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The genetics ofacute myeloidleukemia 43
https://t.me/med1917
that TP53 mutations in treatment- related AML (t- AML) were
the result of DNA damage from cytotoxic chemotherapy.
However, recent studies using genome sequencing of t- AML
patients and chemotherapy- naive adults have shown that TP53
mutations increase with age, occur several years (3–6 years)
before the development of t- AML, and have been found prior
to the exposure of chemotherapy in patients who later developed t- AML. This has led to an alternative hypothesis for the
pathogenesis of t- AML, one in which chemotherapy does not
directly induce TP53 mutations, but instead, TP53 mutations
are age related, occurring in hematopoietic stem cells prior to
treatment with chemotherapy. Mutations in TP53 are postulated to drive leukemogenesis through a dominant negative
effect. The resulting loss of p53 activity favors genetic instability and resistance to chemotherapy, which leads to the development of AML once another genetic aberration is acquired.
Mutation of TP53, most commonly a missense mutation
in the DNA- binding domain, is the most frequent mutation
observed in AML with complex cytogenetics. It is also often
associated with specific copy number variants, including
−5/−5q, −7/−7q, and −17p. TP53 mutations are less fre-
quently associated with alterations in the RAS pathway (4%),
FLT3 (6%), or NPM1 (8%), and to a lesser frequency have
been found to co- exist with single nucleotide variants in
recurrent AML genes like TET2, IDH1/2, and DNMT3A.
The observed frequency of TP53 mutations is ~10% in de
novo AML, 30–40% in t- AML and older patients (age ≥ 70
years) with AML, and 50–70% in AML with complex cytogenetics. TP53 mutations are independently associated with
resistance to chemotherapy, lower CR rates, and shorter OS.
Their association with complex cytogenetics is additive, with
co- occurrence having a particularly poor prognosis. Variant
allele frequency (VAF) is also postulated to have a role in the
prognosis of TP53- mutated AML. A 2020 retrospective study
evaluating the prognostic impact of TP53 mutation VAF
found that a VAF >40% was independently associated with a
significantly higher cumulative incidence of relapse, worse
relapsewith mutated TP53” as a subtype of AML, whereas the WHO
2022 classification does not consider TP53 mutation to
define standalone AML. In the most recent ELN risk classification, TP53- mutated AML with a VAF of ≥10% is listed in
the adverse risk category.
free survival, and OS. The ICC recognizes “AML
WT1
WT1 (Wilms tumor 1) gene, on chromosome 11p, encodes a
zinc- finger transcription factor. The precise role of WT1 in
normal and malignant hematopoiesis remains controversial,
but it has been implicated in the regulation of cell survival,
proliferation, and differentiation. Its role as a tumor suppressor gene has been implicated as mutations in WT1, primarily
in exons 7 and 9, are observed in 10% of normal- karyotype
AMLs. However, WT1 is also overexpressed in various cancers, including AML, suggesting a role as an oncogene. High
expression of WT1 in AML made it an attractive potential
marker for MRD monitoring; however, unlike NPM1, which
shows stability during disease progression, WT1 mutational
status has been found to change often during disease progression and at relapse, decreasing its utility for MRD monitoring. Mutations in WT1 act as an independent negative
prognostic indicator in AML by reducing rates of CR,
increasing rates of relapse, and shortening OS; however, it is
not included in the 2022 ELN prognostic risk categories.
Mutations are noted in heterozygous and homozygous states
and show some association with FLT3- ITD and CEBPA,
though larger studies are required to confirm this.
BCOR
BCL6 corepressor (BCOR) is a transcription factor that is
involved in the control of hematopoietic stem cell development. BCOR is a part of the polycomb repressive complex
(PRC1.1), which mediates transcriptional repression through
epigenetic modification of histones. BCOR also functions as
a corepressor of BCL- 6 that enhances BCL- 6- mediated transcriptional repression, which is needed for the differentiation of CD4+ T cells in follicular helper T- cells.
In AML, BCOR mutations are detected in 3.8–5.0% of adult
de novo AML, 4% of AML with myelodysplasia- related
changes, 1.7% of pediatric AML, and are most often associated
with normal cytogenetics. Mutations are scattered throughout
the BCOR coding sequence, but over half of the mutations
occur in exon 4. The most common type of mutations is
frameshifts (~37%), followed by nonsense and missense mutations (~20% each). BCOR mutations result in the absence of
full- length BCOR protein and either absent expression or
expression of an abnormally truncated BCOR protein. The
disruptive nature of BCOR mutations is consistent with the
tumor suppressor role of BCOR. Co- mutation of DNMT3A
and/or RUNX1 have been reported, whereas BCOR mutations
were mutually exclusive of FLT3 and NPM1 mutations.
BCOR-
mutated AML is associated with poor prognosis
including lower rates of remission after induction chemotherapy and shorter OS. BCOR mutations are also newly recognized as a mutation in the “AML with myelodysplasia- related
gene mutations” category in the 2022 ICC and WHO diagnostic classifications. In the ELN 2022 risk classification
guidelines, BCOR- mutated AML is listed in the adverse risk
categor y.
Spliceosome genes
Spliceosomes are large ribonucleoprotein complexes involved
in the removal of introns and ligation of exons from premessenger RNA (mRNA) to produce mature mRNA and
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44 Molecular Hematology
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protein products. Alternative splicing is a mechanism by
which cells vary how the pre- mRNA is processed and thus
serves to diversify the proteome. However, mutations in
splicing regulatory sequences or mutations in core and/or
accessory components of the spliceosome complex can lead
to pathogenic alternative splicing, which can ultimately
result in cancers such as myeloid neoplasms.
