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Molecular diagnostics andrisk assessment inmyeloid malignancies 53
https://t.me/med1917
the bone marrow and peripheral blood. While capable of
dividing and proliferating, these cells cannot differentiate
into mature hematopoietic cells due to compromising genetic
changes. Cell differentiation is a highly regulated process.
The development of AML involves genetic events that confer
a proliferative advantage to affected cells and genetic events
that interfere with hematopoietic differentiation.
Not unlike other malignant diseases, the genetic alterations in AML include mutation of oncogenes as well as the
loss of tumor suppressor genes. Compared to solid tumors,
however, many hematologic malignancies are associated
with a single characteristic cytogenetic abnormality (e.g. the
Philadelphia chromosome t(9;22) in chronic myeloid leukemia and t(15;17) in acute promyelocytic leukemia, APL).
The identification of recurring chromosomal abnormalities and translocations has largely advanced our understanding of the pathobiology of AML. In addition to having
considerable prognostic significance for patients with AML,
certain cytogenetic abnormalities identified by karyotype
analysis affect the planning of treatment. Abnormalities in
certain genes (e.g. mutations in FLT3, NPM1, KIT) as well as
gene expression profiles confer prognostic significance in
adult patients with AML (Table4.1).
General methodology ofcytogenetic analysis
inhematologic malignancies
The malignant cells in many patients with leukemia, lymphoma, or other malignant hematologic diseases have acquired
clonal chromosomal abnormalities. Some specific cytogenetic
abnormalities are closely, and sometimes uniquely, associated
with morphologically and clinically distinct entities of leukemia or lymphoma, as well as with their prognosis.
Compared to other malignancies and given the ease of
obtaining and processing bone marrow or peripheral blood
samples, significantly more information is available about
the relationship between cytogenetic abnormalities and the
pathogenesis and natural history of the leukemias.
The detection of clonal cytogenetic abnormalities allows
for the differentiation between benign reactive lymphoid or
myeloid hyperplasia versus a monoclonal malignant proliferation and, in some cases, the establishment of specific
diagnoses (e.g. the Philadelphia chromosome in CML). In
addition, the study of cytogenetic abnormalities has provided insight into disease pathogenesis (by identifying genes
controlling cell growth and leukemogenesis), prognosis
(clonal evolution can signify a more aggressive disease
Table4.1 AML risk classification by genetics at initial diagnosis, according tothe European LeukemiaNet (ELN) 2022
Risk category Genetic abnormality
Favorable •
Intermediate • Mutated NPM1 with FLT3- ITD
Adverse • t(6;9)(p23;q34.1)/DEK::NUP214
a
In the absence of other class- defining recurring genetic abnormalities.
b
Excluding core- binding- factor (CBF) AML.
c
For the time being, these markers should not be used as an adverse prognostic marker if they co- occur with favorable- risk AML subtypes.
Source: Adapted from Döhner, H., Wei, A.H., Appelbaum, F.R. etal. (2022). Diagnosis and management of AML in adults: 2022 ELN
recommendations from an international expert panel. Blood 140(12): 1345–1377.
t(8;21)(q22;q22.1)/RUNX1::RUNX1T1
• inv(16)(p13.1q22) or t(16;16)(p13.1;q22)/CBFB::MYH11
• Mutated NPM1 without FLT3- ITD
• bZIP in- frame mutated CEBPA
• Wild- type NPM1 with FLT3- ITD
• t(9;11)(p21.3;q23.3)/KMT2A::MLLT3
• Cytogenetic and/or molecular abnormalities not classified as favorable or adverse
• t(v;11q23.3)/KMT2A- rearranged (exclusive KMT2A- PTD)
• t(9;22)(q34.1;q11.2)/BCR::ABL1
• t(8;16)(p11;p13)/KAT6A::CREBBP
• inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2)/GATA2, MECOM(EVI1)
• t(3q26.2;v)/MECOM(EVI1)- rearranged
• −5 or del(5q); −7; −17/abn(17p)
• Complex karyotype (≥3 unrelated chromosome abnormalities)
excluding loss of X- or Y- chromosomes or one single autosomal monosomy in combination with at least one
structural chromosome abnormalityb)
• Mutated ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, or ZRSR2
• Mutated TP53
a
, monosomal karyotype (two or more monosomies,
c
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54 Molecular Hematology
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course), and treatment planning (certain chromosomal
changes predict for response to specific therapies, or to
choose targeted therapy).
While the detection of specific chromosome translocations and gene fusions allows for a precise diagnosis of leukemia, analysis of recurrent somatic mutations in AML
and MDS adds important genetic information, offering
better risk stratification and selection of targeted
Methods ofdetection
therapies.
Importantly, each technique has its advantages and disad-
The following methods are used for the detection of cytogenetic aberrations: conventional metaphase cytogenetic analysis, FISH analysis, reverse transcription- polymerase chain
reaction (RT- PCR), microarray- based genomic copy number analysis, or DNA or RNA sequencing. An overview of
the methods and their respective advantages and disadvantages is provided in Table4.2.
