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Chapter3
https://t.me/med1917
The genetics ofacute
myeloidleukemia
Amye M. Harrigan and Amy M. Trottier
Department of Medicine, Division of Hematology, QEII Health Sciences Centre, Dalhousie University, Halifax, NS, Canada
Introduction, 33
AML with recurrent cytogenetic abnormalities, 36
Molecular genetic aberrations not detectable by conventional
cytogenetics, 39
Activated signaling, 40
Myeloid transcription factors, 41
Tumor suppressors, 42
Spliceosome genes, 43
Introduction
Acute myeloid leukemia (AML) is a heterogeneous disease
with respect to clinical and morphological features as well as
genetic aberrations. These genetic aberrations can be inherited or, more commonly, acquired over time from age- related
stress hematopoiesis; from other risk factors such as previous
cancer treatments (i.e. radiation or chemotherapy); exposure
to environmental toxins; or arise due to other unknown factors. The culmination of a driver mutation(s) and cooperating mutations within a hematopoietic stem or progenitor cell
can result in leukemogenic stem cells (LSC), which have a
proliferative advantage over normal hematopoietic stem or
progenitor cells and lose their ability to differentiate into a
mature myeloid cell. Ultimately, these LSC can lead to the
development of AML. Despite the advances in the understanding of the molecular aberrations associated with AML,
there is still much to learn about why and how these genetic
aberrations arise in the first place.
Traditionally AML patients were divided into three broad
risk groups based on cytogenetic abnormalities: favorable,
intermediate, and adverse, and each group had different cure
rates. However, approximately 50% of patients have a normal
karyotype (normal- karyotype AML). These patients have
varied clinical outcomes, with some attaining a good
response to conventional chemotherapy and others having
high rates of relapse and disease- related mortality. This led to
the search for recurrent genetic aberrations that are not
detectable by conventional cytogenetics. With the advent
and wider availability of next- generation sequencing
Cohesin complex gene, 44
DNA methylation, 44
Chromatin modifiers, 46
Germline predisposition, 46
AML therapies and MRD monitoring targeted by genetics, 48
Summary, 49
Further reading, 49
(NGS)methods such as wholeexpression profiling, significant progress has been made in
the identification of recurrent genetic mutations at the
molecular level. These “molecular markers” include mutations in specific genes as well as altered gene expression.
The importance of molecular markers was first introduced
in the 2008 update of the World Health Organization (WHO)
classification of AML with the addition of the provisional
entities “AML with mutated NPM1” and “AML with mutated
CEBPA.” Since then, advances in the understanding of the
molecular determinants of AML have increased at a rapid
pace and have led to improvements in the diagnosis,
prognostication, and management of AML. In 2022, the
WHO, along with European LeukemiaNet (ELN) and the
International Consensus Classification (ICC), another group
with expertise in clinical, pathologic, and genetic aspects of
AML, all published updated AML guidelines that emphasize
the importance of cytogenetic and molecular testing in the
diagnosis, prognosis, and classification of AML.
This chapter reviews the prognostically important recurrent genetic aberrations observed in adult AML (Table3.1).
As most currently available data on the impact of genetic
abnormalities in AML are derived from large trials enrolling
younger adult patients (<65 years), the implications for older
adults, who constitute the majority of incident AML cases
(median age at diagnosis is between 65 and 70 years), remain
less clear. Less is known about the molecular landscape of
pediatric AML. However, it appears that the molecular landscape of pediatric AML differs compared to that of adult
AML. In a study published by the Children’s Oncology
genome sequencing and gene
Molecular Hematology, Fifth Edition. Edited by Drew Provan and Hillard M. Lazarus.
© 2024 John Wiley & Sons Ltd. Published 2024 by John Wiley & Sons Ltd.
Table3.1 Important genetic aberrations inadult Acute Myelogenous Leukemia
https://t.me/med1917
Common co- occurring
Genetic aberrancy Type of AML
Cytogenetic
abnormalities
Numerical
chromosomal
abnormalities
NPM1 De novo AML FLT3- ITD Favorable (mutated NPM1
Activated signaling FLT3 ITD De novo AML t(15;17) Intermediate FLT3 inhibitors are approved for
t(8;21)(q22;q22)/
RUNX1::RUNX1T1
inv(16)(p13.1;q22) or t(16;16)
(p13.1;q22)/CBFB::MYH11
t(15;17)(q22;q21) and other
PML RARA
KMT2A and many partners De novo AML NA Intermediate for t(9;11)(p.21.3;q
Deletions (5q, 7q, 12p, 17p),
monosomy 7 or 13,
isochrome 17q, isodicentric
(X)(q13), complex
cytogenetics (i.e. ≥3
cytogenetic abnormalities)
FLT3 TKD De novo AML NA
c- KIT NA
De novo AML Signaling pathway mutations
De novo AML Signaling pathway mutations
APL Deletion 7q and trisomy 8