In myeloid hematologic malignancies such as MDS,
chronic myelomonocytic leukemia (CMML), and AML,
mutations in spliceosome- regulating genes have been identified. Approximately one- third of patients with MDS and
almost 50% of AML arising from MDS have a mutation in a
gene that codes for a component of the spliceosome complex.
Spliceosome mutations are less commonly encountered in de
novo AML (~7%). Of the spliceosome genes, the most commonly mutated are splicing factor 3B subunit 1 (SF3B1), serine and arginine rich splicing factor 2 (SRSF2), U2 small
nuclear RNA auxiliary factor 1 (U2AF1), and zinc finger,
CCCH type, RNA- binding motif and serine and argininerich 2 (ZRSR2). These spliceosome mutations are most often
heterozygous in nature and mutually exclusive to one another.
Mutations in SF3B1, SRSF2, and U2AF1 are gain- offunction mutations, whereas mutations in ZRSR2, which is
located on the X chromosome, are nonsense or frameshift
mutations, resulting in loss- of- function. The most frequently
reported co- occurring mutations are found in RUNX1, TET2,
and ASXL1. Further studies are needed to better describe the
precise pathogenetic mechanisms of splicing deregulation of
each spliceosome gene and how this is influenced by cooperating mutations to promote leukemogenesis.
Clinically, mutations in SF3B1, SRSF2, U2AF1, or ZRSR2
are associated with older age, lower WBC count, and previous
diagnosis of MDS. The presence of spliceosome mutations
confers a shorter OS and EFS. Similar to BCOR mutations,
these four spliceosome genes are listed in the “AML with
myelodysplasia- related gene mutations” category in the 2022
ICC and WHO AML classifications and in the adverse risk
category as per the most recent ELN risk classification.
Cohesin complex gene
Cohesin is a large multiprotein complex composed of
SMC1A, SMC3, RAD21, STAG1, and STAG2. The cohesin
complex’s main role is to maintain the polarity of sister chromatids during mitosis, but it is also involved in doublestranded DNA damage repair and transcriptional regulation.
Recurrent mutations, typically loss- of- function mutations,
in the cohesin complex have been found in 6–13% of de novo
and 20% of AML cases arising from MDS, respectively. In
one large cohort study, 48% of cohesin- mutated AML cases
had a normal karyotype. Cohesin mutations have also been
reported in MDS and are especially prevalent in high- risk
MDS. Murine and invitro models have shed light on the role
of cohesin mutations in leukemogenesis. In these studies,
mutation of cohesin in hematopoietic stem cells led to a differentiation block with increased CD34+ progenitor cells
and a shift toward myeloid lineages, with cohesin knockdown mice later developing features of MPNs. Cohesin
mutations appear to occur early in leukemogenesis and are
insufficient to cause overt leukemia as the sole mutation.
STAG2 mutations are the most common cohesin gene
mutations. STAG2 is the only cohesion complex gene listed
as a myelodysplasia- related gene mutation in both the
2022iterations of the WHO and ICC AML diagnostic criteria. Furthermore, STAG2 is also categorized into the adverse
risk category as per the ELN; however, the evidence to support the adverse risk with STAG 2- mutated AML is limited
and conflicting. Further studies on the prognostic impact of
cohesin mutations in AML are needed to establish the prognostic impact these mutations.
DNA methylation
DNMT3A
DNA methylation is an epigenetic process (a process that
alters gene expression without changing DNA sequences)
that is frequently altered in malignancies, including AML.
Cancer genomes often exhibit global DNA hypomethylation,
but have also been found to display DNA hypermethylation
of the promotor regions of tumor suppressor genes. The
Cancer Genome Atlas Research Network found recurrent
mutations in genes involved in DNA methylation in 44% of
AML cases. Of these, mutation in DNA methyltransferase
3A (DNMT3A) was the most common, seen in 26% of all
AML cases examined.
DNMT3A encodes a DNA methyltransferase enzyme that
functions independently of replication. Whole- genome and
whole- exome sequencing have found that DNMT3A is one
of the most frequently mutated genes in AML, occurring in
12–35% of all cases, and is most often seen in normalkaryotype AML. The most common mutations of DNMT3A
involve missense mutations at residue R882 near the carboxyl terminus of the encoded protein. The precise biological and functional effect of these mutations is not yet fully
understood. However, a study examining clonal relationships in AML found that mutations in DNMT3A, along with
mutations in other genes that encode epigenetic modifiers,
were acquired early on in leukemogenesis, were usually present in the founding clone, and were rarely the sole mutation.
DNMT3A is one of the most commonly mutated genes in cases
of clonal hematopoiesis. DNMT3A is frequently co- mutated
along with NPM1, FLT3, and IDH1/2 (particularly IDH2).
These data suggest that isolated mutations in DNMT3A are
not sufficient to result in overt AML.
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The genetics ofacute myeloidleukemia 45
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The prognostic impact of DNMT3A mutations in AML
has been controversial. In several studies, including two
meta- analyses with over 4000 patients, DNMT3A mutations
have been found to confer a worse prognosis, with reduced
OS and relapse- free survival (RFS) compared to wide- type
DNMT3A, whereas other studies have found no significant
influence on survival outcomes.
IDH1 and IDH2
IDH1 and IDH2 encode for isocitrate dehydrogenases
that catalyze the oxidative decarboxylation of isocitrate to
α- ketoglutarate. IDH1 is cytoplasmic, whereas IDH2 is
mitochondrial in location. IDH1/2 are mutated in several
different cancers, including AML, and share similar features
in these cases. IDH1/2 mutations are predominantly somatic,
heterozygous, and occur at an early stage of tumorigenesis.