Table4.2 Selection ofmethods ofdetection andtheir respective advantages anddisadvantages
Method Description Advantages Disadvantages
Conventional
metaphase
cytogenetic analysis
FISH analysis Employs fluorescently labeled DNA
Reverse-
transcription-
PCR (RT- PCR)
Genomic microarray Uses single nucleotide polymorphisms
Targeted sequencing Highly sensitive at single nucleotide level,
Whole genome
sequencing
Examines entire tumor genome by
inducing cellular division and arresting
it in metaphase
probes to hybridize to metaphase
chromosomes or interphase nuclei and
is most useful when certain
abnormalities are suspected or seem
likely based on other investigations
Messenger RNA (mRNA) is copied into
complementary DNA (cDNA) and
subsequently the fusion gene is
amplified by PCR using specific primers
from each gene to detect
translocations resulting in fusion genes
(SNPs) to identify disease susceptibility
loci, genomic imbalances, and loss of
heterozygosity (LOH)
allows detection of recurring gene
fusion transcripts and mutations in
panel reactions
Sequences the entire genome of an
individual, providing comprehensive
information about all genetic
alterations, including point mutations,
insertions, deletions, and structural
rearrangements
vantages – while molecular methods, such as RT- PCR or
NGS techniques assess the entire (mixed) cell population,
FISH provides analysis at the single cell level. A comprehensive picture of the genetic landscape of leukemia or lymphoma emerges from the combination of conventional
cytogenetic analysis, FISH tests, microarray, and NGS studies, which enables personalized genetic- based treatment.
Particularly important in evaluating
hematologic malignancies with
many potential genetic
abnormalities
Rapid and sensitive, can be employed
with bone marrow/peripheral blood
smears, cytospin slides, fixed and
sectioned tissue, provides analysis
at the single cell level
Highly sensitive, suitable for detecting
measurable residual disease (MRD),
assesses entire cell population
Allow genomic analysis without
sequencing
Highly sensitive at single nucleotide
level, allows detection of recurring
gene fusion transcripts and
mutations; can detect novel gene
fusions, including those defining
specific leukemic entities
Detects known gene mutations and
fusion transcripts and can identify
novel ones; powerful tool for
identifying genetic alterations that
may be missed by other sequencing
approaches, and for advancing
understanding of the molecular
basis of leukemia/lymphoma
Technically labor intensive,
requires dividing cells
Cannot detect additional
abnormalities or multiple
clones alone
Requires DNA sequencing to
confirm identity of amplified
product in certain cases
Analyses only pre- defined
sequences
Generates large amounts of data
and requires specific
bioinformatics systems
Generates large amounts of data
and requires specific
bioinformatics systems
Note: Advantages and disadvantages are not exhaustive and may overlap between methods.
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Molecular diagnostics andrisk assessment inmyeloid malignancies 55
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Genetic andclinical consequences
ofchromosomal translocation
An important role in the process of malignant transformation is alterations in the expression of genes that lie at the
breakpoints of the chromosomal translocations or within
functional domains of the encoded proteins. The genes with
a role in transformation can be grouped into the following
functional classes: (i) tyrosine or serine protein kinases; (ii)
cell surface receptors and growth factors; (iii) proteins that
regulate transcription; and (iv) regulators of apoptosis.
Among these, genes that encode proteins regulating transcription are most commonly involved. Gene transcription is
the process by which the information in genomic DNA is
transcribed to RNA. Transcription factors are central to this
process, recognizing and binding to target sequences in the
regulatory elements of genes or to other DNA- binding proteins in a tissue- specific fashion. Chromosomal translocations can alter gene function by aberrant regulation of gene
expression or via expression of a novel fusion protein. The
latter results from the juxtaposition of coding sequences
from two genes that are normally located on different chromosomes. This produces a fusion mRNA and a chimeric
protein with altered gene function. There are three wellcharacterized fusion proteins: BCR::ABL1, PML::RARA, and
RUNX::RUNXT1.
As a result of the t(9;22) in CML a shortened chromosome
22 (i.e. Philadelphia chromosome) leads to the BCR::ABL1
fusion protein. BCR::ABL1 contains the entire catalytic
domain from the tyrosine kinase ABL1, leading to constitutively increased tyrosine kinase activity and explaining the
abnormal cellular proliferation observed in CML. Imatinib
and other tyrosine kinase inhibitors specifically target this
tyrosine kinase activity and exhibit remarkable activity in all
phases of CML.
In APL, another fusion protein is formed by the t(15;17)
producing a fusion protein involving the promyelocytic leukemia (PML) and retinoic acid receptor- alpha proteins
(PML::RARA). The APL fusion protein is responsible for the
growth arrest of promyelocytes, presumably by aberrant
repression of retinoic acid receptortion through histone deacetylase- dependent chromatin
remodeling. Treatment with two compounds, all- transretinoic acid (ATRA) or arsenic trioxide can bypass the transcriptional repression and activate genes leading to the
terminal differentiation of promyelocytes.