De novo AML NA Adverse for −5 or del(5q); −7;
cytogenetic or molecular
abnormalities
(NRAS, KIT, and FLT3)
Epigenetic modifier mutations
(ASXL1, ASXL2, EZH2)
Cohesin complex mutations
(RAD21, SMC1A, SMC3, STAG2)
NRAS, KIT, and FLT3
Signaling pathways mutations
(FLT3, NRAS, KRAS)
Tumor suppressor mutations (WT1)
Chromatin organization mutations
(ARID1B and ARID1A)
Other oncogene mutations (SALL4,
MED12, NSD1)
ELN 2022
Risk category
Favorable Patients with t(8;21) AML are
NA t(15;17)(q22;q21) has an
23.3)/MLLT3::KMT2A
Adverse for t(v;11q23.3)/KMT2A
−17/abn(17p), complex
karyotype and monosomal
karyoptype
without FLT3- ITD)
Intermediate (mutated NPM1
with FLT3- ITD)
Notable important clinical
implications
older, have lower blood counts
and bone marrow blasts
compared to AML with
inv(16)/t(16;16)
t(8;21) have an inferior prognosis
compared to patients with
AML with inv(16)/t(16;16)
excellent prognosis with
complete remission rates
greater than 90%
Cornerstone of treatment
includes all-
frontline and relapsed or
refractory treatment of AML
with FLT3 mutation
trans- retinoic acid
Myeloid
https://t.me/med1917
transcription
factors
Tumor suppressors TP53 De novo AML, treatment-
Spliceosome SF3B1, SRSF2, U2AF1, ZRSR2 De novo AML, AML arising
Cohesin complex STAG2 AML arising from MDS Adverse
DNA methylation DNMT3A De novo AML NPM1, FLT3, and IDH1/2
Chromatin
modifiers
RUNX1 De novo AML, AML with
minimal differentiation,
AML associated with
MDS, FPD- MM
CEBPA De novo AML, germline
predisposition to AML
related AML
WT1 De novo AML FLT3, CEBPA NA
BCOR De novo AML DNMT3A, RUNX1 Adverse
from MDS
IDH1, IDH2 NPM1 NA IDH1 and IDH2 inhibitors
EZH2 De novo AML, AML arising
from MDS
TET2 De novo AML, AML arising
from MDS
ASXL1 De novo AML, AML arising
from MDS
KMT2A De novo AML Trisomy 13
Trisomy 13 or 21
Mutations in epigenetic modifiers
(ASXL1, IDH2, KMT2A, EZH2),
spliceosome complex (SRSF2,
SF3B1) and others (STAG2,
PHF6, BCOR)
Complex cytogenetics Adverse if VAF ≥ 10%
RUNX1, TET2, ASXL1 Adverse
(particularly IDH2)
RUNX1, ASXL1, NRAS Adverse
NPM1, ASXL1 NA
RUNX1, SRSF2, IDH2 Adverse ASXL1 has also been detected in
FLT3
Adverse Older, male biological sex
Favorable (bZIP in- frame mutated
CEBPA)
NA
NA
approved in certain countries
for frontline and relapsed or
refractory treatment of AML
MPN and CMML
AML, acute myeloid leukemia; CMML, chronic myelomonocytic leukemia; FPDmyeloproliferative neoplasm; VAF, variant allele frequency.
MM, Familial platelet disorder with associated myeloid malignancy; MDS, myelodysplastic syndrome; MPN,
36 Molecular Hematology
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Group, somatic mutations in genes such as MBNL1, ZEB2,
and ELF1 were more prevalent in pediatric patients with
AML compared to adults. Conversely, mutations in DNMT3A
and TP53, which are common in adult AML, were virtually
absent in pediatric AML. Mutations in certain genes, such as
GATA2, FLT3, and WT1 overlap in both adult and pediatric
AML. An extensive review of pediatric AML is beyond the
scope of this chapter.
AML with recurrent cytogenetic
abnormalities
Cytogenetic abnormalities in AML have long been known to
have prognostic relevance and continue to be included in the
classification of AML. Both the 2022WHO and ICC AML
classifications include specific cytogenetic abnormalities as
distinct AML subtypes, including acute promyelocytic leukemia (APL) with t(15;17)(q24.1;q21.2)/PML- RARA, AML
with t(8;21)(q22;q22)/RUNX1::RUN1T1, AML with inv(16)
(p.13.1;q22) or t(16;16)(p.13.1;q22)/CBF::MYH11, AML
with t(9;11)(p22;q23)/MLLT3::KMT2A. However, in these
classification schemes, different blast thresholds are used for
certain cytogenetic abnormalities to define AML. In the
WHO scheme, no minimum blast percentage threshold is
specified, whereas the ICC requires at least 10% blasts in the
bone marrow or peripheral blood to define AML in the presence of a listed cytogenetic abnormality. AML with t(9;22)
(q34.1;q11.2)/BCR::ABL1 is the only cytogenetic abnormality in both these schemes that requires 20% blasts for diagnosis. Several recurrent translocations are associated with a
favorable prognosis when treated with appropriate therapeutic agents, while particular numerical chromosomal abnormalities, such as monosomies of chromosomes 5 and/or 7,
are associated with a poor prognosis. Advances in molecular
genetic research have enabled a better understanding of the
mechanisms by which these translocations cause leukemia.
Importantly, these studies have demonstrated that the pathogenesis of AML is one of a sequential acquisition of genetic
aberrations, with a single aberration being insufficient alone
to cause overt leukemia. On average, cases of de novo AML
contain more than 10 significant gene mutations, many of
which can be grouped into one of eight functional categories
of genes that are thought to participate in leukemogenesis
including: nucleophosmin gene (NPM1), activated signaling,
myeloid transcription factors, tumor suppressor genes, spliceosome genes, cohesinrelated genes, and chromatin- modifying genes.