Mutations in IDH1 and IDH2 have been found in 6–16%
and 8–19% of AML, respectively. They are most commonly
found in normal- karyotype AML, are mutually exclusive of
one another, and are associated with mutated NPM1.
Epigenetic studies have found that IDH1/2 mutations result
in global DNA hypermethylation and impair hematopoietic
differentiation.
The prognostic impact of IDH1 and IDH2 mutations is
less clear. Several studies have found no impact of IDH
mutations on OS, CR, or RFS for normal- karyotype AML;
however, results from the literature are conflicting. Some
studies have found an adverse prognostic impact of particular IDH1 mutations, whereas others found no difference.
The findings for IDH2 mutations have ranged from a favorable prognostic impact with longer OS for R140Q IDH2mutation to an adverse prognosis with worse OS and reduced CR
rates for R172 IDH2mutations, and no significant impact in
other cases.
In recent years, oral IDH1 and IDH2inhibitors for the
treatment of IDH1/2- mutated AML have been developed.
These small molecule targeted inhibitors have shown effective inhibition of the R-
2- hydroxyglutarate oncometabolite, a
product of the IDH mutation, and subsequent restoration of
normal myeloid differentiation. IDH1 and IDH2inhibitors,
Ivosidenib and Enasidenib, respectively, are so far only
approved for clinical use in certain countries. In the United
States, the Food and Drug Administration has approved
both IDH inhibitors for frontline treatment of patients with
IDH1/2- mutated AML who are unfit for intensive chemotherapy and for relapsed or refractory (r/r) IDH1/2- mutated
AML. Enasidenib (IDH2inhibitor) is approved by Health
Canada for the treatment of r/r AML and in Europe, IDH
inhibitors are currently not approved for IDH- mutated AML.
Studies evaluating the benefit of adding IDH1/2inhibitor to
intensive induction chemotherapy for fit patients with
IDH1/2- mutated AML are ongoing.
EZH2
Enhancer of Zeste Homolog 2 (EZH2) is a gene located on
the long arm of chromosome 7 that encodes for a histone
methyltransferase and functional core subunit of the polycomb repressive complex 2 (PRC2), a key epigenetic regulator. Loss of function mutations in EZH2 have been reported
in a variety of myeloid neoplasms including MDS/MPN,
myelofibrosis, MDS, and de novo AML. In an analysis of
1604 patients with newly diagnosed AML, EZH2 mutations
were found in 4% of patients. Mutations were most frequently detected in exons 17 and 18 comprising the SET
domain, which is important for the catalytic activity of the
EZH2 protein. Most of the detected mutations were single
nucleotide variants (SNV) (67% missense and 33% nonsense/frameshift), followed by small indels. Co- mutations in
RUNX1, ASXL1, and NRAS were detected at higher rates in
the EHZ2 mutated patients compared to the wild- type EHZ2
AML patients. Similar to BCOR, spliceosome, and STAG 2
mutations, EZH2 is listed as a myelodysplasia- related gene
mutation in the 2022iteration of the WHO and ICC diagnostic criteria and listed in the adverse risk category of the
ELN classification.
TET2
TET2 (Tet Methylcytosine Dioxygenase 2) encodes a methylcytosine dioxygenase, which is involved in myelopoiesis. In
2009, somatic mutations in TET2 were described for the first
time in various myeloid disorders, including MDS, MPNs,
and AML. Since that time, the frequency of TET2 mutations
and its role in leukemogenesis have been examined. Mouse
models and invitro studies have found that TET2 mutations,
which lead to loss of function, result in widespread DNA
hypermethylation of enhancer elements of tumor suppressor
genes and consequent decreased expression of these protective genes.
Mutated TET2 occurs in 12–17% and 24–32% of de novo
and AML arising from MDS, respectively. Like the other
genes involved in DNA methylation (IDH1/2 and DNMT3A),
TET2 mutations occur early in leukemogenesis and are a
commonly mutated gene in clonal hematopoiesis. TET2-
mutated AML is most frequently associated with a normal
karyotype and is often seen with co- mutations in NPM1 and
ASXL1. TET2 mutations and IDH1/2 mutations are mutually
exclusive.
As with IDH mutations, the independent prognostic significance of TET2 mutations remains unclear, with conflicting evidence. Some groups have reported inferior OS versus
wild- type TET2 for young (age < 60) patients with AML,
whereas others have reported no significant difference.
Other studies have found that mutated TET2 confers worse
CR rates and shorter EFS and OS only for normal- karyotype
patients in the ELN favorable risk category.
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46 Molecular Hematology
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Chromatin modifiers
ASXL1
ASXL1 (Additional Sex Combs Like- 1), located on chromosome 20q, encodes a protein member of the Polycomb group
that is thought to disrupt chromatin in particular areas,
resulting in both epigenetic activation of certain genes and
repression of others. ASXL1 mutations have been found in
MPNs, MDS, CMML, and AML. The majority of ASXL1
mutations are frameshift and nonsense mutations in exon
12. ASXL1 mutations are relatively specific for AML that
arises from MDS, occurring at a frequency of 6.5% of de
novo AML but 30% of AML arising from MDS. ASXL1
mutations are also associated with RUNX1, SRSF2, and IDH2
mutations as well as older age at diagnosis. They are almost
mutually exclusive of NPM1, FLT3- ITD, and DNMT3A
mutations. Like other epigenetic modifiers, mutations in
ASXL1 are believed to occur early in the process of leukemogenesis and are not sufficient alone to result in overt AML.