AML with RUNX1::RUNX1T1 fusion constitutes a clini-
cally distinct subset of AML with characteristic morphology,
portending a favorable prognosis. Molecularly, the t(8;21)
forms a chimeric protein RUNX1::RUNX1T1. The gene
RUNX1 encodes one sub- unit of a heterodimeric transcription factor termed core- binding factor (CBF), shown to be
essential for hematopoiesis. The oncogenic potential of the
mediated gene transcrip-
RUNX1::RUNX1T1 fusion protein is believed to be due to
aberrant recruitment of nuclear transcriptional co- repressor
complexes leading to transcriptional repression of normal
RUNX1 target genes.
All of the above- mentioned fusion proteins are specific to
the disease. Thus, they can be used for the initial diagnosis as
well as for detecting measurable residual disease (MRD).
Karyotypic evolution
Additional cytogenetic abnormalities may help to detect
transformation to a more advanced disease stage or to a more
aggressive malignancy. As evidenced by subclones on cytogenetic analysis, clonal heterogeneity and evolution are frequent in AML and constitute an independent adverse
prognostic marker in cytogenetically adverse- risk AML.
Clinical implications
Both the clinical course as well as the likelihood of responding to particular treatments can be predicted by specific
genetic abnormalities (e.g. retinoic acid or arsenic trioxide in
APL). The prognostic information derived from cytogenetic
analysis is often independent of other clinical features.
Patients who have less favorable clinical or cytogenetic characteristics may be better treated with more intensive or
investigational therapies, while those with more favorable
prognostic features may be treated with standard regimens.
Moreover, the disappearance of a chromosomal abnormality
present at diagnosis signifies complete remission following
treatment, while its reappearance invariably indicates relapse
of the disease.
Treatment selection
The optimal timing of hematopoietic cell transplantation
(HCT) in the treatment of AML and MDS is still under debate
for some biologic subtypes. Cytogenetic analysis before the
commencement of treatment can guide the choice among
post-
remission therapies that differ in effectiveness, acute and
chronic morbidity, as well as cost. The majority of AML
patients with favorable cytogenetic features (e.g. t(8;21) and
inv(16) or t(16;16)), can be successfully treated solely with
intensive consolidation chemotherapy. Patients who relapse
can be salvaged with HCT in early relapse or second remission. On the other hand, patients with a chromosome abnormality that portends a bad prognosis (e.g. complex karyotype
[≥3 abnormalities] in AML) are rarely successfully treated
with standard therapy and can have drug- resistant disease.
These patients may benefit from transplantation in first
remission or investigational drugs.
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56 Molecular Hematology
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Recurrent cytogenetic abnormalities in MDS
The WHO classification differentiates between MDS with
defining genetic abnormalities and MDS, defined solely by
morphology – thus, genetic testing is as essential for the
diagnosis of MDS as the evaluation of the bone marrow and
peripheral smear.
At the time of diagnosis, approximately half of patients
with MDS have clonal chromosomal abnormalities. These
chromosomal changes can manifest as structural abnormalities involving one chromosome (e.g. inversion, interstitial
deletion), numerical changes (e.g. monosomy or trisomy), or
be a balanced translocation between two chromosomes.
About 15% of patients have multiple abnormalities and thus
exhibit complex karyotypes (≥3 or more abnormalities).
During the course of the disease, additional chromosomal
aberrations may develop on top of previous ones or emerge
in patients with no previous karyotypic abnormality. These
changes are invariably associated with poor prognosis and
progression to acute leukemia. In contrast to AML, none of
the chromosomal abnormalities are specific to MDS and are
also found in other myeloid diseases.
The karyotypic abnormalities that are most commonly
found in MDS are del(5q), del(7q) or monosomy 7 (−7), trisomy 8 (+8), del(20q), as well as loss of the Y chromosome.
Although the same cytogenetic lesions also occur in de novo
AML, the balanced translocations typically found in AML are
very uncommon in MDS. Rather, the chromosomal abnormalities in MDS are usually unbalanced, indicating gain or
loss of chromosomal material. Cytogenetic abnormalities are
even more prevalent in therapy- related MDS/AML, as up to
70% of cases have chromosomal abnormalities, particularly
deletions of chromosomes 5 and 7 or chromosome 17p.
Deletion of the long arm of chromosome 5with or without
additional karyotypic abnormalities is not only the most frequent chromosomal aberration in MDS, it is the only one
constituting its own entity in the WHO classification.
The term “MDS- 5q” refers to a distinctive type of MDS that is
defined by isolated del(5q) or del(5q) plus one other abnormality
(with the exception of −7/del(7q)). A characteristic hallmark on
morphology examination is the finding of megakaryocytes with
hypolobated nuclei. The deletions on chromosome 5 are large
interstitial deletions resulting in retention of only one normal
allele of all genes contained within the deleted segment– this is
referred to as a haploinsufficient state. Numerous genes on the
long arm of chromosome 5have been implicated in MDS pathogenesis and its response to specific therapies.
MDS cases with del(5q) as the sole chromosomal abnormality carry a relatively good prognosis and frequently
respond to treatment with lenalidomide. In contrast, monosomy 5 or del(5q) with more than one additional chromosomal change is associated with advanced MDS and portends
a worse prognosis.
With the notable exception of MDS with isolated del(5q),
other chromosomal aberrations in MDS have not correlated
with specific morphological or clinical entities. However,
they do play a substantial role in the pathogenesis and prognosis and have a major impact on the clinical management of
patients.
