, peripheral blood; BM, bone marrow; NA, not available; RQ-PCR, real-time quantitative polymerase chain
reaction; cDNA, complimentary DNA; HR, hematological relapse; CCR, continuous complete remission; NGS, next-generation sequencing; HSCT, hematopoietic stem cell transplantation;
CR, complete remission; TP, timepoint; ddPCR, digital droplet PCR; MFC, multiparametric flow cytometry; CHT, chemotherapy.
Int. J. Mol. Sci. 2018, 19, 3492 8 of 33
The consensus document from the ELN MRD Working Party indicates that AML patients with
NPM1 mutations, such as patients with RUNX1-RUNX1T1, CBFb-MYH11 or PML-RARA fusion
transcripts, should have molecular MRD assessment at informative clinical timepoints. During the
active treatment phase, MRD assessment for these molecular lesions is recommended at minimum at
diagnosis, after 2 cycles of induction/consolidation chemotherapy, and at the end of treatment [74].
Furthermore, after the end of treatment, samples for MRD analyses should in general be collected
every 3 months for 24 months. Thereafter, monitoring beyond 2 years of follow-up should be based on
the relapse risk of the patient and decided individually [74]. Specifically regarding NPM1-mutated
AML, with or without other concomitant mutations, monitoring of NPM1 transcripts is recommended
in BM and PB, if possible. If NPM1-mutated MRD remains negative in PB but positive in BM after
the end of treatments, transcripts should be closely monitored every 4 weeks for at least 3 months,
in order to evaluate any MRD increase. Conversely, if a rising MRD is not confirmed or MRD becomes
undetectable, then MRD retesting may be regularly performed at 3-month intervals for at least the first
2 years after the end of treatment [74].
At first glance, the clinical management of NPM1-mutated AML patients according to these
recommendations could appear clearly defined. However, several controversial issues remain,
especially with regard to the most clinically significant timepoints and MRD thresholds to be
considered, but also with respect to the best source to be analyzed, namely BM or PB samples,
and correlation of MRD with other known prognostic indicators (Table 2) [8].
Int. J. Mol. Sci. 2018, 19, 3492 9 of 33
Table 2. Clinical impact of MRD monitoring in NPM1-mutated AML patients.
Median Time
Intensive Correlation with
Significant MRD since Molecular
Reference CHT/HSCT Prognostic Timepoints Other Molecular Clonal Evolution Clinical Relevance
Threshold to Morphologic
(No. of Patients) Markers
Relapse (Range)
- NPM1-mutated copies closely
Gorello et al., NA (3/5 cases with MRD correlated with clinical status
Yes/NA NA NA NA NA
2006 [30] <1% long-term survivors) and predicted impending
hematologic relapse in 2 cases
- Any rise of mutant signals
during follow-up increased
relapse risk
Chou et al., 38 Yes/11 End of consolidation; FLT3-ITD 4.9 months - MRD < 0.1% predicted longer
0.1% No RFS and OS
2007 [31] alloHSCT follow-up worsened RFS (1–12.3)
- Failure to achieve 2 logs
reduction after consolidation
predicted shorter RFS and OS
- In selected patients, it was
NA (median log10
In 2/21 relapses possible to correlate the
Papadaki et al., 50 Yes/11 reduction of 2.48 post
NA NA (9.5%) NPM1 NA changes of the NPM1 mutation
2008 [32] alloHSCT induction correlated with
mutation was lost. A levels with the clinical course
response to therapy)
of the disease
- MRD negativity or
NA (in 19 patients in CCR Expression levels maintenance of very low levels
MRD increase 1 to
a median 3% MRD after of WT1 and NPM1 of NPM1 mutation in patients
Barragan et al., 5 months before
24 Yes/NA induction was shown. NA showed strong No in continuous CR
2008 [33] relapse in 4/6
Median MRD level after positive
cases - Increase in NPM1 transcripts
consolidation 0.3% correlation.
before or at the time of relapse
- After HSCT 10/14 cases (71%)
All 4 patients (29%) with Correlation with PCR-negative, of which 4
Bacher et al., achieved stable CR.
-/13 alloHSCT NA persistent MRD positive molecular No 24 days (12–38)
2009 [34]
after HSCT relapsed chimerism. - MRD increase preceded
morphologic relapse
- early assessment
- MRD for NPM1 mutation
(days 18–60)
0.01% during 1st line assessed at 4 different time
- days 60–121 FLT3-ITD intervals after start of therapy
Schnittger et al., 252 Yes/53 treatment.0.1% after
- days 121–365 prognostic factor No 62 days (15–221) (mainly after day 60) is an
2009 [35] alloHSCT HSCT and during 2nd
- longer than 1 year affecting EFS independent and highly
line treatment.
after start predictive parameter for EFS.
of treatment
Int. J. Mol. Sci. 2018, 19, 3492 10 of 33
Table 2. Cont.
Median Time
Relapse (Range)
- Concordant results in BM and
High rate of PB in 60% of sample pairs.
Stahl et al., congruent results - Cases with <0.01% MRD in BM
-/25 alloHSCT NA Post-HSCT follow-up No NA
2010 [36] with chimerism were negative in PB.
analysis - Higher MRD levels in BM
(>0.2%) predicted PB positivity
Reappearance of NPM1 - Molecular relapse preceded

mutation or one order hematological relapse in 80% of
Dvorakova et al., 25 Yes/4
NCN increase in patients NA NA No 97 days (12–141) evaluable patients.
2010 [37] alloHSCT
with persistent positivity - Strong correlation between PB
at any timepoint. and BM samples
- Mathematical model to
More rapid MRD 120 days without determine the frequency of
Ommen et al., 0.005% (threshold to relapse detection
NA/NA NA growth in No FLT3-ITD, 65 days
2010 [38] define molecular relapse) - Sampling every 4 and 6 months
FLT3-ITD+ cases in FLT3-ITD+ cases
suggested in FLT3-ITD+ and
negative cases, respectively
- All relapses were associated