Mutated ASXL1 has been associated with a poor prognosis
in AML, with lower CR rates and shorter OS and EFS
compared to wild- type ASXL1. ASXL1 is also another
myelodysplasia- related gene mutation listed by the 2022
WHO and ICC and is associated with adverse risk as per
theELN 2022 risk classification. AML with co- mutation in
ASXL1 and SRSF2 has a particularly poor prognosis compared to individuals with AML with only one of ASXL1 or
SRSF2 mutations.
KTM2A (formerly MLL)
As with RUNX1, the KMT2A gene was first noted to be
aberrantly regulated in AML through translocations.
Subsequently, KMT2A partial tandem duplications (PTDs)
were reported in 3–11% of normal- karyotype AML patients.
These mutations are more frequent in AML patients with
normal karyotype or with trisomy 11, and are often associated with FLT3 mutation. KMT2A-
mutation shown to negatively affect prognosis in normalkaryotype AML patients, with most large studies confirming
these initial findings. Similar to the other genes involved in
DNA methylation and chromatin modification previously
discussed, KMT2A- PTDs are interesting because the pres-
ence of a heterozygous KMT2A- PTD has been associated
with silencing of the wild- type allele in AML blasts. The
mechanism for this silencing appears to involve epigenetic
modifications rather than direct mutational effects. Activity
of DOT1L, a histone methyltransferase protein, has been
found to cause aberrant hypermethylation in KMT2A-
rearranged AML, and appears to be vital in the pathogenesis
and maintenance of KMT2A- rearranged leukemia. This suggests that epigenetic events may be sufficient to serve as
cooperating events in leukemogenesis.
PTD was the first gene
Germline predisposition
AML with germline predisposition is an important clinical
entity first introduced in the 2016WHO update on myeloid
malignancies and recently expanded upon in the 2022 ICC
and WHO classifications of AML. Although individually, each
germline predisposition disorder is rare, collectively, they
account for a significant proportion of all AML with prevalence ranging from 4% to 14% in a variety of studies. Genes for
which pathogenic or likely pathogenic variants are known to
predispose to AML have been broken down into various
categories based on their association with or without other
hematologic or organ system manifestations (Table3.2). For
example, like for RUNX1, as previously discussed, deleterious
germline mutations in ANKRD26 and ETV6 are associated
with a constitutional platelet disorder in addition to a predisposition to AML. Germline mutations in other genes, such as
CEBPA, are associated solely with AML without other organ
system manifestations. Identification and recognition of AML
with germline predisposition have important implications for
management, especially when allogeneic hematopoietic stem
cell transplant is being considered, as the majority of these
syndromes are inherited in an autosomal dominant manner
and therefore may be shared by a prospective related donor.
Additionally, patients with certain predisposition syndromes,
such as those associated with telomere biology disorders
and/or bone marrow failure, are at increased risk for severe
morbidity and/or mortality with the use of ionizing radiation
and particular chemotherapy agents. Identification of these
germline predisposition mutations in currently unaffected
family members is also important to enable the implementation of cancer screening and surveillance strategies that are
specific for the particular gene involved.
DDX41 has been found to be one of the most common
germline- mutated genes in adult AML. DDX41 encodes a
member of the DEAD- box helicase family implicated in
mRNA splicing and is essential for myeloid differentiation of
hematopoietic stem and progenitor cells. Germline loss of
function mutations (nonsense, frameshift, or those affecting
the initiation codon) accounts for greater than 60% of deleterious germline DDX41 mutations. Like AML with germline
CEBPA mutations, an additional acquired mutation in the
same gene (i.e. DDX41) is commonly observed at the time
ofAML diagnosis. The clinical features of AML with germline DDX41 mutation are distinct from other AML with
germline predisposition in that patients with AML with a
germline DDX41 mutation typically do not develop AML
until their 60–70s, which is similar to that of de novo AML
and later than most other AML germline predisposition syndromes. Other clinical features of AML with germline
DDX41 mutation include a strong male predominance and
incomplete penetrance. The prognosis for this subtype of
AML is overall favorable, with high response rates to
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Table3.2 Genes forwhich deleterious germline variants predispose toacute myeloid leukemia
https://t.me/med1917
Mode of
Category Syndrome name Gene
inheritance Associated phenotype
The genetics ofacute myeloidleukemia 47
Associated
hematologic
malignancy
Germline
predisposition
without a
preexisting
platelet disorder
or organ
dysfunction
Germline
predisposition
associated with
a preexisting
platelet disorder
Germline
predisposition
with potential
other organ
dysfunction
Germline
predisposition
associated with
bone marrow
failure syndromes
CEBPA AD NA AML
DDX41 AD NA AML
Fraumeni
Li-
syndrome
Bloom Syndrome BLM AR Prenatal growth deficiency, mild
Neurofibromatosis
type 1
Noonan
Syndrome
Fanconi anemia Mutations in the FANC
TP53 AD NA AML
RUNX1 AD Thrombocytopenia, mild bleeding
diathesis
ANKRD26 AD Thrombocytopenia, mild bleeding
diathesis
ETV6 AD Aplastic anemia, Thrombocytopenia,
mild bleeding diathesis
GATA2 AD Cutaneous warts and lymphedema
(Emberger syndrome)
Immunodeficiency
Mycobacterial/atypical infections
(MonoMAC syndrome)
Monocytopenia
SAMD9L
SAMD9
NF1 AD Café au lait spots, neurofibromas AML