Certain cytogenetic abnormalities in MDS are used in the
prediction of survival and progression to AML. The presence
or absence of specific chromosomal aberrations are incorporated into several prognostic scoring systems for MDS, such
as the original, the revised, and the molecular International
Prognostic Scoring Systems (IPSS, IPSS-
R, IPSS- M), the
WHO Prognostic Scoring System, and the MD Anderson
Cancer Center MDS model. The recently published system,
IPSS- M, is shown in Figure4.1.
Importantly, cytogenetic analysis cannot predict individual MDS patient outcome due to the fact that many patients
succumb to persistent pancytopenia, irrespective of progression to AML.
Recurrent cytogenetic aberrations in AML
A key component in the evaluation of all patients with newly
diagnosed or suspected AML is the cytogenetic analysis of
metaphase cells. In some cases, specific cytogenetic abnormalities correspond to morphologically and clinically distinct subsets of the disease. Considering clinical, molecular/
genetic, and pathological factors, the WHO classification of
tumors of the hematopoietic and lymphoid tissues divides
AML into two major subgroups: “AML with defining genetic
abnormalities” and “AML, defined by differentiation.” While
the latter group includes cases that lack defining genetic
abnormalities, the former group contains cases with defining
gene fusions, rearrangements, and mutations.
About 50–60% of patients with de novo AML have abnormal karyotypes if standard banding techniques are used. The
most common abnormalities seen on karyotype are t(15;17)
(q24.1;q21.1), trisomy 8, t(8;21)(q22;q22.1), rearrangements
of 11q, and inv(16)(p13.1q22)/t(16;16)(p13.1;q22).
According to the WHO, AML with defining genetic abnormalities are diagnosed regardless of blast count inthe following
subgroups with defining cytogenetic abnormalities:
APL with PML::RARA fusion. Highly specific for APL, the
balanced translocation t(15,17)(q24;q21) results in a novel
PML::RARA fusion protein that silences genes that are
critical for cell differentiation, resulting in a differentia-
tion block and an expansion of myeloid progenitors. APL
constitutes a medical emergency carrying a high rate of
early mortality, often due to hemorrhage from dissemi-
nated intravascular coagulation. Treatment should be
started as soon as the diagnosis is suggested by cytologic
criteria. Cytogenetic and molecular genetic analysis can
then confirm the diagnosis. If prompt treatment with
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Molecular diagnostics andrisk assessment inmyeloid malignancies 57
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Clinical data
• Bone marrow blasts [%]
• Hemoglobin [g/dL]
• Platelet count [x10
IPSS-R cytogenetics category
• Very good | -Y, del(11q)
• Good | normal, del(5q), del(12p),
• Intermediate | del(7q), +8, +19, i(17q),
• Poor | -7, inv(3)/t(3q)/del(3q),
• Very poor | Complex: > 3 abnormalities
Figure4.1 International Prognostic Scoring System- Molecular (IPSS- M). Hemoglobin, number of thrombocytes, bone marrow blast
percentage, IPSS- R cytogenetics category, and molecular data on 31 genes are scored and used to assign MDS patients to one of six risk groups
exhibiting significant differences in median survival and probability of developing AML. Based on Bernard, E., Tuechler, H., Greenberg, P.L. etal. (2022).
Molecular international prognostic scoring system for myelodysplastic syndromes. NEJM Evid. 1(7).
9
/L]
del(20q), double incl.
del(5q)
any other single or double
independent clones
double including -7/del(7q),
complex: 3 abnormalities
Prognostic favourable factor:
• SF3B1 in the absence of comutations in:
Risk factors
• TP53 multihit
• KMT2A PTD
• FLT3 ITD or FLT3 TKD
• SF3B1 in the presence of isolated del(5q)
• NPM1
• RUNX1
• NRAS
• ETV6
• IDH2
• CBL
• EZH2
• U2AF1
• SRSF2
• DNMT3A
• ASXL1
• KRAS
• Nres
(# of mutations in the listed genes,
variable with possible values 0, 1 or 2)
Molecular data
BCOR, BCORL1, RUNX1, NRAS,
STAG2, SRSF2, and del(5q)
Nres genes
BCOR, BCORL1, CEBPA, ETNK1, GATA2,
GNB1, IDH1, NF1, PHF6, PPM1D, PRPF8,
PTPN11, SETBP1, STAG2, and WT1
ATRA or arsenic trioxide is started, these patients have an
excellent prognosis.
AML with RUNX1::RUNX1T1 fusion (previously AML
with t(8;21)(q22;q22.1)). The t(8;21)(q22;q22.1) is found
in 5–7% of AML cases and is the most frequent abnormality in children with AML. The RUNX1::RUNX1T1 fusion
blocks myeloid differentiation but requires other cooperating pathogenic alterations for leukemic development
and progression. This cytogenetic abnormality has a distinct morphological phenotype and heralds a favorable
prognosis in adults, but a poor prognosis in children.