No (NPM1 with high levels of
Reoccurrence of NPM1 NPM1 mutation is
mutation stability. NPM1 mutation
Kristensen et al., mutation at any time superior to WT1
20 Yes/NA NA Karyotype 46 days (20–182) - Detectable NPM1 mutation
2011 [39] was associated with expression levels
evolution in 56% following a CR period was
relapse. as MRD marker.
of relapses). accompanied by morphologic
relapse in all cases
- Observation of higher
NPM1-mutated transcript
- -MRD negativity In 5 patients levels after double induction or
after 2 FLT3-ITD NPM1 mutation after completion of
Kronke et al., 245 Yes/80 induction cycles significant factor was not detectable 2.6 months consolidation therapy was a
2% significant factor for higher risk
2011 [40] alloHSCT - -After completion for inferior at relapse (9% of (0.4–23.6)
of therapy survival. evaluable relapse of relapse and death.
- -During follow-up samples) - Serial post-treatment MRD
assessment allowed early
detection of relapse
Int. J. Mol. Sci. 2018, 19, 3492 11 of 33
Table 2. Cont.
Median Time
Relapse (Range)
- Parallel assessment of MRD by
NGS and RQ-PCR was
NA NPM1 mutation concordant in 95% of
Thol et al., (mean allelic ratio at not detectable in analyzed samples
10 Yes/NA NA NA NA
2012 [41] diagnosis 0.37, 1/4 patients at
- NGS as a potentially highly
range 0.29–0.46) relapse.
flexible and reliable tool to
assess MRD
Similar kinetics of
FLT3-ITD, NPM1
- The 3 MRD markers tested
Abdelhamid and WT1 NA for NPM1
20 Yes/NA NA After induction therapy No showed comparable kinetics in
et al., 2012 [42] expression for mutation
17/20 (85%) cases
predicted clinical
trend.
MRD for FLT3-ITD - MRD negativity predicted
NA (samples collected as sensitive as lasting remission independent
54 (30 NPM1-
Schiller et al., at diagnosis, during other MRD
mutated) Yes/7 NA NA NA of alloHSCT or non-alloHSCT
2012 [43] treatment and parameters as
alloHSCT - FLT3-ITD analyses equivalently
follow-up) NPM1 mutations
sensitive in PB samples
or MLL-PTD
- 121 days
Prognostic role of - Rising of MRD was associated
(70–172) for
-MRD level >1% after MRD remained with increased risk of relapse.
Shayegi et al., 155 Yes/40 After intensive CHT MRD > 1%
conventional CHT-MRD significant after No - DFS and OS analyses revealed
2013 [44] alloHSCT and after HSCT - 66 days
level >10% after alloHSCT adjustment for significantly worse outcomes in
(34–98) for
FLT3-ITD status patients with rising MRD levels
MRD > 10%
Concordance of
quantitative - In 5/6 patients, the kinetics of
Jeziskova et al., 8 Yes/4 detection of IDH2 IDH2 quantification was nearly
NA NA NA NA
2013 [45] alloHSCT and NPM1 identical to the kinetics of
mutations, except NPM1 mutations
for on case.
Int. J. Mol. Sci. 2018, 19, 3492 12 of 33
Table 2. Cont.
Median Time
Relapse (Range)
- As a proof of principle, NGS
In 2 patients
documented MRD in all
genetically distinct
Salipante et al., samples deemed negative by
6 Yes/NA NA NA NA NPM1 tumor NA
2014 [46] flow cytometry, without the
subclones were
need for
detected
mutation-specific probes
- Assessment of MRD levels after
Prognostic role of NPM1 mutation
induction was significant to
Hubmann et al., 158 Yes/30 cut-off ratio 0.01 and 3-log MRD regardless of not detectable in 3
After induction CHT 58 days (20–98) identify patients in CR with
2014 [47] alloHSCT reduction ELN risk of 45 (6.7%)
high risk of relapse. There was
stratification. patients at relapse
also a trend for OS
- NPM1 mutation levels by
- days 18–60 digital and conventional
RQ-PCR significantly
Bacher et al., - days 61–120
99 Yes/NA 0.01% NA No NA correlated a diagnosis and
2014 [48] - days 121–365 during follow-up
- >day 365 - digital PCR threshold of 0.01%
prognostically relevant.
- WT1 MRD associated with
increased hazard of relapse and
After adjustment shorter OS from CR.
- after for FLT3-ITD
- Positive NPM1-mutated MRD
Lambert et al., induction CHT status, the effect of
77 Yes/NA 0.1% (in BM samples) No NA predicted higher risk of relapse,
2014 [49] - at the end NPM1 MRD
but did not influence OS
of treatment appeared to be
- Achievement of negative NPM1
similar.
mutation MRD significantly
more frequent in GO arm
Analysis of - IDH1/2 mutational status by
- post correlation One patient with NGS predicted relapse or
induction CHT between NPM1 IDH2 mutation disease evolution in 100% cases
Debarri et al.,
31 Yes/NA 0.1% - post first and mutations and developed a NA - DNMT3A mutations not
2015 [50]
second IDH1/2 or NPM1-negative correlated with disease status
consolidation courses DNMT3A MDS (a preleukemic clone persisted
mutations in 40% of cases in CR)
Int. J. Mol. Sci. 2018, 19, 3492 13 of 33
Table 2. Cont.
Median Time
Relapse (Range)
One of 8 relapsing - RQ-PCR of NPM1 A type
Pettersson et al., patients developed mutation was more sensitive
15 Yes/5 HSCT 0.1% During follow-up NA NA
2016 [51] a NPM1-negative and reliable than MFC for
relapse determination of MRD
- older age and pre-transplant
FLT3-ITD MRD had independent
4 months (3–13) prognostic impact on EFS
positivity had no
from HSCT to and OS
pre-transplant (less than adverse effect on
Karas et al., relapse, especially
-/60 alloHSCT 0.1% 1 week prior to start HSCT outcome in No - 3-year relapse rate, EFS and OS
2016 [52] in patients with
conditioning regimen) patients with AML were 6%, 72%, 75% with
high preHSCT
with NPM1 low-level MRD and 48%, 35%,
MRD
mutation in CR 40% in patients with higher
MRD levels
- Relapse occurred in 6 (54.5%)
Alizad patients, whose NPM1
11 Yes/6
Ghandforoush <5 log reduction during follow-up NA No NA mutation levels showed less
alloHSCT
et al., 2016 [53] than 5 log reduction
after treatment
- Persistence of NPM1 mutated
Presence of transcripts, observed in 15% of
FLT3-ITD or patients after second CHT cycle,
NPM1 mutations associated with greater risk of
DNMT3A
Ivey et al., 346 + 91 Yes/82 after second CHT cycle detectable in 133 days (BM), 87 relapse after a 3-year follow-up
0.01% mutations did not
2016 [54] alloHSCT (PB samples) 69/70 (99%) days (PB) and lower survival rate
provide additional
patients at relapse
prognostic (24% vs. 75%)
information - MRD as the only independent
prognostic factor for death
- Significant difference in OS
after alloHSCT between
pre-transplant MRD-positive
and MRD-negative patients
FLT3-ITD status (estimated 5-year OS rates
Kayser et al., 40% vs. 89%)
-/67 alloHSCT 1% prior to alloHSCT had no impact on No NA
2016 [55]
prognosis - Outcome of patients with
preHSCT MRD positivity as
poor as that of cases
transplanted with
refractory disease
Int. J. Mol. Sci. 2018, 19, 3492 14 of 33
Table 2. Cont.
Median Time
Relapse (Range)
- For mutation load of NPM1,
good correlation between
results from deep sequencing
Malmberg et al., CPS1, FAM193A, and RQ-PCR
NA/NA 0.001% NA NA NA
2017 [56] ITGB7
- Targeted deep sequencing more
sensitive for MRD
quantification than MFC
Abnormal - Patients without early MRD
karyotype, reduction had higher incidence
FLT3-ITD, PB of relapse and shorter OS
Balsat et al., 152 Yes/44 4-log reduction in PB post-induction CHT
MRD associated No NA - In patients with non favorable
2017 [57] alloHSCT MRD cycle
with higher AML, HSCT improved
relapse incidence outcomes only in case of <4-log
and shorter OS. reduction PB-MRD
FLT3-ITD did not - Molecular MRD monitoring of
Schieppati et al., 68 Yes/9
0.5% TP1 (BM), TP3 (PB) impact on No NA crucial importance in detecting
2017 [58] alloHSCT
relapse risk relapse at an early stage
- Novel ddPCR technique
composed of massively
multiplex pools of
insertion-specific primers that
Mencia-Trinchant selectively detected virtually all
3 Yes/- NA NA NA No NA
et al., 2017 [59] NPM1 mutations in a manner
that was robust to clonal
heterogeneity and did not
require NPM1
sequence information
- Mutations in DNMT3A, TET2,
JAK2 less likely to be cleared
than NPM1 (negative in 8/9
Getta et al., cases), IDH1/2, FLT3-ITD
-/104 alloHSCT <5% VAF before HSCT NA No NA
2017 [60]
- MRD detected concurrently
with MFC and NGS conferred
the highest relapse risk
Int. J. Mol. Sci. 2018, 19, 3492 15 of 33
Table 2. Cont.
Median Time
Relapse (Range)
- 17/51 (33.3%) patients were
Adverse MRD positive before HSCT
prognostic role of
101 days after - The 2-year cumulative
NPM1 mutation
Bill et al., HSCT (median incidence of relapse was 64.7%
51 Yes/51 HSCT 0.01% prior to HSCT MRD independent NA
2018 [61] time to relapse for vs. 6% translating into OS
of other known
all patient cohort) 38.8% vs. 71.7% in pre HSCT
prognostic
MRD+ and
markers
MRD-negative, respectively
- mutations persisted in 51.4% of
samples obtained patients during CR
during a defined period - Persistent DTA mutations not
Jongen-Lavrencic of remission, between correlated with increased
430 (168) Yes/ 2.5% allele frequency - NA NA
et al., 2018 [62] 21 days and 4 months relapse rate
after start of second - Persistent non-DTA mutations
treatment cycle conferred higher relapse rates
and lower survival rates
- Before HSCT MRD detected by
MFC was the most significant
Peri-HSCT MRD risk factor for relapse
Zhou et al., pre-HSCT and post
-/59 alloHSCT 0.01% independent No NA
2018 [63] HSCT (around day +28) - NGS testing of NPM1-mutated
prognostic factor
post-HSCT improved the risk
assessment of relapse
- molecular CR obtained in
73.7% of NPM1-mutated cases
MRD negativity at any
Zappasodi et al., 201 Yes/4 - molecular CR at the end of
NA time, during or after the NA No NA
2018 [64] alloHSCT treatment prognostic factor for
end of 1st line treatment
DFS and OS in
NPM1-mutated AML
MRD was an - Post-HSCT deep sequencing
4.5 months (3.5–11)
Delsing independent risk MRD status was significantly
for MRD+; 7.7
Malmberg et al., -/29 alloHSCT 0.02% pre and post-HSCT factor associated No associated with clinical
months (7–32) for
2018 [65] with clinical outcome (3-year OS 20%
MRD-cases
outcome vs. 89%)
Int. J. Mol. Sci. 2018, 19, 3492 16 of 33
Table 2. Cont.
Median Time
Relapse (Range)
NPM1 MRD - Outcome of patients who
negativity became MRD negative or
associated with remained positive but had 2017
lower relapse rate ELN favorable risk profile was
Kapp-Schwoerer 611 Yes/162 post two CHT cycles superior after high-dose AraC
2% and better OS No NA
et al., 2018 [66] alloHSCT (BM) consolidation than
independent of
DNMT3A and after alloHSCT
FLT3-ITD - MRD monitoring useful to
mutations inform post-remission therapy
- molecular analysis had higher
Caprioli et al., sensitivity than MFC
-/27 alloHSCT 0.01% pre-alloHSCT NA No NA
2018 [67] - Low-level or negative MRD
associated with improved LFS
- NGS was an useful test for
prediction of relapse
NGS-MRD for and survival
1-log cut-off between post NPM1 mutations
Patkar et al., post induction and post - NGS and MFC may be
83 Yes/- induction and post as the most No NA
2018 [68] consolidation complementary, with patients
consolidation independent
MRD-negative by both
prognostic factor
techniques having
excellent outcome
- MRD+ status post induction
and post consolidation
Higher risk of associated with lower OS and
0.1% post death in subjects shorter DFS and OS (33% vs
Onecha et al., 50 Yes/7 post induction and post
induction/0.025% post with advanced age, No NA 81%), respectively
2018 [69] alloHSCT consolidation
consolidation MRD+ status or
- NGS improved the capacity to
with FLT3-ITD
predict AML outcome over
MFC or RQ-PCR
Int. J. Mol. Sci. 2018, 19, 3492 17 of 33
Table 2. Cont.
Median Time
Relapse (Range)
- ddPCR more sensitive than
NPM1 mutation NGS for IDH1/2 MRD detection
MRD more
Petrova et al., - In the absence of more sensitive
22 Yes/NA NA NA sensitive than NA NA
2018 [70] markers, IDH1/2 mutations can
IDH1/2-based
be used as reliable markers for
MRD
MRD monitoring
- Overall response rate 45% (CR
>3 log NPM1 MRD No factors, in 23.5% of patients)
0/0 reduction observed in including type of
- Median OS 280 days
(34 patients 4/6 patients after HMA, FLT3 and
Prata et al., - No difference in OS between
received upfront NA 6 cycles, but three of IDH1/2 status, NA NA
2018 [71] cohorts with or without NPM1
therapy with them relapsed within were prognostic of
mutations suggesting limited
HMA) <6 months of this response or
therapeutic impact of HMA in
reduction. survival
NPM1-mutated AML
- NPM1-mutated transcripts
Evaluation of levels correlated with disease
Ottone et al., correlation with course, as a reliable
177 Yes/NA NA NA NA NA MRD marker
2018 [72] DNMT3A and
FLT3 mutations - DNMT3AR882H levels did not
reflect AML status
In multivariate
analysis including - Persistence of non-DNMT3A
age, leukocyte mutations was significantly
count and genetic associated with higher risk of
risk, residual AML relapse and shorter RFS,
Gaksch et al., After at least one
34 Yes/- VAF <0.5% disease positivity NA NA
2018 [73] consolidation cycle with a trend for shorter OS.
remained
- Strong concordance between
statistically
NGS and ddPCR for
significant as an
NPM1 mutations
adverse factor
for RFS
MRD, minimal/measurable residual disease; AML, acute myeloid leukemia; CHT, chemotherapy; HSCT, hematopoietic stem cell transplantation; NA; not available; alloHSCT, allogeneic
HSCT; RFS, relapse-free survival; OS, overall survival; CR, complete remission; EFS, event-free survival; BM, bone marrow; PB, peripheral blood; NCN, normalized copy number;
NGS, next-generation sequencing; RQ-PCR, real-time quantitative polymerase chain reaction; DFS, disease-free survival; ELN, European Leukemia Net; GO, gemtuzumab ozogamycin;
MFC, multiparametric flow cytometry; TP, timepoint; ddPCR, digital droplet PCR; DTA (DNMT3A, TET2, ASXL1); VAF, variant allele frequency; LFS, leukemia-free survival, HMA,
hypomethylating agents (azacitidine, decitabine, guadecitabine).
Int. J. Mol. Sci. 2018, 19, 3492 18 of 33
4. Prognostic MRD Thresholds and Relevant Timepoints for NPM1 Mutations