PTPN11, NRAS, KRAS AD Facial dysmorphism, cardiomyopathy,
genes affecting DNA
repair pathway
AD Ataxia- pancytopenia (AXPC)
syndrome
Myelodysplasia, infection, growth
restriction, adrenal hypoplasia,
genital phenotypes, and
enteropathy (MIRAGE syndrome)
immunodeficiency, excessive
photosensitivity, type II diabetes,
hypogonadism
chylothorax, hygroma, short stature
AR except
FANCB,
which is
X- linked
Aplastic anemia
Short stature, café au lait spots,
abnormal thumbs, absent radii,
microcephaly, micro- ophthalmia,
structural renal anomalies
MDS
CMML
ALL (hypodiploid)
AML
MDS
AML
B-
ALL
AML
CMML
MDS
AML (monosomy
7)
MDS
CMML
AML
Hypocellular MDS
(monosomy 7)
Myeloid and
lymphoid
leukemias
Lymphomas
JMML
AML
ALL
AML
MDS
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Shwachman-
Diamond
syndrome
Telomere biology
disorder
SBDS, DNAJC21, EFL1 AR Exocrine pancreatic insufficiency,
skeletal dysplasia, hepatomegaly,
multilineage cytopenia
Mutations in at least
14known genes
identified including
TERT, TERC, DKC1,
andothers
AD, AR,
X- linked
Premature graying of hair, nail
dystrophy, pulmonary fibrosis,
idiopathic liver cirrhosis,
macrocytosis, arteriovenous
malformations
AML
MDS
AML
MDS
(continued)
48 Molecular Hematology
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Table3.2 (Continued)
Category Syndrome name Gene
Mode of
inheritance Associated phenotype
Associated
hematologic
malignancy
Severe congenital
neutropenia
Diamond-
Adapted from the 2022 ELN recommendations and previous reviews on inherited predisposition to hematopoietic malignancies. Only
predisposition genes listed in both the 2022WHO and ELN classifications are included in this table; however, this list is nonare many other genes that are known to predispose to acute myeloid leukemia. AD, autosomal dominant; ALL, acute lymphoblastic leukemia;
AML, acute myeloid leukemia; AR, autosomal recessive; CMML, chronic myelomonocytic leukemia; JMML, juvenile myelomonocytic leukemia;
MDS, myelodysplastic syndrome; NA, not applicable.
Blackfan
anemia
chemotherapy and prolonged progression- free survival in
comparison to age- matched DDX41 wild- type AML. An
extensive review of germline predisposition to AML is
beyond the scope of this chapter.
Mutations in many genes
involved in neutrophil
differentiation including
ELANE, HAX1, CSF3R,
and others
Mutations in ribosomal
subunit genes including
RSP19
AD, AR Variable depending on the underlying
mutation
AD, AR Short stature and phenotypic
anomalies including craniofacial,
musculoskeletal, and cardiac
malformations
Pure red cell aplasia
AML
MDS
AML
MDS
exhaustive as there
DNA methylation (e.g. DNMT3, TET2, EZH2) have been
identified as key drivers of AML. The identification of these
driver mutations served as the biological rationale for how
HMA could become a treatment option for AML. Studies
exploring different AML subtypes and their responsiveness
to HMA have yielded interesting results as mutations in
AML therapies and MRD monitoring
targeted by genetics
genes involved in DNA methylation were not predictive of
responsiveness to HMA. However, larger cohorts of patients
with these mutations need to be studied to confirm these
Advances in molecular technologies have led to the discovery of numerous driver mutations in AML. These advances
have also provided insight into new targets for directed therapies in AML as well as for MRD monitoring. Although a
comprehensive review of targeted therapies and MRD monitoring is beyond the scope of this chapter, we will highlight
some key agents and clinically important points.
Targeted therapies are becoming increasingly used in the
clinical setting and have improved outcomes in AML. As
mentioned in their respective sections, the use of ATRA in
APL, FLT3inhibitors in FLT3- mutated AML and IDH inhib-
itors in IDH- mutated AML are examples of targeted therapies that block the oncogenic mutation and restore the
normal hematopoietic cell maturation and differentiation.
Another example of how the improved understanding of
aberrant molecular drivers in AML has led to new treatment
is illustrated by the introduction of hypomethylating agents
(HMA; e.g. azacitidine and decitabine), alone or in combination with venetoclax (a BCL- 2inhibitor) for the treatment of
AML in patients who are older and/or unfit for intensive
chemotherapy. Multiple mutations in genes responsible for
findings. In a relatively short period of time, targeted therapies for AML have changed the landscape of treatment
options for patients with AML. Further research into improving upon these targeted therapies as well as the development
of additional targeted agents continues at a staggering pace
and is driven by the increased knowledge of the molecular
genetic landscape of AML.
MRD is an important prognostic indicator in AML and
how to best measure MRD is an area in which there are many
ongoing studies. RT- qPCR for the detection and monitoring
of AML in patients with PML- RARα, CBF mutations, and
NPM1 mutations has been established and studies have identified potential thresholds of prognostic significance. Multiparameter flow cytometry is another established technique
for MRD assessment; however, its utility in clinical practice
is limited by lack of standardization and the need for an
experienced pathologist for the interpretation of the results.
Another MRD technology that is being explored is targeted
NGS of a driver mutation found at AML diagnosis. However,
poor sensitivity, particularly at low VAF; lack of standardization between laboratories and available platforms; slower
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The genetics ofacute myeloidleukemia 49
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turnaround time; and higher cost compared to RT- qPCR
and multi- parameter flow cytometry limit the current utility
of the technology for MRD assessment.