Factors like a high mutation burden (≥2) or the presence
of KIT mutations adversely affect prognosis. Besides this,
hereditary variants of RUNX1 are responsible for familial
platelet disorder and are associated with an inherited AML
predisposition.
AML with CBFB::MYH11 fusion. Representing approxi-
mately 5–8% of cases in younger patients, its classical bone
marrow morphology typically shows monocytic and granulocytic differentiation with abnormal eosinophils. The
prognosis of AML with CBFB::MYH11 is favorable.
Patients with inv(16) generally respond well to intensive
chemotherapy.
AML with DEK::NUP214 fusion. Seen in about 1% of adult
patients with newly diagnosed AML and 0.6–1.7% of
AML in children. The bone marrow is morphologically
associated with multilineage dysplasia, including nuclear
lobulation in erythroblasts and myeloid cells, ring sideroblasts, hypogranular myelopoiesis, and micromegakaryocytes. FLT3- ITD is present frequently and does not impact
overall survival but appears to be associated with faster
relapse kinetics. Patients generally have a poor response to
standard therapy.
AML with RBM15::MRTFA fusion. This subtype occurs in
<1% of all AML and 10–12% of pediatric acute megakaryoblastic leukemia (AMKL). The RBM15::MRTFA fusion
gene modulates HOX- induced differentiation and extracellular signaling pathways, associated with leukemogenesis. Cases are defined by karyotype-
based evidence of
t(1;22)(p13.3;q13.1) or molecular confirmation of inframe fusion between RBM15 and MRTFA.
AML with KMT2A rearrangement. KMT2A rearrange-
ments are the most common abnormality in children,
constituting 20% of all AML cases. In adults, they are
found in 2–3% of patients with AML. More than 80 fusion
partners of KMT2A are described, with MLLT3, AFDN,
ELL, and MLLT10 being the most common fusion partners. Morphologically, most cases show monocytic,
monoblastic, or myelomonocytic features. The prognosis
depends on the fusion partner. Cases with KMT2A::MLLT3
fusion have an intermediate risk of relapse. Other KMT2A
rearrangements are associated with a poor prognosis.
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58 Molecular Hematology
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AML with MECOM rearrangement. MECOM describes the
myelodysplastic syndrome 1 (MDS1) and ecotropic viral
integration site 1 (EVI1) complex locus (MECOM) on
chromosome 3q26 that encodes several alternative transcripts through intergenic splicing. EVI1 is an oncogenic
transcription factor whose expression in neoplastic
myeloid cells disrupts myeloid differentiation, cell cycle
regulation, and cell signaling pathways. MECOM rearrangements occur in all age groups. In young adults, they
occur in ~1.2% of de novo AML. The most common
(~40%) cytogenetic alterations involving MECOM include
inv(3)(q21.3q26.2) and t(3;3)(q21.3;q26.2). In bone marrow, multilineage dysplasia is common and most pronounced in megakaryocytes. Erythroid and granulocytic
dysplasia is also common and marrow eosinophils, basophils, and/or mast cells may be increased. The disease
course is aggressive and even worsened by a complex karyotype, additional monosomy 7, and ≥2 additional
mutations.
AML with NUP98 rearrangement. More than 40 fusion
partners of NUP98, categorized into HOX and non- HOX
genes, are already described. In children, the incidence is
2–4% of AML, in adults 2.3%. Most cases have a high blast
count in the bone marrow and blasts may or may not show
maturation. If present, it tends to be myelomonocytic or
monocytic. Dysplastic changes may be seen. In up to 34%
of patients under 3 years of age, leukemic blasts have
megakaryoblastic differentiation. The prognosis of this
AML subtype is poor and even worse when FLT3- ITD is
present.
While the above cytogenetic aberrations constitute a
diagnosis of AML irrespective of blast count, there are
additional subtypes of AML where certain genetic abnormalities bear prognostic significance. However, these
require bone marrow blast counts of at least 20% for diagnosis of AML:
AML with BCR::ABL1 fusion. The BCR::ABL1 fusion is rare
in AML; the subtype accounts for <0.5% of all AML cases
and <0.5% of all cases of acute and chronic leukemia with
BCR::ABL1. 50–60% of cases have additional chromo-
somal alterations. Compared to the myeloid blast phase of
CML, patients with AML with BCR::ABL1 usually show a
higher proportion of blasts and reduced relative and abso-
lute numbers of basophils in bone marrow. The blast crite-
rion still applies in this AML subtype to avoid overlap with
CML. Overall, the response to standard AML induction
chemotherapy is poor and treatment with single-
tyrosine kinase inhibitors does appear to be ineffective.
AML with other defined genetic alterations. This entity
includes emerging or provisional AML subtypes with
distinct genetic features. Overall, these subtypes tend to
have an unfavorable prognosis or a high risk of relapse.
agent
The following aberrations constitute subtypes of this
entity:
• AML with RUNX1T3(CBFA2T3)::GLIS2
• AML with KAT6A::CREBBP
• AML with FUS::ERG
• AML with MNX1::ETV6
• AML with NPM1::MLF1
Therapy- related chromosomal
aberrations in AML post cytotoxic
therapy
According to the WHO classification from 2022, there are two
categories of MDS or AML post cytotoxic therapy (pCT): (i)
cases following exposure to alkylating agents and / or ionizing
radiation and (ii) those following exposure to topoisomerase II
inhibitors. Patients who develop MDS or AML pCT have distinct clinical presentations and genetic alterations (Table4.3).