As above mentioned, several clinical trials have so far validated the clinical relevance of molecular
MRD monitoring in NPM1-mutated AML patients (Table 2), trying to identify prognostic MRD
thresholds and most relevant timepoints for collecting either BM or PB samples [30–73]. Schnittger et al.
retrospectively analyzed 1227 diagnostic and follow-up samples in 252 NPM1-mutated AML patients
by RQ-PCR assays [35]. A total of 47 relapses were predictable due to NPM1 mutation levels increase
of at least 1 log or in 15 cases because of a less than 3-log reduction in NPM1 mutation levels.
High prognostic value for MRD monitoring was documented at four different intervals after therapy
initiation (namely 18–60 days, 61–120 days, 121–365 days, >365 days), with levels of residual NPM1
transcripts as the most relevant factor affecting event-free survival (EFS) in multivariate analysis,
also in the subset of patients undergoing hematopoietic stem cell transplantation (HSCT). Moreover,
MRD thresholds of 0.01% and 0.1% NPM1-mutated/ABL1 obtained during first-line treatment and after
HSCT or second-line therapy, respectively, discriminated between prognostic subgroups, but without
influencing overall survival (OS) [35]. Another German-Austrian group evaluated MRD prognostic
role in 245 intensively treated adults with NPM1-mutated AML [40]. Achievement of RQ-PCR
negativity after double induction therapy identified patients with low 4-year cumulative incidence of
relapse (CIR), namely 6.5% compared with 53%, for subjects with persisting MRD positivity. This also
translated into significant differences in OS (90% versus 51%, respectively; p = 0.001). Furthermore,
according to Kronke et al., NPM1-mutated transcript levels were an independent prognostic factor also
when analyzed after completion of consolidation therapy (4-year OS 80% and 44% for MRD-negative
and MRD-positive patients, respectively) and during follow-up, when serial MRD monitoring allowed
early prediction of AML relapse in patients exceeding an arbitrary cut-off value of greater than 2%
NPM1-mutated/ABL1 [40]. Shayegi et al. subsequently investigated the prognostic impact of different
NPM1-mutated MRD cut-off values, among 155 AML patients treated within intensive treatment
protocols [44]. They found that MRD levels >1% after completion of conventional chemotherapy
were associated with increased relapse risk and significantly worse survival outcomes, whereas
for patients tested after having undergone HSCT, the best prognostic cut-off value for disease-free
survival (DFS) and OS was 10% NPM1-mutated/ABL1 [44]. Interestingly, Hubmann et al. monitored
molecular MRD during aplasia, after induction and consolidation chemotherapy, and during follow-up
in 158 patients with NPM1-mutated AML [47]. The assessment of MRD, with a cut-off ratio of 0.01 for
absolute NPM1-mutated ratios and a 3-log relative reduction, after induction therapy resulted the most
appropriate checkpoint in order to identify patients in CR at high risk of relapse. Indeed, the authors
observed 2-year CIR 77.8% and 26.4% for MRD-positive and MRD-negative after induction, respectively.
In this series, early molecular response thus seemed to be more prognostically relevant than persistent
MRD positivity after consolidation treatments [47]. Subsequently, Ivey et al. reported serial prospective
NPM1 mutation MRD monitoring in 346 AML patients enrolled in NCRI AML17 trial [54]. The major
finding of this large clinical study was that persistence of NPM1-mutated transcripts in PB samples
exceeding a value of 0.01% NPM1-mutated/ABL1 after the second chemotherapy cycle was associated
with a greater risk of relapse at a 3-year follow-up than was the absence of such mutated transcripts
(82% versus 30%, respectively; p < 0.001). Notably, MRD positivity after two treatment cycles was
observed in 15% of patients and also translated into lower OS rates in these subjects compared with
MRD-negative patients (24% versus 75%, respectively; p < 0.001). Additionally, the presence of MRD
at this testing timepoint was the only independent prognostic factor for death in multivariate analysis
and superseded other adverse baseline cytogenetic or molecular factors, such as FLT3-ITD or DNMT3A
mutations [54]. More recently, Balsat et al. reported on MRD evaluation in 152 NPM1-mutated AML
patients, showing that patients who did not achieve a 4-log reduction in NPM1-mutated transcripts in
PB samples after induction therapy (45% of study cohort) had a higher 3-year CIR (65.8% versus 20.5%)
and a shorter 3-year OS (40.8% versus 91.2%) compared with patients who obtained an adequate
MRD reduction [57]. Interestingly, the outcome of patients with a positive PB or BM MRD but with
>4-log reduction in PB MRD after induction treatment had a similar risk of relapse to subjects with
Int. J. Mol. Sci. 2018, 19, 3492 19 of 33
both MRD negativity and >4-log reduction in PB MRD. Notably, in the subgroup of patients with
FLT3-ITD, only age, white blood cell (WBC) count and <4-log reduction in PB NPM1-mutated MRD
after induction, but not FLT3-ITD allelic ratio, were identified as prognostic factors. Among the
71 patients with non-favorable risk AML according to ELN risk stratification, DFS and OS were
significantly improved by allogeneic HSCT only in those with a <4-log reduction in PB MRD, whereas
this benefit was not documented in those with an MRD clearance >4-log, thereby suggesting the
potential use of early MRD investigation in PB as a predictive factor for HSCT indication [57].
Collectively, the results of the studies presented above confirmed that RQ-PCR is a reliable
molecular tool to assess MRD in most patients with NPM1-mutated AML [35,40,44,47,54,57]. The MRD
monitoring, by either a kinetic measure [32,35,47,53,57,68] or the obtainment of an absolute threshold of
NPM1-mutated transcripts [31,33,35,38,40,44,47–52,54–56,58,61,63,65–67,69], was overall predictive of
relapse and survival outcomes (Table 2). However, due to differences in patients’ cohorts and the lack
of standardization of NPM1-mutated RQ-PCR MRD assays and analytical methods, any comparison
between different studies remains extremely difficult, and evidence-based conclusions on the best MRD
thresholds and timepoints cannot be definitely drawn [8,44,75]. Interestingly, in the study by Balsat et
al., a positive MRD determination in BM and concurrent negativity in PB samples was found in 24.6%
of cases [57], in concordance with the 14.5% reported by Shayegi et al. in MRD samples collected at
different timepoints [44]. Absolute differences in NPM1-mutated RQ-PCR sensitivity generally favor
BM over PB on the order of 0.6–1.5 log10 [44,54,57,75], so that BM aspirate was initially considered
to be the best source to investigate MRD [75]. Also, in the series by Ivey et al., serial monitoring of
PB and BM paired samples confirmed that the BM analysis increased the rate of detection of MRD,
affording a median 1–log10 increment in sensitivity [54]. However, the presence of NPM1-mutated
transcripts, with a 0.01% absolute threshold to define positivity, in PB of patients after the second
chemotherapy cycle was highly discriminatory and was the only significant prognostic factor [54].
It has thus been hypothesized that MRD assessment in BM could overestimate the quality of response
due to the risk of BM samples dilution by PB, thereby justifying PB MRD monitoring at least at definite
timepoints [2,44,54,75], perhaps with 0.01% cut-off value to define MRD negativity, even though using
a >4-log reduction could also be considered reasonable [75]. Monitoring MRD in PB samples also
offers other advantages, such as decreased procedural morbidity and the possibility of more frequent
testing [8].
5. NPM1 Mutation Levels at AML Diagnosis