Summary
Recurrent cytogenetic abnormalities are frequent in AML
and many molecular aberrations have also been discovered
that are undetectable by conventional karyotyping. The
advent of NGS technologies has shed much light on leukemogenesis and has uncovered numerous driver mutations commonly found in AML. Some of these driver mutations are
initiating events, others are cooperating mutations, and several have prognostic impact. Studies suggest that at least two,
and often more, driver mutations are required for the development of overt AML. Various patterns of mutual cooperativity as well as exclusivity have been found among these
mutations. The vast majority of identified driver mutations
fall within one of eight functional categories, including
NPM1, activated signaling, myeloid transcription factors,
tumor suppressor genes, spliceosome genes, cohesin- complex
genes, DNA methylation- related genes, and chromatinmodifying genes.
Patients with AML who are at high risk of relapse are recommended for allogeneic stem cell transplantation, a treatment with a relatively high morbidity and mortality. The
decision of whether to proceed to transplantation is a difficult one for both the patient and the physician. Many of the
newly discovered driver mutations have prognostic implications that can help risk- stratify patients to make treatment
decisions clearer. These mutations have also led to the discovery of novel targeted treatment options that can be used
alone or in combination with induction chemotherapy or
in patients who are not fit for intensive chemotherapy to
improve outcomes and/or to reduce treatment- related toxicity. While AML continues to have a dismal prognosis, the
improved understanding of the molecular and cytogenetic
aberrancies that drive AML has brought new excitement
to the field of leukemia research and has identified new
avenues for the prognostication, treatment, and monitoring
of patients with AML.
Further reading
Genetics of AML
Arber, D.A., Orazi, A., Hasserjian, R.P. et al. (2022). International
consensus classification of myeloid neoplasms and acute leukemias:
integrating morphologic, clinical, and genomic data. Blood 140:
1200–1228.
Bolouri, H. et al. (2018). The molecular landscape of pediatric acute
myeloid leukemia reveals recurrent structural alterations and agespecific mutational interactions. Nat. Med. 24 (1): 103–112.
Cancer Genome Atlas Research Network (2013). Genomic and epig-
enomic landscapes of adult de novo acute myeloid leukemia. N. Engl.
J. Med. 368: 2059–2074.
Döhner, H., Wei, A.H., Appelbaum, F.R. etal. (2022). Diagnosis and man-
agement of AML in adults: 2022 recommendations from an international expert panel on behalf of the ELN. Blood 140: 1345–1377.
Jamani, K. and Owen, C. (2015). Update on recurrent genetic aberra-
tions in acute myeloid leukemia. Int. J. Hematol. Oncol. 4: 179–190.
Khoury, J.D., Solary, E., Abla, O. etal. (2022). The 5th edition of the
World Health Organization classification of haematolymphoid
tumours: myeloid and histiocytic/dendritic neoplasms. Leukemia 36:
1703–1719.
Papaemmanuil, E., Gerstung, M., Bullinger, L. etal. (2016). Genomic
classification and prognosis in acute myeloid leukemia. N. Engl. J.
Med. 374: 2209–2221.
Core- binding factor leukemias
Jahn, N., Terzer, T., Sträng, E. etal. (2020). Genomic heterogeneity in
core- binding factor acute myeloid leukemia and its clinical implication. Blood Adv. 4: 6342–6352.
NPM1
Falini, B., Brunetti, L., Sportoletti, P., and Paola- Martelli, M. (2020).
NPM1- mutated acute myeloid leukemia: from bench to bedside.
Blood 136: 1707–1721.
Activated signaling
Chen, W., Xie, H., Wang, H. etal. (2016). Prognostic significance of KIT
mutations in core- binding factor acute myeloid leukemia: a systematic review and meta-
Daver, N., Venugopal, S., and Ravandi, F. (2021). FLT3mutated acute
myeloid leukemia: 2021 treatment algorithm. Blood Cancer J. 11: 1–9.
analysis. PLoS One 11: e0146614.
Myeloid transcription factors
(RUNX1,CEBPA)
Al- Harbi, S., Aljurf, M., Mohty, M. et al. (2020). An update on the
molecular pathogenesis and potential therapeutic targeting of AML
with t(8;21)(q22;q22.1);RUNX1-
Taube, F., Georgi, J.A., Kramer, M. etal. (2022). CEBPA mutations in
4708 patients with acute myeloid leukemia: differential impact of
bZIP and TAD mutations on outcome. Blood 139: 87–103.
RUNX1T1. Blood Adv. 4: 229–238.
Tumor suppressors (BCOR, TP53)
Short, N.J., Montalban- Bravo, G., Hwang, H. et al. (2020). Prognostic
and therapeutic impacts of mutant TP53 variant allelic frequency in
newly diagnosed acute myeloid leukemia. Blood Adv. 4: 5681–5689.
Sportoletti, P., Sorcini, D., and Falini, B. (2021). BCOR gene alterations
in hematologic diseases. Blood 138: 2455–2468.
Spliceosome genes
Lachowiez, C.A., Loghavi, S., Furudate, K. etal. (2021). Impact of splic-
ing mutations in acute myeloid leukemia treated with hypomethylating agents combined with venetoclax. Blood Adv. 5: 2173–2183.
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50 Molecular Hematology
https://t.me/med1917
Cohesin complex genes
Eckardt, J.N., Stasik, S., Röllig, C. et al. (2023). Alterations of cohesin
complex genes in acute myeloid leukemia: differential coclinical presentation and impact on outcome. Blood Cancer J. 13: 1–9.