Recurrently mutated genes in MDS
The recent advances in less expensive high- throughput
genome sequencing technologies have enabled the discovery
of genetic lesions that drive the pathogenesis of the majority
of human cancers, including the MDS.
Based on panels of genes that were previously found to be
mutated in other myeloid diseases, a number of studies have
described the mutational landscape of somatic point mutations in MDS. According to current data, up to 90% of
patients bear mutations in at least one recurrently mutated
gene and it is believed that the remaining 10% of patients
have mutations in genes yet to be identified.
MDS is the result of the sequential accrual of somatic
mutations in hematopoietic stem cells. Based on calculations, these stem cells amass approximately 1.3mutations in
exons every decade, but the majority of these are innocent
bystanders with no functional alteration. However, this calculation presumes that the rate of mutation is constant– in
reality, the process of mutational accrual could be dynamic
mutations.
The spectrum of recurrent somatic mutations in MDS and
other myeloid diseases can be grouped according to their
biologic pathways.
Mutations insplicing factors
A hallmark of eukaryotes, the alternative splicing of premRNA enables the generation of different proteins from the
same gene. The pre- mRNA is an intermediate product that
occurs during the translation of DNA into proteins. It is the
messenger ribonucleic acid (mRNA) immediately after its
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Molecular diagnostics andrisk assessment inmyeloid malignancies 59
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Table4.3 Characteristic clinical andgenetic features ofAML andMDS pCT according toWHO classification 2022
AML- pCT
Type 1
Frequency 60–70% 20–30%
Cytotoxic agents Alkylating agents and
radiation
Latency (after exposure) 5–7 years 1–3 years
Presentation and
diagnosis
Typical genetic
alterations
Outcome Unfavorable Favorable; comparable to de
a
The typical genetic alterations of MN- pCT include mutations in TP53 and PPMID. The biology of cases with de novo type genetic alterations,
such as CBF gene rearrangements and NPM1 mutations may be different, although prognosis is still worse than de novo cases. Hence, these
cases should still be categorized under myeloid neoplasms post cytotoxic therapies.
b
IPSS- R applicable to t- MDS in predicting AML transformation and overall survival.
Source: Adapted from 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(7), 1703–1719.
a
AML, with a pre- MDS phase Overt AML, no prior MDS
• Unbalanced, often complex
karyotypes
• Loss of 5q, 7q, 17p
• Loss of 3p, 11q, 12p, 13q, 18q,
21q
• Trisomy 8 and 11
•
Frequently TP53 deletions +TP53
mutations
/or ionizing
AML- pCT
Type 2 MDS- pCT
DNA topoisomerase II
inhibitors
phase
Balanced translocations
•
• KMT2A fusions
• t(15;17)(q24;q21)
• inv(16)(p13q22)
• t(8;21)(q22;q22)
• t(3;21)(q26;q22)
novo AML if the respective
therapy can be applied
Alkylating agents and /or ionizing
radiation
MDS, mostly high- and very- high-
risk according to IPSS- R
• >70% aberrant karyotype
• Typically unbalanced
chromosome aberrations
• Loss of 5q, 7q, 17p
• Loss of 12p, 13q, 18q, 20q
• Gains of 1q, 21q
•
Frequently TP53 deletions +TP53
mutations
Poor; low to intermediate IPSS- R
subgroups show inferior outcome
compared with de novo MDS
b
formation in the transcription phase of protein biosynthesis,
when it has not yet been processed. The pre- mRNA is
modified by adding a cap structure (at the 5′ end), a poly(A)
tail, and by splicing the introns in the nucleus. Interestingly,
one of the hallmarks of cancer is aberrant alternative splicing. Recurrent mutations in genes that are part of the spliceosome have been found in up to 60% of cases of MDS.
The mutations in SF3B1, U2AF1, and SRSF2 are invariably
found within defined hotspots of these genes leading to loss
of function, altering the operation of the splicing machinery.
Mutations within the splicing componentry most commonly affect SF3B1 and occur in up to 90% of MDS patients
with ring sideroblasts. It is believed that the ring sideroblast
phenotype is the result of the aberrant splicing of genes central to iron homeostasis. In this context, SF3B1 mutations are
associated with a good prognosis, leading to the recognition
of the MDS subtype MDS with low blasts and SF3B1 muta-
tion. However, research indicates that the genetic landscape
influences the prognosis in SF3B1 mutated MDS. According
to the IPSS-
M (see also Figure4.1), SF3B1 in the context of a
del(5q) or a co- mutation in either BCOR, BCORL1, RUNX1,
NRAS, STAG2, or SRSF2 represents a genetic risk factor.
The second most frequent splicing factor mutation in
MDS affects the SRSF2 gene; this gene is commonly mutated
in the overlap syndromes of MDS/MPN. Mutations in
SRSF2 were demonstrated to alter binding of the protein to
splice enhancers with resulting mis- splicing of several
important downstream genes one of which is EZH2. In
10–15% of MDS cases mutations are found in U2AF1.