In most series, NPM1-mutated transcript levels at diagnosis did not correlate with the type of
NPM1 mutation or with other clinical characteristics, namely age, sex, WBC count, BM blast count,
lactate dehydrogenase level, ELN risk stratification or FLT3 gene mutational status [7,9,12,19,26].
On the contrary, Balsat et al. found higher median NPM1-mutated baseline levels in cases with WBC
count greater than 50 × 109 /L or with FLT3-ITD positivity, whereas no significant correlation was
documented with age, karyotype or other concurrent gene mutations [29]. However, it should be
noted that pretreatment NPM1-mutated expression levels detected by RQ-PCR did not influence
CR rate after induction chemotherapy [19], CIR [19] and survival parameters, such as EFS and
OS [7,12,19,26]. Conversely, Patel et al. recently reported targeted sequencing data from 109 patients
with NPM1-mutated AML to retrospectively evaluate the potential significance of NPM1 variant
allele frequency (VAF) at diagnosis, comutations and clinical features on patient outcomes [76].
Interestingly, high NPM1 VAF (≥0.44) observed on NGS correlated with shortened median OS
(12.1 months versus not reached, p < 0.0001) as well as median EFS (7.5 versus 65.4 months, p < 0.0001).
High NPM1-mutated allele burden at diagnosis was an independent predictor of unfavorable clinical
outcomes, particularly in patients who received HSCT in first CR and in the subgroup of subjects
with concomitant DNMT3A mutations. Because of BM multilineage involvement of NPM1 mutations
not being restricted to cells with blast morphology [12,16,20], the VAF could in fact reveal the true
clonal disease burden. Higher NPM1 mutant allele burden may be less amenable to eradication by
Int. J. Mol. Sci. 2018, 19, 3492 20 of 33
intensive chemotherapy, potentially resulting in a higher likelihood of MRD persistence. Alternatively,