Thol, F., Kade, S., Schlarmann, C. etal. (2012). Frequency and prognos-
tic impact of mutations in SRSF2, U2AF1, and ZRSR2in patients
with myelodysplastic syndromes. Blood 119: 3578–3584.
mutations,
DNA methylation (EHZ2, TET2, IDH1/IDH2,
DNMT3A)
Figueroa, M.E., Abdel- Wahab, O., Lu, C. etal. (2010). Leukemic IDH1
and IDH2mutations result in a hypermethylation phenotype,
TET2 function, and impair hematopoietic differentiation. Cancer
Cell 18: 553–567.
Im, A.P., Sehgal, A.R., Carroll, M.P. etal. (2014). DNMT3A and IDH
mutations in acute myeloid leukemia and other myeloid malignancies: associations with prognosis and potential treatment strategies.
Leukemia 28: 1774–1783.
Stasik, S., Middeke, J.M., Kramer, M. etal. (2020). EZH2mutations and
impact on clinical outcome: an analysis in 1,604 patients with newly
diagnosed acute myeloid leukemia. Haematologica 105: e228.
disrupt
Chromatin modifiers
Richardson, D.R., Swoboda, D.M., Moore, D.T. etal. (2021). Genomic
characteristics and prognostic significance of co- mutated ASXL1/
SRSF2 acute myeloid leukemia. Am. J. Hematol. 96: 462–470.
Issa, G.C., Zarka, J., Sasaki, K. etal. (2021). Predictors of outcomes in
adults with acute myeloid leukemia and KMT2A rearrangements.
Blood Cancer J. 11: 162.
Germline predisposition
Duployez, N., Largeaud, L., Duchmann, M. etal. (2022). Prognostic
impact of DDX41 germline mutations in intensively treated acute
myeloid leukemia patients: an ALFA756–768.
Harrigan, A.M. and Trottier, A.M. (2023). Hereditary acute myeloid
leukemia associated with CFamilial Cancer 22: 331–339.
Klco, J.M. and Mullighan, C.G. (2021). Advances in germline predispo-
sition to acute leukaemias and myeloid neoplasms. Nat. Rev. Cancer
21: 122–137.
Trottier, A.M. and Godley, L.A. (2021). Inherited predisposition to hae-
matopoietic malignancies: overcoming barriers and exploring
opportunities. Br. J. Haematol. 194: 663–676.
terminal CEBPA germline variants.
FILO study. Blood 140:
AML therapies and MRD monitoring
targeted by genetics
Aitken, M.J.L., Ravandi, F., Patel, K.P., and Short, N.J. (2021). Prognostic
and therapeutic implications of measurable residual disease in acute
myeloid leukemia. J. Hematol. Oncol. 14: 1–15.
Kayser, S. and Levis, M.J. (2022). Updates on targeted therapies for
acute myeloid leukaemia. Br. J. Haematol. 196: 316–328.
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Chapter4
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Molecular diagnostics andrisk
assessment inmyeloid malignancies
Christian Scharenberg1 and Torsten Haferlach
1
Department of Hematology, Skaraborgs Hospital Skövde, Skövde, Sweden
2
MLL Munich Leukemia Laboratory, Munich, Germany
Introduction, 51
Methods of detection, 54
Karyotypic evolution, 55
Clinical implications, 55
Treatment selection, 55
Therapy- related chromosomal aberrations in
AML post- cytotoxic therapy, 58
Mutations in splicing factors,
58
Introduction
Within a little more than a decade, high- throughput genome
sequencing technologies have enabled the discovery of
genetic lesions that drive the pathogenesis of the majority of
human cancers. This progress allowed for the identification
of recurring chromosomal abnormalities and gene alterations. Not only have these technological advancements tremendously evolved our understanding of the pathobiology of
myeloid neoplasms, but they also paved the way for significant improvement in disease diagnostics and management.
The ever- increasing importance of (molecular) genetics is
especially reflected by two classification systems, published
in 2022:
• the 5th edition of the World Health Organization
Classification of Haematolymphoid Tumours (Khoury
et al. 2022, https://tumourclassification.iarc.who.int/
welcome)
• the International Consensus Classification (ICC) (Arber
etal. 2022)
In contrast to prior classification efforts, genetics take
center stage in defining the respective disease entities in both
classifications. Morphological or histopathological features
are only used to classify entities in the absence of defining
genetic characteristics. This classification principle is particularly well exemplified in the diseases myelodysplastic
neoplasm (MDS) and acute myeloid leukemia (AML).
Epigenetic regulators,
Histone modification, 60
DNA methylation, 60
Cohesin complex, 60
Transcription factors, 60
Cell signaling genes, 60
References, 65
Further reading,
A disease continuum
While previously considered related albeit distinct entities,
MDS and AML are now thought of as part of a disease continuum with a recognizable pre- malignant state. In addition
to the usefulness of molecular analyses in diagnosis and
prognosis, the discovery of such mutations has offered
genetic tools to study clonal diversity and disease evolution.
Clonal hematopoiesis of indeterminate potential (CHIP)
While cancer is now known to result from the stepwise accumulation of somatic mutations, it has long proved difficult to
define the initial stages that precede the development of
overt hematologic malignancies.
Using existing datasets of exome sequencing on peripheral
blood samples from more than 30,000 patients without
known hematological cancers, three pioneering studies found
evidence for somatic mutations in blood DNA. Despite being
rare (less than 1%) in people under 40 years of age, somatic
mutations increase with age, with frequencies up to 10% and
20% in patients over the age of 65 and 90, respectively. To
describe this phenomenon, the acronym CHIP for “Clonal
Hematopoiesis of Indeterminate Potential” has been adopted.