Resulting loss- of- function is reported to lead to increased
exon skipping. SRSF2 and U2AF1 have not been associated
with a similar favorable prognosis as SF3B1. Despite the
aforementioned findings, no single mis- spliced isoform has
hitherto been implicated in the pathogenesis of MDS.
Moreover, studies using mouse models of splicing genes
have not yielded similar patterns of altered splicing, neither
between mice and men nor between individual mice. The
role that these mutations play in MDS pathobiology is perhaps unrelated to their known function within the splicing
machinery.
Epigenetic regulators
Another set of genes found to be recurrently mutated in
MDS affect genes that are involved in epigenetic regulation.
Epigenetic regulation is the result of post- translational modification of either histones or the DNA itself.
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Histone modification
Enzymes can covalently modify the tails of histones leading
to changes in the structure of chromatin and affecting the
binding of protein involved in gene regulation. Among the
central regulators of transcriptional silencing in development are the polycomb repressive complexes (PRCs) composed of two separate protein complexes, PRC1 and PRC2.
Their action on histones H2A and H3 results in the compaction of chromatin. Genes that are part of both protein complexes have been found to be mutated in MDS. Mutation in
two components of PRC1, i.e. BCOR and BCORL1, have been
shown to be recurrently mutated in 5% of MDS cases and
found to portend an unfavorable prognosis. EZH2 is mutated
in about 5% of MDS patients; the resulting protein is part of
the catalytic subunit of PRC2 and studies in transgenic mice
have confirmed that loss of Ezh2 results in MDS.
While in itself ASXL1 is not part of the PRC, it has been
shown to indirectly affect PRC2 leading to deubiquitylation
of histone H2A. Approximately 20% of MDS patients carry
recurrent mutations in ASXL1. Mutations in BCOR, EZH2,
and ASXL1 have been shown to disrupt normal hematopoi-
etic differentiation likely explaining their putative role in
dysplasia and cytopenia.
DNA methylation
The regulatory regions of the DNA can either be accessible
or inaccessible. Which state is favored is largely controlled by
the methylation of cytosines in repetitive CpG elements.
DNA methyltransferases (DNMTs) add methyl groups while
the TET family of proteins demethylate in a multistep process. Mutations in DNMT3A leading to loss- of- function of
the protein are found in approximately 15% of MDS cases.
TET2 is one of the most frequently mutated genes found in
up to 30% of MDS cases. The state of hypermethylation of
cytosines at enhancer sites is believed to repress several genes
with important roles in myeloid differentiation. Of note,
DNMT3A and TET2 are both expressed in hematopoietic
stem cells– in a complementary, interconnected, yinyang kind of fashion– having essential roles in self- renewal
and myeloid differentiation.
An initially puzzling observation was how mutations in
enzymes involved in the citric acid cycle play a role in MDS
and leukemia pathogenesis. It was found that cells with
mutations in IDH1 and IDH2 accumulated a novel oncome-
tabolite, 2- hydroxyglutarate (2- HG), instead of alphaketoglutarate. 2- HG was shown to diffuse to the nucleus
leading to inhibition of– among others– TET2 and thereby
also affecting DNA methylation. Mutations in IDH1 or IDH2
occur in ~5% of patients with MDS and portend an unfavorable clinical outcome. While the oncometabolite was
and-
shown to be sufficient to promote leukemogenesis, the
effects were found to be reversible. This has led to the development of 2- HG inhibitors, which are currently being evaluated in clinical trials.
Cohesin complex
The formation of a specific protein complex that holds
together sister chromatids, preventing the collapse of the
replication fork is an important part of DNA repair. This
protein complex is made up of the so- called cohesins, i.e.
STAG2, SMC3, SMC1A, and RAD21. In aggregate, inactivat-
ing mutations in cohesin genes are found in 15% of MDS
cases. Perhaps somewhat counterintuitive given their function, cohesin mutations have not been associated with chromosomal abnormalities. Rather, cohesin mutations are now
believed to lead to altered gene expression due to a failure to
stabilize DNA loops that normally facilitate the interaction
of promoters and distant enhancers.
Transcription factors
A small group of hematopoietic transcription factors have
been found to be recurrently mutated in MDS. Since germline mutations in RUNX1, GATA2, and ETV6 had been
linked to the development of MDS and AML in inherited
bone marrow failure disorders, these genes were also
included in targeted sequencing panels for the study of MDS.
Found in approximately 10% of MDS cases, mutations in
RUNX1 are often associated with severe thrombocytopenia.
The protein forms the sub- unit of the core binding factor
and is responsible for DNA binding. RUNX1has several target genes with important roles in hematopoiesis.
GATA2 is a zinc finger protein highly expressed in HSCs
and essential for normal hematopoietic differentiation.
Somatic mutations in GATA2 account for only 1% of MDS
cases. The transcription factor Wilms tumor protein WT1has
been shown to recruit TET2 to specific loci and is mutated in
fewer than 5% of cases of MDS. There are additional transcription factors with mutation frequencies below 1%.