Core- binding factor leukemias
The core- binding factor (CBF) is a key regulator of hematopoiesis and the most frequent target of chromosomal translocations associated with leukemia. This transcription factor
complex genes, DNA methylation-
is composed of two heterodimeric components: the αencoded by RUNX1 (also called AML1 or CBFA) and the β
subunit encoded by CBFB. Homozygous loss of function
of either RUNX1 or CBFB in genetically engineered mice
results in a complete lack of definitive hematopoiesis and
embryonic death, indicating that both components of CBF
are necessary for normal hematopoietic development. CBF
AML is a relatively frequent subtype of adult AML,
accounting of ~15% of adult AML cases, and is classified in
the favorable risk category. CBF AML includes the balanced
translocations t(8;21)(q22;q22), in which the RUNX1 gene
is fused to RUNX1T1 (ETO), and inv(16)(p13.1;q22) or
t(16;16)(p13.1;q22), in which CBFB is fused to MYH11.
TheCBF partner gene in each case has constitutive activity
leading topersistent expression of the RUNX1–RUNX1T1
(AMLETO) or CBFβ–MYH11 fusion protein, causing recruitment of the corepressor complex and undergenes regulated by the CBF complex.
Data from invitro studies and transgenic animal models
suggest a dominant- negative role for these fusion genes.
RUNX1–RUNX1T1 acts as a dominant- negative inhibitor of
the wild- type CBF, with the fusion transcript retaining
RUNX1’s DNA binding and heterodimerization domains,
but lacking the C- terminal transcription activation domain.
The mechanism of dominant- negative activity is less clear
for CBFβ–MYH11, but both prevent transactivation of CBF
targets. Despite this understanding, the precise mechanism
by which these fusion proteins exert their leukemogenic
effect remains to be elucidated. However, it is clear that these
translocations alone are not sufficient to result in overt
leukemia. For example, conditional alleles of RUNX1–
RUNX1T1 expressed in adult hematopoietic progenitors are
not sufficient to cause AML, but result instead in a myeloproliferative phenotype. Experience from mouse models has
shown that expression of these fusion proteins resulted in
aberrant self- renewal but, by themselves, were insufficient to
result in leukemia, with exposure to chemical mutagens
being necessary to induce overt AML.
Although CBF AML is classified in the favorable risk category, there is heterogeneity within this group as ~40% of
patients will relapse after standard induction treatment. NGS
techniques have allowed for better characterization of cooperating mutations that may contribute to leukemogenesis
and outcomes of CBF AML. Recent studies looking at the
molecular profile of CBF AML have found that certain mutations (mainly mutations in signaling pathway genes such as
NRAS, KIT, and FLT3) are common to both AML with (8;21)
and inv(16)/t(16;16). Mutations in genes encoding for epigenetic modifiers (ASXL1, ASXL2, EZH2) and cohesin complex (RAD21, SMC1A, SMC3, STAG 2) are common in AML
with t(8;21) but rare in AML with inv(16)/t(16;16). Overall,
these findings suggest that AML with inv(16)/t(16;16) is
largely driven by alterations in signaling pathways, whereas
subunit
expression of
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The genetics ofacute myeloidleukemia 37
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the pathogenesis of AML with t(8;21) is less clearly understood
given the co- mutations in genes from different functional
pathways.
The different molecular profiles of CBF AML manifest
with differing clinical characteristics. Patients with t(8;21)
AML tend to be older and have lower white blood cell (WBC),
platelet, and hemoglobin counts, as well as lower bone
marrow blasts compared with those with inv(16)/t(16;16).
Patients with AML with t(8;21) also have an inferior prognosis compared to patients with AML with inv(16)/t(16;16).
Furthermore, the presence of specific additional genetic aberrations such as trisomy 8 as well as mutations in FLT3 and
KIT exon 17mutations appear to also confer a worse prognosis in cases of CBF AML, whereas mutations in NRAS and
WT1 confer a more favorable prognosis.
The aberrant fusion transcript present in the CBF AML
leukemic clone is stable throughout states of active disease
and at relapses. Therefore, this makes the aberrant fusion
transcript (i.e. inv(16)/t(16;16) and t(8;21)) an ideal biomarker for measurable residual disease (MRD). Reverse
transcription- quantitative polymerase chain reaction
(RT- qPCR) can be used to detect MRD in CBF AML and
studies have reported cutoff values that are helpful in
predicting therisk of relapse as well as favorable outcomes
based on the RT- qPCR results at the end of induction and/or
end of first consolidation.
Acute promyelocytic leukemia
As with the CBF genes, there are also multiple chromosomal
translocations that involve the retinoic acid receptor RARα
locus on chromosome 17. The subgrouping of patients with
RARα translocations is particularly important because of the
distinctly better prognosis that most of these translocations
confer. In t(15;17)(q22;q21), the RARα gene fuses with a
nuclear regulatory factor called the promyelocytic leukemia
gene (PML), giving rise to the PML–RARα gene fusion
product. Rarely, the PML–RARα fusion can result from
cryptic or complex cytogenetic rearrangements and lack
t(15;17)(q22;q21) on routine cytogenetic testing. Several
infrequent variant translocations with RARα also occur
involving the ZBTB16 (previously known as PLZF), NPM1,
NuMA, STAT5B, or PRKAR1A genes. Each of these is associated with an APL characterized by a block in differentiation
at the promyelocyte stage of hematopoietic development.
The t(15;17)(q22;q21) is the most frequent translocation
causing APL and has been extensively studied. PML–RARα
expression is associated with a block in differentiation due
to aberrant recruitment of the nuclear co- repressor complex,similar to observations in the context of the RUNX1–
RUNX1T1 and CBFβ–MYH11 fusions.
APL with PML- RARα is considered to have an excellent
prognosis (with a complete remission rate greater than 90%).