high NPM1-mutated allele burden may indicate the presence of disease in different hematopoietic
cell populations, belonging to the NPM1-mutated leukemic clone regardless of blast morphology,
which could be intrinsically resistant to chemotherapy and could subsequently foster leukemia relapse,
resulting in adverse clinical outcomes [76].
6. Sequential MRD Monitoring and Molecular Prediction of Relapse

Several studies have investigated whether NPM1-mutated MRD monitoring during follow-up
of AML patients in CR after the end of intensive treatments could foresee hematologic
relapse [31,33–35,37–40,44,47,52,54,61,65]. Notably, post-treatment serial testing may portend
morphologic recurrence either when, in previously MRD-negative patients, NPM1-mutated transcripts
again become detectable or, alternatively, when rising MRD levels are observed in patients with
persistent low-level MRD positivity [75]. Ommen et al. documented that kinetics of molecular
relapse was markedly different among AML with NPM1 mutations or with rearrangements such
as PML-RARA, RUNX1-RUNX1T1 or CBFb-MYH11, and subsequently developed a mathematical
model to predict the time elapsing between molecular and hematologic relapse [38]. According to
this model, AML positive for CBFb-MYH11 displayed a slower clone regrowth than AML with other
molecular signatures, including NPM1 mutations [38,77]. In many patients who previously obtained
molecular CR, reoccurrence of NPM1-mutated transcripts may be detectable by molecular techniques
over 2–3 months before overt morphologic relapse occurred [1,31,33–35,37–40,44,47,52,54,61,65],
as summarized in Table 2, with significant differences observed between patients with concurrent
FLT3-ITD positivity or negativity (65 versus 120 days, respectively) [38]. Furthermore, median
time since molecular to hematologic relapse could also have a broad range, as documented by
Kronke et al. (median 2.6 months, range 0.4 to 23.6) [40], and may significantly vary according
to the NPM1-mutated/ABL1 cut-off value considered for MRD positivity, as shown in the study
Shayegi et al. [44]. Finally, Ivey et al. observed a longer time to overt relapse if molecular MRD was
first detected in BM rather than in PB samples (133 versus 87 days, respectively) [54]. These data
collectively suggest that serial molecular MRD assessment during follow-up may provide a timely
prediction of impending morphologic relapse in NPM1-mutated AML patients, potentially allowing
pre-emptive therapeutic approaches, preferably in the context of clinical trials [1,75,77].
7. MRD Levels and Allogeneic Hematopoietic Stem Cell Transplant in NPM1-Mutated AML
The use of alloHSCT in patients with NPM1-mutated AML is still controversial. In the
retrospective study by Rollig et al., a significant improvement in RFS was documented in the
donor group, indicating a beneficial effect of alloHSCT in NPM1-mutated AML patients in first
CR, also observed in the favorable subgroup without FLT3-ITD [78]. Even in the absence of a
significant difference in OS, most likely as a result of the fact that NPM1-mutated AML patients
experiencing relapse often achieved response to salvage treatments, alloHSCT could represent a
valuable consolidation option for NPM1-mutated AML patients in first CR if a sibling donor is
available, especially in young patients, with a relatively low risk of non-relapse mortality [78].
Unfortunately, prospective MRD monitoring was not performed within this trial [78]. Notably, several
subsequent studies investigated the prognostic significance of pre-transplant NPM1-mutated MRD
levels (Table 2) [52,55,57,60,61,63,65,67]. In detail, Karas et al., among 60 NPM1-mutated AML patients
undergoing alloHSCT, identified a significantly negative prognostic impact on EFS and OS, only for
age above 63 years and for pre-transplant MRD positivity, immediately before starting the conditioning
regimen, with cut-off 0.1% [52]. The estimated probabilities of 3-year relapse, EFS and OS were
6%, 72% and 75%, compared with 48%, 35% and 40% for patients with lower and higher levels
of MRD, respectively [52]. Kayser et al. reported on 67 NPM1-mutated AML patients, receiving
alloHSCT in either first (31 cases) or second (20 cases) CR or with refractory disease (16 cases) [55].
Overall, patients transplanted in CR had significantly longer OS as compared to patients with
Int. J. Mol. Sci. 2018, 19, 3492 21 of 33
refractory disease. However, for patients undergoing alloHSCT in CR, there was a highly significant
difference in OS between pre-transplant MRD-negative and MRD-positive cases, with estimated
5-year OS rates of 89% versus 40%, respectively. Interestingly, these latter patients had an OS rate
comparable to that observed in patients receiving alloHSCT with refractory disease (38%). According
to Kayser et al., pre-transplant NPM1-mutated MRD positivity (>1%) was a significant predictor
of poor outcome after alloHSCT, independently of other variables, such as FLT3-ITD mutational
status, BM blast count at diagnosis and age [55]. More recently, Bill et al. applied highly sensitive
ddPCR to quantify pre-transplant NPM1-mutated MRD in 51 patients [61]. The 2-year CIR was
64.7% versus 6%, translating into OS rate 38.8% versus 71.7% in pre-transplant MRD-positive and
MRD-negative cases, respectively. Thus, pre-transplant MRD persistence independently predicted
adverse outcomes also in this cohort, including a large proportion of older patients (86.3% of
cases, with median age 62 years), mainly receiving non-myeloablative conditioning regimens [61].
Interestingly, the recommendation to perform alloHSCT only based on other known prognostic factors,
without considering pre-transplant NPM1-mutated MRD levels, is currently questionable [52,55,61].
According to Balsat et al., pre-transplant MRD assessment demonstrated that a survival benefit from
alloHSCT was not seen for patients with >4-log NPM1-mutated MRD clearance, whereas it was
maintained for those with insufficient MRD clearance, suggesting the relevant use of MRD evaluation
in transplant selection, to be further validated in clinical studies [57,75]. However, it is so far uncertain
whether patients with persistent MRD positivity could benefit from additional chemotherapy cycles
before alloHSCT or, alternatively, from intensification of conditioning regimen, whenever possible.
Prospective clinical trials are warranted to identify treatment options to improve the prognosis of
NPM1-mutated AML patients resulting MRD-positive prior to alloHSCT [61].
Furthermore, since the first observation in a small patient cohort by Bacher et al. [34],
post-transplant NPM1-mutated MRD monitoring has been shown to be predictive of relapse in several
large retrospective series [35,36,44,63,65]. Schnittger et al. reported a significant effect on survival of
NPM1 mutation levels, with relevant threshold 0.1%, for samples analyzed between days 61 and 120
and between 121 and 365 after alloHSCT [35]. Similarly, Shayegi et al. documented that an increasing
MRD burden exceeding the threshold of 10% NPM1-mutated/ABL1 on RQ-PCR assay was strongly
associated with an increased risk of relapse, therefore resulting in significantly worse outcomes in
terms of DFS and OS [44]. Most recently, Zhou et al. retrospectively investigated the application of
NGS to improve prediction of post-alloHSCT relapse in 59 adults with NPM1-mutated AML [63].
While pre-transplant MFC testing identified a subset of high-risk patients in whom additional therapy
before HSCT could potentially be useful; high-sensitivity NGS analysis of NPM1-mutated around
day 28 after alloHSCT predicted morphologic relapse in 15 of 18 cases (83%). Peri-transplant MRD
evaluation, improving the risk assessment for relapse, may identify high-risk patients who could
benefit from pre-emptive treatments after alloHSCT [63]. Moreover, Delsing Malmberg et al. reported
that MRD analysis using targeted deep sequencing 3 months after alloHSCT was a strong independent
predictor of both relapse risk and OS in adult NPM1-mutated AML patients [65]. Specifically, the 3-year
OS was 20% ± 17.9% versus 88.6% ± 7.8% (p < 0.001) for MRD-positive and MRD-negative patients,
respectively [65]. The above-presented data clearly suggest that NPM1-mutated AML patients
undergoing alloHSCT should be carefully monitored by MRD assays after transplant in order to early
detect imminent morphologic relapse [55,61]. However, the use of early pre-emptive strategies, namely
tapering of immunosuppression, donor lymphocyte infusions, administration of hypomethylating
agents or other targeted drugs, in patients with persisting or re-occurring NPM1-mutated MRD
positivity after alloHSCT, are not currently standardized [55,61,75,79]. Based upon the pivotal
RELAZA phase 2 study, which evaluated the role of 5-azacitidine in 20 either AML or myelodysplastic
syndrome (MDS) patients with MRD positivity after alloHSCT [80], Platzbecker et al. recently
reported the results of RELAZA2 trial, which investigated MRD monitoring in 205 patients with
either advanced MDS (27 cases) or AML (178 cases, including 31 with NPM1 mutations), in CR
after either conventional chemotherapy only (58 cases) or consecutive alloHSCT (147 cases) [81].
Int. J. Mol. Sci. 2018, 19, 3492 22 of 33
In 53 patients who became MRD-positive at a median of 100 days after start of screening, pre-emptive
5-azacitidine was administered, resulting in overall response rates of 71% and 48% in patients with
and without antecedent alloHSCT, respectively. At a median follow-up of 13 months, OS rate
was 76%, suggesting that MRD-guided therapy with an hypomethylating agent may prevent or
substantially delay morphologic relapse in high risk MDS or AML patients [81]. Moreover, Sockel et al.
pre-emptively treated with 5-azacitidine 10 NPM1-mutated AML patients in first or second CR after
intensive chemotherapy or autologous or allogeneic HSCT (3 cases), who experienced either molecular
relapse or persistently detectable MRD positivity in sequential RQ-PCR analyses [82]. Molecular
response, with at least 1-log decrease in the MRD level compared with pretreatment value, was obtained
in 7 of the 10 patients. At a median follow-up of 10 months (range 2–12), a median of 5 cycles of
5-azacitidine were administered and the 7 patients remained in morphologic CR, suggesting a potential
efficacy of 5-azaciditine in controlling NPM1-mutated MRD [82]. Although post-transplant early
cellular or pharmacologic preemptive strategies to enhance graft-versus-leukemia effect or to eradicate
persistent MRD have preliminarily demonstrated improved outcomes, their definitive clinical efficacy
should be further investigated and determined in randomized clinical trials [79].
8. MRD Assessment in Elderly Patients with NPM1-Mutated AML