Individuals in whom mutations were detected had usually
only a single mutation. Remarkably, mutations in only three
genes, i.e. DNMT3A, TET2, and ASXL1, explained the major-
ity of driver mutations sustaining clonal hematopoiesis.
2
59
65
Molecular Hematology, Fifth Edition. Edited by Drew Provan and Hillard M. Lazarus.
© 2024 John Wiley & Sons Ltd. Published 2024 by John Wiley & Sons Ltd.
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51
52 Molecular Hematology
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The initial hit occurs in a single stem cell, and it is believed
that the subsequent expansion of the formed clone is quite
substantial (median clone size measured in the peripheral
blood was approximately 18%). In contrast to previous studies that could detect certain chromosomal translocations
only transiently in healthy individuals, CHIP appears to persist for years with no indication of spontaneous resolution.
Direct evidence for the evolution from clonal hematopoiesis
to hematologic malignancy was possible in a small number
of patients who developed AML as the leukemic samples
were found to contain the somatic mutations at high allele
fractions. On the basis of these findings, CHIP is now viewed
as a precursor myeloid disease state and recognized as such
in the 5th edition of the WHO classification. A diagnosis of
CHIP requires the detection of somatic mutations of myeloid
malignancy- associated genes in the absence of unexplained
cytopenia or a diagnosed hematologic disorder.
In addition to the risk of developing myeloid neoplasia,
which is below 1% annually, CHIP also confers an increased
risk of cardiovascular disease. In fact, CHIP is on par as a risk
factor with established cardiovascular risk factors, such as
smoking.
Aside from an obvious role of certain mutations in myeloid expansion, the mechanisms for this are likely complex
and are beginning to be unraveled, e.g. macrophages carrying TET2 mutations have been demonstrated to be hyperinflammatory, thus offering an explanation for their role in
atherosclerotic disease.
Clonal cytopenia of undetermined significance (CCUS)
Further along the disease continuum, clonal cytopenia of
undetermined significance (CCUS) is also characterized by
the presence of clonal hematopoiesis. The presence of
cytopenia(s) that are not explained by hematologic or nonhematologic conditions distinguishes CCUS from CHIP.
As with CHIP, CCUS is recognized as a precursor myeloid
disease entity in the 5th edition of the WHO classification. In
CCUS, the presence of gene mutations or non-
diseasedefining chromosomal abnormalities diagnostically confirms clonal hematopoiesis. Aside to the demonstration of
clonal hematopoiesis, a diagnosis of CCUS requires the presence of otherwise unexplained, persisting cytopenia(s) and
the exclusion of myeloid neoplasia based on a bone marrow
examination.
While the risk of progression to myeloid neoplasia is generally increased in CCUS patients, the individual progression
risk strongly varies between patients. Studies identified the
number of mutations, the mutational load, and the gene(s)
affected as major influencing variables. With respect to the
affected genes, research has shown that certain mutations or
mutation patterns are highly predictive for the development
of myeloid neoplasia. Among isolated mutations are splicing
factors (see also recurrently mutated genes in MDS) as well as
RUNX1 and JAK2. The combination of a DNMT3A, TET2, or
ASXL1 mutation with at least one other mutation is also con-
sidered as highly predictive. There is preliminary evidence
that the clinical presentation of CCUS patients carrying a
highly predictive mutation or mutational pattern might not
differ from patients with diagnosed myeloid neoplasia.
MDS
The MDS comprise a series of hematologic conditions of
abnormal cellular maturation leading to chronic cytopenias
(i.e. anemia, neutropenia, thrombocytopenia). As a result,
patients with MDS are at risk for symptomatic anemia, infection, and bleeding, as well as progression to AML, which is
often refractory to standard treatment.
The complexities of the pathobiology of MDS are beginning to be elucidated. The development of MDS occurs via a
series of genetic changes in a hematopoietic stem cell. These
changes alter normal hematopoietic growth and differentiation, resulting in an accumulation of abnormal, immature
myeloid cells in the bone marrow, resulting in impairment of
normal hematopoiesis. Advances in the identification of
recurring chromosomal abnormalities and gene alterations
have provided insight into the pathobiology of MDS.
It is well established that specific cytogenetic abnormalities
identified by karyotype analysis or fluorescence in situ hybridization (FISH) analysis bear prognostic significance for
patients with primary MDS and affect the planning of treatment. Certain gene mutations also confer independent prognostic significance in adult patients with MDS, and these
changes are increasingly incorporated into treatment planning. Even those few patients without obvious abnormalities
detected by karyotypic analysis, FISH, or gene mutation analyses likely have acquired copy number alterations or abnormalities in gene expression profiles, which may help to identify
genes with important roles for the pathogenesis of MDS.
The development of secondary, therapy- related MDS or
AML is associated with characteristic chromosomal abnormalities in patients who developed MDS or AML after radiation therapy and/or chemotherapy for an earlier disease, e.g.
lymphoma or a solid tumor, or even non-
malignant disorders, such as rheumatoid arthritis, or following organ
transplantation.
AML
At first glance, the development of AML resembles that of
MDS. However, after a series of genetic changes generate
pre- AML stem cells, the final transformative event leading to
overt development of AML occurs in a hematopoietic precursor rather than in hematopoietic stem cells. By altering
normal hematopoietic growth and differentiation, large
numbers of abnormal, immature myeloid cells accumulate in
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