It has a particular sensitivity to all-
trans- retinoic acid
(ATRA), a ligand for RARα, which has proven to be a very
effective therapy for this subtype of AML. The efficacy of
ATRA in the treatment of APL is related to the ability of
ATRA to bind to the fusion protein, with resultant dissociation of the nuclear co- repressor complex. Promyelocytes are
then able to engage normal hematopoietic differentiation
programs that ultimately result in apoptotic cell death.
However, for some variant RARα translocations, including
cases with the ZBTB16- RARα and STAT5B- RARα, the
fusion products are resistant to ATRA, suggesting they have
different functional effects than the PML–RARα translocation. To stress the importance of the PML- RARα fusion,
including cryptic rearrangements, both the WHO and ICC
AML classifications include APL with t(15;17)(q22;q21);
PML- RARα as a distinct subtype of AML. The ICC classifies
AML with other, non- PML- RARα, RARα rearrangement as
another subtype of AML.
Like the CBF leukemias, expression of PML–RARα is not
sufficient to induce AML. There are several lines of evidence
supporting this assertion, including transgenic murine models of PML–RARα- induced AML. In this model, although
the fusion gene is present in the germline and is expressed
during embryonic and adult development, the animals do
not develop AML until 3–6months after birth, and even
then, the penetrance is modest (15–30%) and often there is
also the acquisition of secondary cooperating genetic events.
Almost half of pediatric and adult APL patients harbor further chromosomal alterations in addition to the t(15;17)
identified by conventional cytogenetics, with deletion 7q and
trisomy 8 being the most prevalent alterations. Nextgeneration sequencing of APL cases has revealed that about
70% of patients with APL harbor a mean of 1 somatic mutation. Mutations in the FLT3 gene, including FLT3 internal
tandem duplication (ITD) and FLT3- D835mutations are the
most frequent co- occurring mutations in pediatric and adult
APL, representing up to 40% of cases. These cases often
present with an elevated WBC count. Other co-
occurring
genetic aberrations include mutations in signaling pathways
(FLT3, NRAS, KRAS), tumor suppression (WT1), chromatin
organization (ARID1B and ARID1A), and oncogenes (SALL4,
MED12, NSD1). Although mutations in epigenetic modifiers
(DNMT3A, TET2, IDH1, IDH2, and ASXL1) represent less
than 6% of APL cases, the presence of these mutations has
been associated with a poor prognosis with regard to overall
survival (OS) and disease- free survival.
KMT2A gene rearrangements (11q23)
Abnormalities of 11q23 containing the KMT2A gene (formerly known as the mixed lineage leukemia, MLL, gene) are
found in 3–7% of AMLs. These abnormalities can occur
at any age but are particularly common in children and
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38 Molecular Hematology
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therapy- related AML after treatment with DNA topoisomerase II inhibitors. The KMT2A gene is the most promiscuous of genes as it is involved in over 120 different
translocations in AML with more than 70 different partner
genes now identified. These translocations lead to fusion
genes containing the N- terminus of KMT2A fused in- frame
to the partner gene, most of which have subsequently been
noted to have roles in normal hematopoiesis. Molecular
studies have also shown that KMT2A is frequently rearranged by cryptic translocations that are not detectable by
conventional cytogenetics. KMT2A encodes for a histone
methyltransferase involved in the regulation of gene transcription via chromatin remodeling. It is important for
embryonic development and regulation of hematopoietic
differentiation. Similar to RUNX1 and CBFB, homozygous
disruption of KMT2A in mice is embryonic lethal because
of disordered hematopoiesis. KMT2A is thought to function
as both a transcriptional activator and a transcriptional
repressor, using different domains within the protein.
Importantly, the repression domain is more N- terminal to
the activation domain, such that oncogenic KMT2A fusion
proteins are thought to retain their repression, while losing
their transcriptional activation capability. The prognostic
impact of KMT2A translocations is variable and depends on
the particular fusion partner gene.
The most frequent KMT2A translocation detectable by
conventional cytogenetics in AML is t(9;11)(p22;q23),
involving MLLT3 (AF9). AML with t(9;11)(p22;q23) is rec-
ognized as a distinct AML subtype by the ICC but not the
WHO classification, which instead lists AML with KMT2A
rearrangement as a subtype of AML. AML with t(9;11)
(p22;q23) falls into the intermediate risk category in the
ELN risk classification. In general, the other KMT2A translocations confer an intermediate to poor prognosis. Less is
known about the mutational landscape of AML with
KMT2A rearrangement, but retrospective studies have
shown that there is a relative paucity of mutations in the leukemic clones at diagnosis and relapse. This supports the
finding that the KMT2A rearrangement is a potent driver in
leukemogenesis.
Other rare chromosomal translocations
Other rare recurrent chromosomal translocations, including
t(6;9)(p23;q34.1), inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2),
and t(1;22)(p13.3;q13.3), are found in AML and are listed as
distinct entities in the 2022WHO and ICC classifications.
t(6;9)(p23;q34.1) results in the formation of a chimeric fusion
gene DEK- NUP214. The DEK- NUP214 protein alters nuclear
transport and acts as an aberrant transcription factor. t(6;9)
(p23;q34.1) is a rare, adverse risk cytogenetic abnormality
found in 0.7–1.8% of AML. Characteristics of AML with
t(6;9)(p23;q34.1) include occurrence in children and young
adults, association with basophilia and multilineage dysplasia
and associated with a poor prognosis. t(6;9)(p23;q34.1) is
usually the sole karyotypic abnormality and is commonly
associated with FLT3-
ITD mutations.
Inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2) is the most common abnormality of chromosome 3q in AML, results in
MECOM activation by repositioning of a GATA2 enhancer,
and leads to GATA2 haploinsufficiency. Inv(3)/t(3;3) is found
in 1–2% of AML, is seldom seen in pediatric AML, and is
anadverse cytogenetic risk factor conferring a short OS and
low complete remission rate. AML (megakaryoblastic) with
t(1;22)(p13.3;q13.3); RBM15- MKL1 occurs in <1% of AML,
is found in infants and young children (≤3 years) with acute
megakaryoblastic leukemia without Down syndrome, and is
suggested to have a poor prognosis, though this is uncertain
due to the low number of cases reported.
Numerical chromosomal abnormalities
In addition to translocations, recurrent numeric chromosomal abnormalities are frequently noted in AML. Many of
these chromosomal abnormalities are listed as myelodysplasiarelated, AML- defining cytogenetic abnormalities. These
include deletions (5q, 7q, 12p, 17p), monosomy 7 or 13,
isochrome 17q, isodicentric (X)(q13) and complex cytogenetics (i.e. ≥3 cytogenetic abnormalities). Most of these are
also listed in the ELN risk stratification scheme as adverse
risk. In particular, partial or complete loss of chromosome 5
and/or 7 confers a very poor prognosis. In de novo AML,
acquired trisomy of chromosome 8 or 21 can also be seen and
this carries an intermediate prognosis. Abnormalities of chromosome 7, usually in the form of monosomy or partial deletion of the long arm, constitute the second most frequent
numerical chromosomal abnormality in de novo AML, after
trisomy 8.
Many investigators have attempted to narrow the region of
interest on chromosomes 5 and 7 to determine the deleted
genes that result in leukemia, but no single culprit gene has
been conclusively identified. However, investigations into
myelodysplastic syndromes (MDS) involving deletion of the
long arm of chromosome 5 (5q–) have provided the most
insight into how deletion in chromosome 5 can predispose to
AML. Studies have revealed that haploinsufficiency of several
genes (e.g. RPS14, CSNK1A1, APC, EGR1, miR-
145, and
miR- 14a) located on the commonly deleted regions of
chromosome 5 contribute to the pathogenesis and hematological phenotype associated with MDS with deletion 5q.
Furthermore, the presence of other cooperating mutations,
such as TP53, increases the rate of development of AML, but
does not necessarily cause overt leukemia. These results suggest that deletion of a number of different genes on chromosome 5 along with cooperating mutations in tumor suppressor
genes may be required to result in leukemia.
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The genetics ofacute myeloidleukemia 39
DNA methylation-related genes (44%)
Chromatin-modifying genes (30%)
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Molecular genetic aberrations not
detectable by conventional
cytogenetics
The development of NGS technology has allowed for rapid
and relatively inexpensive DNA and RNA sequencing,
including whole- genome sequencing, and has revolutionized
the understanding of the complex genetic landscape in AML.
These technologies have uncovered hundreds of novel recurrent genetic mutations at the molecular level; however, only
a small subset of these have been found to contribute to leukemogenesis and/or to clearly impact prognosis.
The increased use of NGS technologies in research and
clinical practice has also led to the recognition that genetically distinct clonal subpopulations of myeloid cells with
mutations in genes known to be associated with AML can
be found in otherwise healthy, AML- free, individuals. This
finding is termed clonal hematopoiesis of indeterminate
potential (CHIP) and is most prevalent in older individuals, with studies reporting CHIP in up to 10% of individuals
aged >65 years and in as many as 18% of those over the age
of 90 years. The most commonly reported genes mutated in
CHIP include DNMT3A, TET2, and ASXL1. The clinical
significance of CHIP remains to be better elucidated;
however, CHIP has been reported to be associated with
decreased OS, increased risk of hematologic malignancy
development, and a greater risk for cardiovascular disease,
compared with age- matched members of the general
population.
Driver gene mutations
The subset of recurrent gene mutations that have been implicated in leukemogenesis are known as driver mutations.
Themajority of mutations found in AML are not involved
directly in leukemogenesis, but rather are believed to occur
in unstable leukemic cells or in hematopoietic stem cells prior
to an AML-
initiating event. These are called passenger mutations and do not impact the course of disease. Some driver
mutations are thought to initiate the leukemogenic process
(the initiating or founding clone), whereas others act in a
cooperating fashion, accelerating or encouraging the leukemogenic process, and in some cases, contributing to chemotherapy resistance and/or risk of relapse. Various patterns of
cooperativity as well as mutual exclusivity between different
genes have also been found. It is thus clear that one mutation
alone is not sufficient to transform a normal cell into a leukemic blast. An exception to this is chronic myelogenous leukemia (CML), a myeloproliferative neoplasm (MPN) defined at
a molecular level by a translocation between chromosome 9
and 22, resulting in the expression of an oncogenic tyrosine
kinase, BCR- ABL. While most individuals with CML present
in the chronic phase, a small percentage can present as an
acute leukemia (i.e. blast phase CML).
Using whole-
genome and whole- exome sequencing of 200
cases of de novo AML, a landmark study by the Cancer
Genome Atlas Research Network published in 2013 generated a genomic database and found that nearly all cases of
AML had at least one driver mutation in one of eight functional categories relevant in the pathogenesis of AML,
including the nucleophosmin gene (NPM1) (27% of cases),
activated signaling (59%), myeloid transcription factors
(22%), tumor suppressor genes (16%), spliceosome genes
(14%), cohesin- complex genes (13%), DNA methylationrelated genes (44%), and chromatin- modifying genes (30%).