Incidence of AML is highest among older subjects, with a median age at disease onset of
67 years [83,84]. Despite the use of intensive chemotherapy in fit elderly patients, clinical outcomes
remain generally dismal, with lower CR rates (around 40–50%) and few long-term survivors compared
with younger patients [85,86]. Indeed, even when short-term benefits have been demonstrated,
very few patients older than 60 years actually become long-term survivors, with OS rates reported
to be <10% at 3 years and <5% at 5 years [85,86]. In addition to comorbidities, which commonly
hamper intensive treatment approaches, it should be noted that the genetic profile of AML in the
elderly is often unfavorable, with a high incidence of adverse-risk karyotypes and a lower frequency
of good-risk molecular features [83]. Accordingly, NPM1 mutations are found in only 20–25% of older
AML patients, and in 35–40% of those with normal karyotype [83,87]. The possibility of obtaining CR
remains relatively high (60–80%) in elderly NPM1-mutated AML patients [83,88]. However, while
NPM1-mutated AML in younger adults is generally associated with favorable clinical outcomes,
especially in the absence of FLT3-ITD, in older patients the risk of relapse is markedly higher (1-year
CIR 71% in patients aged >65 years) and survival significantly lower (2-year OS 19%) [88]. In this
disappointing clinical setting, NPM1-mutated MRD monitoring may potentially be useful for patients
who obtain morphologic CR in order to define the best personalized post-remissional treatment
approach and to early detect relapse [83]. However, although multiple series evaluating NPM1-mutated
MRD also enrolled some patients aged >60 years (Table 1) [31–37,39,42–45,47–56,58,60–63,65,67,69,71],
no study so far specifically focused on application and clinical significance of MRD monitoring
in elderly patients with NPM1-mutated AML. Since the most predictive timepoints and MRD
thresholds appear to be highly variable even between cohorts of younger adult patients, they cannot
be easily applied to elderly subjects with poor prognosis and future studies are needed to further
address these underexplored issues [83,89]. However, older AML patients frequently receive low to
moderate-intensity treatments, mainly with hypomethylating agents, able to prolong survival even
without the achievement of morphologic CR, thereby raising further controversies about the relevance
of MRD studies in this clinical context [71,83].
9. Clonal Evolution and Loss of NPM1 Mutation at AML Relapse