The relative frequency of mutations in these functional categories in de novo AML is shown in Figure3.1. With the
increasing use of next- generation sequencing in research
and clinical practice since the time of that landmark publication, additional genetic mutations in these functional categories have been identified as clinically significant for the
diagnosis, prognosis, and/or management of AML.
Figure3.1 Frequency of driver mutations within eight distinct
functional categories in de novo AML as found by the Cancer
Genome Atlas Research Network using whole genome
sequencing.
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NPM1 (27%)
Myeloid transcription factors (22%)
Spliceosome genes (14%) Cohesin-complex genes (13%)
Activated signaling (59%)
Tumor suppressor genes (16%)
40 Molecular Hematology
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A summary of the now well- established molecular markers that have been incorporated into routine care of AML
(e.g. NPM1, FLT3, and CEBPA) as well as other clinically sig-
nificant genes in AML is presented in what follows, focusing
on driver gene mutations associated with possible prognostic
and/or therapeutic impacts in patients with AML.
NPM1
NPM1 mutations were first reported in 2005 as the most frequent genetic alteration in patients with normal- karyotype
AML. NPM1 is a nucleolar phosphoprotein that continuously shuttles between the nucleus and the cytoplasm, but
with predominant nucleolar localization. NPM1 acts as a
molecular chaperone in the nucleolus and is also involved in
cell cycle progression and regulation of the alternate reading
frame (ARF)/TP53 tumor suppressor pathway. Mutations in
NPM1 occur in exon 12, with more than 95% consisting of a
4- bp insertion at position 960. This 4- bp insertion results in
a frameshift within the C- terminal region of the NPM1 protein, leading to loss of nucleolar localization and gain of a
nuclear export signal. The mutant NPM1 protein (called
NPM1c+) therefore acquires aberrant cytoplasmic localization, which is readily detected by immunohistochemistry.
This cytoplasmic localization is thought to be necessary for
the leukemogenic activity of NPM1c+. Although details of
the exact mechanism are still not fully elucidated, it is
hypothesized that NPM1c+ induces cytoplasmic delocalization of several nuclear proteins involved in apoptosis, DNA
repair, and differentiation as well as transcription factors.
Mutant NPM1c+ also interacts with exoportin- 1 (XPO1),
causing aberrant cytoplasmic dislocation of NPM1c+ and
promotes increased expression of homeobox (HOX) genes,
which are critical for maintaining the leukemic state of
NPM1- mutated cells.
NPM1 mutations occur predominantly in the normal-
karyotype AML subgroup, with about 50% of this group
affected. A 2016landmark study, the cancer genome project,
used cytogenetics and gene sequencing to evaluate driver
mutations in 1540 AML patients. They examined patterns of
co-
mutation and mutually exclusive mutations. In this study,
NPM1 mutations were almost never found in isolation.
Commonly co- occurring mutations in DNMT3A, FLT3,
NRAS, and TET2 were found. Of these patterns of co- mutation,
the most extensively studied has been AML with mutated
NPM1 also carrying the FLT3- ITD mutation, which coexists
in ∼40% of NPM1- mutated cases.
The prognostic relevance of NPM1 mutations has been
clarified in several large studies, with clear evidence that
patients with mutated NPM1/wild- type FLT3 have an
improved prognosis over other normal- karyotype AML
patients. With conventional chemotherapy, these individuals have an event- free survival (EFS) and OS similar to
those with CBF leukemias. In most studies, the favorable
outcome predicted by NPM1 mutations is restricted to
patients who lack FLT3- ITD. Interestingly, initial studies
suggest that NPM1 mutations are very stable between
diagnosis and relapse, and thus are likely to constitute an
initiating rather than cooperating event in leukemogenesis.
Subsequent acquisition of FLT3 or other cooperating
mutations is thought to be necessary to trigger overt AML
development. Newer evidence has proposed that other
co- occurring mutations in NPM1- mutated AML may have
an impact on prognosis. Patients with NPM1/N- RAS,
NPM1/RAD21, or NPM1/FLT3- TKD seem to have a relatively good prognosis. Conversely, cases with NPM1/WT1
double- mutated or NPM1/FLT3- ITD/DNMT3A triple-
mutated are associated with a particularly adverse outcome.
In the ELN 2022 risk classification, mutated NPM1 without
FLT3- ITD or adverse cytogenetic abnormalities remains
listed in the favorable risk category.
Given the stability of NPM1 mutations, their high frequency, and that they are not found in individuals with clonal
hematopoiesis, RT- qPCR assays for NPM1 mutations have
been developed for MRD monitoring and found to be predictive for relapse. In mutated NPM1- AML, achieving MRD
negativity in peripheral blood or bone marrow is predictive
of a low risk of AML relapse and good survival.
Activated signaling
FLT3
FLT3 (FMS- like tyrosine kinase 3) encodes a membranebound receptor tyrosine kinase with important roles in
hematopoietic stem/progenitor cell survival, differentiation,
and proliferation. FLT3 is constitutively activated by
acquired mutation in approximately 20–40% of cases of
AML. In 20–25% of cases, ITDs are noted in the juxtamembrane domain, ranging in size from 3 to 400 nucleotides,
with the resultant transcripts always being in adding to the length of the protein. FLT3 is normally
expressed in myeloid and lymphoid progenitor cells and
expression is lost as hematopoietic cells differentiate. In
comparison, ITDs of FLT3 result in constitutive activation of
the tyrosine kinase. This results in subsequent activation of
downstream signaling targets, including the phosphatidylinositol 3- kinase (PI3K)/AKT, Ras/MAPK, and JAK2/STAT
pathways. Mutations in FLT3 also occur in the activation
loop of the tyrosine kinase domain (TKD) in about 5–10% of
AML cases. These also result in constitutive kinase activation, but there is increasing evidence that the downstream
signaling events are different in the setting of TKD versus
ITD, such that their clinical implications are not equivalent.