As expected for founder genetic lesions, NPM1 gene mutation is generally stable throughout the
course of the disease and has long been considered almost invariably present in patients experiencing
AML relapse [14,90,91]. Notably, it has been detected at relapse even many years after the initial
diagnosis, in patients experiencing more than one relapse and in relapses occurring in extramedullary
sites [14,92,93]. However, after the first observation by Papadaki et al. that two among 21 (9.5%)
Int. J. Mol. Sci. 2018, 19, 3492 23 of 33
NPM1-mutated AML patients experiencing relapse, lost NPM1 mutation at leukemia recurrence [32],
many groups investigating NPM1-mutated molecular MRD provided further information on stability
of NPM1 mutation at relapse, as summarized in Table 2. While in several cohorts, clonal AML evolution
at disease recurrence was not observed [31,33–39,42,43,48,49,52,53,55,57–60,63–69], in the remaining
studies the frequencies of patients experiencing AML relapse with undetectable NPM1 mutations were
extremely variable, ranging from 1% in the prospective trial by Ivey et al. [54] to 25% documented
in smaller series [41,94]. Notably, cases with loss of NPM1 mutation at disease reoccurrence were
initially considered as secondary therapy-related myeloid neoplasms rather than true relapses from the
previously found NPM1-mutated leukemic clone [14,32,40]. Interestingly, to address the role of clonal
evolution in relapsed NPM1-mutated AML, Kronke et al. applied high-resolution SNP-array profiling
and performed comprehensive gene mutation screening in 53 paired BM/PB samples obtained at
diagnosis and relapse [95]. High stability between primary and relapse samples was observed for
mutations in DNMT3A (97%), IDH2 (92%) and NPM1 (91%) genes, whereas FLT3-ITD (75%) and
IDH1 (75%) were less stable. Interestingly, DNMT3A mutations were consistently documented in
primary and relapse samples from all the 5 cases with NPM1 mutation loss at relapse, suggesting
that DNMT3A mutation most likely preceded NPM1 mutation in the pathogenesis of the disease
and a common ancestral DNMT3A-mutated clone with NPM1 wild-type gave rise to both primary
and relapsed AML [95]. It should be noted that MRD monitoring based on RQ-PCR for NPM1
mutations may be hampered in the rare cases where relapse develops from an ancestral NPM1-negative
clone, probably arising from pre-existing non-malignant clonal hematopoiesis, therefore resulting in
false-negative MRD results [81,94,95]. Furthermore, a switch of the NPM1 mutation subtype from
D to A has been recently described in a patient with late NPM1-mutated AML relapse, 8 years after
the first diagnosis [96]. Clinical outcome of patients developing secondary AML with a switch of
the NPM1 mutation subtype may be misinterpreted by follow-up strategies only focusing on rising
MRD levels alone, which assume recurrence of the NPM1 mutation subtype previously documented
at initial diagnosis [96]. These rare circumstances demonstrated potential limitations of RQ-PCR in
MRD monitoring.
10. Newer Molecular Technologies to Assess MRD in NPM1-Mutated AML