The clinical implications of TKD mutations remain controversial and larger studies are required to define if these also
frame and
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The genetics ofacute myeloidleukemia 41
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confer a poor prognosis, though they are probably not independently prognostic.
The frequency of FLT3 mutations varies considerably in
different cytogenetic subgroups of AML, with a particularly
high frequency of 30–35% in normal- karyotype and t(15;17)
AML cases. Historically, AML with FLT3- ITD has been
reported to have a poor prognosis in most studies of children
and adults. While the rates of complete remission (CR) after
induction chemotherapy are not significantly different in
AML with FLT3- ITD versus AML with FLT3 wild type, the
relapse rate (RR) is higher and both EFS and OS are shorter.
FLT3- ITD mutation can also be seen in AML with t(15;17),
as previously discussed, but it does not appear to be an independent prognostic factor for patients with APL.
There has been much controversy and debate over the
prognostic significance of the size of FLT3- ITD clones in
AML over recent years. FLT3- ITD- mutated AML with a high
allelic ratio (>0.5) and co- occurring NPM1 mutation was
classified as intermediate risk in the 2017 ELN risk classification, whereas FLT3- ITD- mutated AML with a high allelic
ratio (>0.5) and wild- type NPM1 was classified as adverse
risk. This is in comparison to FLT3- ITD with a low allelic
ratio (<0.5), which was classified as a favorable risk if there
was the presence of a co- occurring NPM1 mutation or intermediate risk if NPM1 was wild type. However, risk stratification based on the allelic ratio of FLT3- ITD mutations was
subsequently removed from the most recent 2022 ELN classification due to lack of a standardized assay to measure the
allelic ratio and evidence for improved outcomes, including
OS, seen with the addition of midostaurin, a first generation
FLT3inhibitor active against both FLT3- ITD and TKD muta-
tions, to standard induction “3 + 7” and consolidation chemotherapy irrespective of the allelic ratio. FLT3- ITD AML
without adverse- risk genetic aberrations with or without the
presence of NPM1 co- mutation is now categorized as intermediate risk irrespective of the allelic ratio. With the success
of first- generation FLT3 inhibitors, second- generation
FLT3inhibitors, which are more selective and potent with
fewer offa second- generation FLT3inhibitor active against both ITD
and TKD mutations, has been approved for relapsed or
refractory FLT3 mutated AML in many countries in North
America (United Stated Food and Drug Administration,
Health Canada), Europe (European Medicines Agency) and
Asia (China National Medical Products Administration and
Japanese Pharmaceuticals and Medical Devices Agency).
ated interest for its use as a marker of MRD. However, studies
have shown that the mutational status of FLT3 may change
between diagnosis and relapse, with about 9% of patients losing their FLT3- ITD status. The length of the FLT3- ITD has
also been noted to vary between diagnosis and relapse. This
discordance between diagnosis and relapse suggests that
target toxicities, are also being studied. Gilteritinib,
The relatively high frequency of FLT3- ITD initially gener-
FLT3 mutations are not sufficient for the development of
overt AML, but may be secondary, cooperating events in leukemogenesis. This is further demonstrated by murine bone
marrow transplantation and “knock- in” models, in which
FLT3- ITD induces an MPN but does not cause AML. The
FLT3- ITD phenotype is similar to that reported in the
murine bone marrow transplantation assay for other constitutively activated tyrosine kinases associated with myeloproliferative phenotypes in humans, including BCR- ABL,
TEL- PDGFβR, TEL- ABL, and TEL- JAK2. Taken together,
these data indicate that constitutive activation of tyrosine
kinases is sufficient to induce a myeloproliferative phenotype, but not AML.
c- KIT
The KIT gene, on chromosome 4q, encodes c- KIT, a member
of the type III receptor tyrosine kinase family, the same family
as FLT3. c- KIT’s ligand, stem cell factor (SCF), promotes
c- KIT dimerization and phosphorylation, activating downstream signaling pathways important for proliferation,
differentiation, and migration of hematopoietic stem
cells. Gain- of- function mutations in c- KIT cause ligandindependent constitutive kinase activation and are found in
5–10% of all AML. However, a much higher frequency of
12–45% is observed in the CBF leukemias. Interestingly, c- KIT
mutations are not seen in APL. Thus, a significant functional
redundancy is noted between c- KIT and FLT3 mutations, with
c- KIT mutations arising in CBF leukemias in which FLT3
aberrations are absent, and FLT3 mutations arising in APL in
which c- KIT mutations are absent. The occurrence of c- KIT
and FLT3 mutations within the same patient is also very rare,
suggesting mutual exclusivity. Like FLT3- ITD in normal-
karyotype AML, c- KIT mutations, particularly in exons 8, 10,
11, and 17, confer a higher risk of relapse and possibly inferior
OS in CBF leukemia. More recently, studies have suggested
that c- KIT mutations have the greatest prognostic impact in
patients with RUNX1- RUNX1T1, but not in those with CBFB-
MYH11. Further studies are needed to confirm these findings.
While the presence of c- KIT mutation does not alter the risk
classification as per the ELN 2022, it is still recommended
that molecular testing for KIT mutations be done at AML
diagnosis, especially for CBF AML.
Myeloid transcription factors
RUNX1
RUNX1 is commonly dysregulated in AML, by translocations in t(8;21) CBF leukemia, copy number variations, and
point mutations. Although point mutations occur in only
6–10% of de novo AML, they are much more frequent
in specific sub- groups, arising in about 20% of AML with
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42 Molecular Hematology
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minimal differentiation and about 25% of AML associated
with MDS, including refractory anemia with excess blasts,
AML with multilineage dysplasia, and AML following MDS.
These point mutations are yet more frequent in therapyrelated MDS/AML and in radiation- associated MDS/AML
(tested in survivors of the atomic bomb in Hiroshima).
RUNX1 mutations are also frequently associated with trisomy 21 (Down syndrome) with an increased incidence of
AML, and AML with trisomy 13.
Further evidence of RUNX1’s involvement in the pathogenesis of AML has come from the analysis of pedigrees with
an inherited predisposition to MDS/AML. Familial platelet
disorder with associated myeloid malignancy (FPD- MM) is
an autosomal dominant disorder caused by pathogenic or
likely pathogenic germline mutations in the RUNX1 gene.
Individuals with FPD- MM have a variable risk of developing
overt MDS/AML (20–60% in different pedigrees) and a large
range in age of presentation of MDS/AML (age 6–75 years).
This latency suggests that other cooperating driver mutations are necessary to trigger overt MDS/AML.
RUNX1 mutations appear to be mutually exclusive of
NPM1 and CEBPA mutations and often co- occur with a com-
plex pattern of gene mutations, such as mutations in epigenetic modifiers (ASXL1, IDH2, KMT2A, EZH2), components
of the spliceosome complex (SRSF2, SF3B1), and others
(STAG 2, PHF6, BCOR). Clinically, RUNX1 mutations are
more common in older and biologically male AML patients.
AML with RUNX1 mutation is classified as an adverse risk in
the 2022 ELN criteria and is associated with a lower CR rate,
shorter EFS, and shorter OS compared to AML with wildtype RUNX1. RUNX1 is one of nine genes listed in the “AML
with myelodysplasia- related gene mutation” category in the
ICC classification. However, the 2022WHO criteria does not
list RUNX1 as a myelodysplasia- defining mutation because of
the overlap with a broad range of other molecular aberrations, calling the specificity of RUNX1 mutations by themselves to define a standalone AML subtype into question.
CEBPA
CEBPA (CCAAT/enhancer- binding protein α) is a transcription factor that regulates genes involved in myeloid differentiation, particularly by inducing granulocytic development of
bipotential myeloid progenitors. The protein consists of
N- terminal transactivating domains, a DNA- binding domain,
and a C- terminal leucine- zipper region (bZIP), necessary for
dimerization. The gene contains two translational start sites,
yielding a 42- kDa and a smaller 30- kDa isoform. Somatic
CEBPA mutations are noted in 5–14% of AML cases and 70%
of these mutations are detected in patients with normalkaryotype AML. Two classes of mutations commonly occur.
One involves N- terminal mutations, which prematurely
stoptranslation and abolish the production of the full- length
42- kDa isoform, while translation of the 30- kDa isoform
remains intact. The second class of mutations involves inframe mutations in the bZIP region and may impair DNA
binding homodimerization, and heterodimerization. Most,
but not all, AML cases with mutated CEBPA are biallelic, with
a combination of an N- terminal and a bZIP mutation. Others
involve a homozygous CEBPA mutation and fewer still are
monoallelic, with a single heterozygous mutation and retained
expression of a wild- type allele.
Previously, it was thought that biallelic mutated CEBPA
was associated with a favorable prognosis compared to
monoallelic mutations. However, several studies have subsequently shown that it is the location of the mutation
within the CEBPA gene that confers the better prognosis.
Specifically, AML with a CEBPA in- frame bZIP mutation,
whether mono- or biallelic, has a favorable prognosis
including a higher chance of achieving CR, better OS, and
lower risk of relapse. AML with in- frame bZIP mutated
CEBPA is a new AML subtype recognized by the 2022WHO
and ICC classifications and falls into the favorable risk category of the 2022 ELN risk classification.
CEBPA mutations have also been observed in pedigrees
with familial AML. Most of the reported pedigrees were
noted to exhibit CEBPA germline N- terminal mutations, but
there are also a small number of cases of familial AML with
CEBPA germline C- terminal mutations. The nature and timing of CEBPA mutations in familial AML provide further
insight into the sequence of molecular events in the development of leukemia. This insight comes particularly from second mutations targeting the CEBPA gene, most often in the
bZIP domain, of the remaining wild- type allele. These appear
necessary to cause overt leukemia in familial CEBPA-
associated AML. As with RUNX1 FPD- MM, the age of pres-
entation of the disease is variable in familial CEBPA- associated
AML but typically occurs at a younger age (median age 25
years) than de novo AML. Interestingly, the cases of familial
AML with germline N- terminal CEBPA mutations appear to
have a nearial AML with germline C- terminal CEBPA mutations have
incomplete penetrance. Further studies are needed to better
understand the differences between these two types of familial AML and their prognostic impact.
complete penetrance, whereas the cases of famil-
Tumor suppressors
TP53
TP53 (Tumor protein 53), located on chromosome 17p,
encodes p53, a tumor suppressor protein that contains DNA
binding, transcriptional activation, and oligomerization
domains. p53 acts as a tumor suppressor through several
mechanisms including regulation of DNA repair, cell cycle
arrest, and initiation of apoptosis. Until recently, it was believed
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