A few studies have so far investigated the potential clinical application of newer molecular
methods, namely ddPCR [27,48,59,61] and NGS [41,46,56,60,62,63,65,68,69,73], to monitor MRD
in NPM1-mutated AML patients. As discussed above, at least 55 frameshift insertion NPM1
mutations have so far been recognized, and several other different gene lesions are theoretically
possible, rendering relative quantification of rare NPM1 mutations by conventional RQ-PCR highly
challenging [59]. Limitations of the application of RQ-PCR-based MRD assays are their dependence
on specific gene mutations, requiring individual reference standard curves based on target serial
dilutions, with commercial plasmid standards being widely available for only the three most common
NPM1 mutation types [59,77]. ddPCR, a high-throughput technology, instead generates absolute
quantification, can clonally amplify target nucleic acids, and does not require a reference standard
curve [10,77]. More specifically, in ddPCR assays the sample is separated in a large number of
partitions, and PCR reactions are compartmentalized to single oil droplets, which are then analyzed
for presence or absence of a target sequence based on fluorescence. Through the use of Poisson
statistics, the starting concentration of the target sequence is measured based on the number of
individual reactions in which it is found to be present [2,10,97]. According to the first few clinical
experiences in small NPM1-mutated AML patient series, ddPCR demonstrated excellent sensitivity and
agreement with RQ-PCR, also allowing for the detection of a variety of rare NPM1 mutation subtypes
(Tables 1 and 2) [48,59]. Notably, Mencia-Trinchant et al. created oligonucleotides with degenerate
sequence at known insertion sites and combined these massively multiplex pools of insertion-specific
primers into a single assay, which may selectively detect virtually all known and potential NPM1
mutations, but not wild-type NPM1 sequences [59]. Even if the precision of ddPCR assay, combined
Int. J. Mol. Sci. 2018, 19, 3492 24 of 33
with its broad applicability also in patients ineligible for RQ-PCR MRD assessment, should be
recognized, it should also be noted that RQ-PCR actually still performs more than adequately for
MRD detection in most NPM1-mutated AML patients, is less expensive, and many clinical laboratories
already possess the equipment and technical expertise for this molecular analysis [98]. In addition,
primer mixes to detect multiple NPM1 mutation could also be used in RQ-PCR assays [35,37,54,99],
while Mencia-Trinchant et al. do not report whether their primer mix can potentially be utilized in
RQ-PCR assays [59]. However, the multiplexing of ddPCR could allow a future wider use of this
molecular tool in NPM1-mutated MRD monitoring, after further validation in prospective clinical
trials, at least in cases showing rarer mutations [97].
Furthermore, multi-gene NGS-based MRD monitoring has recently been applied to AML
patients, including those showing NPM1 mutations (Tables 1 and 2) [41,46,56,60,62,63,65,68,69,73].
NGS technologies, which allow parallel and repeated sequencing of millions of small DNA fragments
across many loci in order to evaluate either a few genes or an entire genome, may overcome some
of the limitations of single genes analysis based on RQ-PCR assays [10,75,77,100]. The ability of
NGS platforms to study large numbers of mutated genes could help to trace the evolution of
malignant clones [77]. However, the genetic clonal heterogeneity at AML diagnosis and during
the course of the disease could, conversely, complicate the MRD monitoring, because the predominant
leukemic clone at presentation may not be the same clone that determines clinical relapse and
mortality [77]. Moreover, intratumoral heterogeneity relies not only on the presence of different
somatic mutations in distinct leukemic subclones, but also on heterogeneity of transcriptional and
epigenetic states [101–103]. Unlike population-level approaches, single-cell RNA-sequencing platforms
enable transcriptomic analysis of an individual cell. Through the combination of high-throughput
sequencing and bioinformatic tools, single-cell RNA sequencing can detect more than 10,000 transcripts
in one cell to distinguish cell subsets and dynamic cellular changes, therefore revealing intratumoral
heterogeneity, with possible applications in identifying treatment-resistant leukemic cells and MRD
monitoring [101–103]. Another significant limitation of NGS technology initially was the intrinsic
error rate that limited its sensitivity for most single-nucleotide variants to 1–2% of all reads [1,104].
This notwithstanding, recent refinements of NGS technology, such as the introduction of random
barcodes that anneal to target sequence fragments prior to amplification or unique molecular indexes,
have eliminated the error rate of sequencing itself and markedly improved the assay sensitivity,
which is actually comparable or even superior to that of RQ-PCR or MFC [75,105]. Thol et al. first
conducted parallel assessment of MRD by NGS and RQ-PCR in a small series of NPM1-mutated AML
patients, observing concordance in 95% of the 38 analyzed samples [41]. As mentioned above, Zhou
et al. recently compared MRD testing by MFC and NGS before and after alloHSCT and showed that
NGS was approximately 10-fold more sensitive than MFC, also demonstrating the clinical utility of
high-sensitivity deep sequencing in risk assessment of relapse, especially after HSCT [63]. Moreover,
Onecha et al. optimized and validated a high-sensitivity NGS method to detect and quantify NPM1,
IDH1/2 and FLT3 mutated sequences, with survival analyses showing that MRD-positive status
tested by NGS was associated with higher risk of relapse and death, whereas MRD-negativity
at post-consolidation correlated with a longer DFS and OS [69]. Although NGS assay currently
shows a similar sensitivity to that of RQ-PCR, it does not require oligonucleotides that hybridize
specifically to a particular sequence, so all the nucleotides in the amplified region can be studied [69].
Consequently, the NGS test is capable of simultaneously detecting all NPM1 mutation types in a single
assay [41,46,56,65]. Therefore, the application of either NGS or multiplex ddPCR may be indicated for
follow-up quantification of rare NPM1 mutations subtypes, or alternatively in anecdotal cases of a
switch of the NPM1 mutation subtype during the course of the disease [69,96]. Despite these potential
advantages, NGS technology is still computationally demanding, time-consuming and expensive,
so that the ELN MRD Working Party suggests that NGS techniques for MRD measurement are actually
best reserved for clinical trials for further validation [74,77].
Int. J. Mol. Sci. 2018, 19, 3492 25 of 33
11. Persistence of Pre-Leukemic Clones at AML Remission: What about NPM1 Mutations?
In the seminal analysis of comprehensive genomic data on samples collected from 50 patients
at AML presentation and remission after induction chemotherapy, Klco et al. documented persistent
leukemia-associated mutations in at least 5% of BM cells at remission in 48% of cases, which were
associated with increased relapse risk and reduced OS. Interestingly, contrary to other genes,
NPM1 mutations were cleared below the VAF 2.5% threshold at day +30 after chemotherapy in all the
18 NPM1-mutated AML analyzed cases [106]. Accordingly, Hirsch et al. reported, in a 69-AML-patient
cohort, that the detection of two or more events in >0.4% of cells by NGS assay after one treatment
course was strongly associated with lower survival in all cytogenetic profiles, especially in cases with
intermediate-risk cytogenetics [107]. Furthermore, Rothenberg-Thurley et al. characterized paired
pre-treatment and remission samples from 126 AML patients for mutations in 68 leukemia-associated
genes. Fifty cases (40%) retained, based on NGS analysis, at least 1 mutation during remission at a
VAF of ≥2%, with mutation persistence more frequently observed for DNMT3A (65%), SRSF2 (64%),
TET2 (55%) and ASXL1 (46%), whereas NPM1 mutation was rarely persisting (1/57 patients, 2%) [108].
Interestingly, among 46 patients with NPM1 mutation, there was a non-significant trend towards higher
molecular MRD levels by RQ-PCR in patients with compared to those without persisting pre-leukemic
lesions. A correlation was observed between persistence of pre-leukemic clones during first remission
and inferior OS and higher incidence of relapse, which was abrogated, in this series, by alloHSCT,
suggesting that mutation persistence may guide post-remission treatments [108]. More recently, further
information on the prognostic role of different persisting molecular lesions in AML patients at remission
has been provided [62,109]. Specifically, among 131 previously untreated AML patients, Morita et al.
observed frequent persistence of somatic mutation at CR in genes that are often pre-leukemic and
associated with clonal hematopoiesis of undetermined significance (CHIP), namely DNMT3A, TET2,
SRSF2, ASXL1 and TP53, whereas mutations in NPM1 gene, transcription factors or receptor tyrosine
kinase genes were often cleared [109]. Although the optimal VAF cut-off remains to be determined,
patients who achieved mutation clearance with a VAF <1% at day +30 after therapy, had significantly
better survival and lower risk of relapse in this series. These prognostic associations were more
pronounced when CHIP-related gene lesions were removed from the analysis [109]. In a larger cohort
of 482 AML patients, targeted NGS found persisting gene mutations in 51.4% of patients during CR,
with VAF ranging from 0.02% to 47%. The detection of persistent mutations in DNMT3A, TET2 and
ASXL1 genes (DTA mutations) was not correlated with an increased relapse rate. Only after exclusion
of DTA mutations from the analysis, the documentation of molecular MRD display significantly
higher relapse rates and lower survival rates, thereby confirming that persistent mutations in genes
associated with age-related CHIP did not have prognostic value in adult patients within a 4-year
time frame [62]. Overall, these studies provided information on the use of a genome-wide approach
to evaluate the impact of persistent multiple gene mutations on treatment outcomes [62,106–109].
The monitoring of specific single-gene MRD levels, including quantification of NPM1 transcripts
generally absent in pre-leukemic clones, could potentially be integrated with evaluation of persisting
pre-leukemic molecular lesions at remission. The combination of these two variables may ultimately
provide complimentary prognostic information and it could potentially be argued that monitoring both
leukemic and pre-leukemic clones after therapy could, in perspective, complement or even supersede
pre-treatment genetic factors for AML prognostic stratification [108].
12. Conclusions
Even though several published clinical studies (Tables 1 and 2) support the independent
prognostic significance of molecular MRD monitoring in NPM1-mutated AML patients, and the
Consensus Document from ELN MRD Working Party clearly indicates that patients showing NPM1
mutations should have molecular assessment of MRD, we have discussed that several controversies
have been raised, and further prospective clinical trials are warranted to standardize the optimal
material, timepoints, molecular tools and cut-offs for a meaningful application of MRD monitoring
Int. J. Mol. Sci. 2018, 19, 3492 26 of 33
to inform treatment decisions [31,74,110]. Caution is still needed when using MRD results alone,
outside of a clinical trial, to make decisions with regard to treatment approaches with potentially high
morbidity and mortality, mainly alloHSCT, in otherwise genetically favorable-risk AML patients [8].
Interestingly, the application of pre-emptive, MRD-directed treatment strategies, potentially including
pharmacological agents targeting NPM1-mutated protein with induction of protein degradation or
drugs inhibiting NPM1 cytoplasmic translocation, such as selinexor, which acts through inhibition of
nuclear export and resulting in NPM1 protein nuclear relocalization, should be warranted and needs
to be prospectively investigated [111–113].
Author Contributions: F.F. designed the study, reviewed the literature and wrote the manuscript; P.C., R.M.,
L.P. and M.L. supervised the study, analyzed data and critically revised the manuscript. All authors approved the
final version of the manuscript.
Funding: This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC),
Milan, Italy (IG 14797-2013) (ML); Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico
San Matteo [Ricerca Corrente 08069113 (PC)].
Acknowledgments: We would like to thank Chiara Quadrelli (RIGENERAND srl. Medolla, Italy) for her technical
support during development of RQ-PCR assay for NPM1-mutated transcripts and Rossana Maffei, Silvia Martinelli
and Patrizia Zucchini, for implementing and validating this assay and other diagnostic molecular techniques for
hematologic malignancies, at our Institution.
Conflicts of Interest: F.F. served on advisory boards for Jannsen on the clinical use of decitabine in AML patients,
M.L. served on advisory boards for Novartis on the clinical use of midostaurin. The other authors declare no
potential conflicts of interest.
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International Journal of

Keywords: NPM1-mutated acute myeloid leukemia; molecular minimal/measurable residual disease

Int. J. Mol. Sci. 2018, 19, 3492; doi:10.3390/ijms19113492 www.mdpi.com/journal/ijms

2. NPM1 Mutations in AML: Biological and Clinical Features

3. ELN Recommendations for MRD Assessment

Table 1. MRD monitoring in NPM1-mutated AML series: review of the literature.

Table 2. Clinical impact of MRD monitoring in NPM1-mutated AML patients.

Reappearance of NPM1 - Molecular relapse preceded

- All relapses were associated

4. Prognostic MRD Thresholds and Relevant Timepoints for NPM1 Mutations

5. NPM1 Mutation Levels at AML Diagnosis

intensive chemotherapy, potentially resulting in a higher likelihood of MRD persistence. Alternatively,

6. Sequential MRD Monitoring and Molecular Prediction of Relapse

8. MRD Assessment in Elderly Patients with NPM1-Mutated AML

9. Clonal Evolution and Loss of NPM1 Mutation at AML Relapse

10. Newer Molecular Technologies to Assess MRD in NPM1-Mutated AML

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