Cell signaling genes
Genes for components of cell signaling networks were found
to be mutated in a host of other myeloid malignancies and
were thus obvious candidates to study in MDS. However,
compared to AML, CMML, or PV/ET, the frequencies with
which these genes are mutated in MDS are quite low.
Mutations in components of the MAPK pathway, e.g. NRAS,
KRAS, NF1, and PTPN11, are the most prevalent in MDS,
and together account for approximately 10% of MDS cases.
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In general, most mutations in signaling pathway components
lead to the constitutive activation of the protein product. Of
note, these mutations usually occur late in the course of the
disease in more aggressive subclones that signify the transition to secondary AML.
TP53
The single most frequently mutated tumor suppressor gene
among all human cancers is tumor protein 53 (TP53). In the
clinical condition known as the Li- Fraumeni syndrome,
humans are born with a single mutant allele of TP53 and
these individuals have a drastically increased risk for many
types of cancer, including MDS and AML. In patients with
MDS, somatic mutations of TP53 are usually associated with
high blast counts, low platelet counts, a complex karyotype,
and previous exposure to chemotherapy. Dubbed “guardian
of the genome,” p53 functions as a mediator of cellular stress.
In the appropriate context, p53increases proapoptotic genes
and mediates cell cycle arrest. Accumulation of additional
genomic aberrations and chromosomal instability together
with chemotherapy resistance observed in patients with
inactivation of the p53 pathway is likely due to inadequate
activation of the DNA damage response and loss of cell cycle
arrest measures, respectively.
The negative prognostic impact of TP53 mutations had
previously been demonstrated in a number of hematological
malignancies, including MDS. While TP53 mutations are
strongly associated with high- risk and secondary (therapyrelated) MDS, the overall incidence in all MDS subgroups
ranges between 7% and 16%. TP53 mutations occur frequently in the context of complex karyotypes, and have also
been associated with disease progression and poor response
to therapy. Research on factors that influence prognosis (e.g.
the mutational load and the karyotype) in TP53 mutated
MDS is ongoing, but the allelic state of TP53 has emerged as
a key prognostic marker. The presence of two or more TP53
mutations, a mutation and a deletion (corresponding to 17q
deletion), or a mutation with a copy-
neutral loss of heterozygosity (CN- LOH), is referred to as biallelic hit or multihit. Biallelic events portend an especially poor prognosis,
represent the factor with the highest risk associated in the
IPSS- M and define the MDS subtype “MDS with biallelic
TP53inactivation.”
Recurrently mutated genes in AML
The observation that the mutational landscapes of MDS and
AML show a significant overlap underlines the notion that
MDS and AML represent stages of a disease continuum. As
with MDS, factors that contribute to splicing, transcription,
epigenetic regulation, cohesion, and cell signaling are frequently found mutated in AML, as is the gene TP53. However,
there are some mutations that represent late events in AML
pathogenesis that are mostly not reported in MDS cases,
notably mutations in NPM1, CEBPA, and FLT3.
Nucleophosmin 1 (NPM 1) participates in many different
cellular processes. It acts as a histone chaperonin and interacts with BRCA2 to regulate centromere duplication. It is
also implicated in proliferation and cell survival, due to its
regulation of the tumor suppressor factors ARF and TP53, as
well as the proapoptotic protein kinase R. Mutations usually
occur in exon 12 of NPM1.
CEBPA is a myeloid transcription factor. Mutations in
CEBPA result in the inhibition of granulocytic maturation.
Since the introduction of “AML with mutated CEBPA” as a
provisional entity in 2008 (4th edition of the WHO classification), it has become evident that cases with mono- allelic
and cases with bi- allelic CEBPA mutation differ in terms of
gene expression and prognosis. The overall favorable outcome was only observed when both CEBPA alleles were
mutated, which led to an adjustment of the disease category
in the WHO classification of 2017. For the current edition of
the WHO classification, published in 2022, the definition of
the disease category was changed again. It now includes biallelic (biCEBPA) as well as single mutations located in the
basic leucine zipper (bZIP) region of the gene (smbZIP-
CEBPA), as a favorable prognosis associated with smbZIP-
CEBPA was demonstrated in different cohorts.
Although FLT3 mutations do not define an AML disease
category, it is strongly recommended to investigate the FLT3
mutation status at diagnosis. FLT3 encodes a receptor tyros-
ine kinase, which acts as receptor for the cytokine FLT3LG.
Upon ligand activation, FLT3 regulates factors of both the
JAK–STAT and the RAS signaling pathway. Mutations of
FLT3 can occur within the tyrosine kinase domain (TKD)
and internal tandem duplications (ITD) of FLT3. Both ren-
der FLT3 constitutively active, which promotes proliferation
and cell survival. In the current recommendations of the
European LeukemiaNet (ELN) of 2022, FLT3- ITD mutations
are generally assigned to the intermediate- risk group. The
prognostic impact of FLT3-
TKD mutations depends on
additional mutations. Aside from its prognostic value, early
detection of a FLT3 mutation has clinical implications, as
patients may benefit from FLT3 inhibitors, such as
midostaurin.
Mutations in AML are more prevalent than in MDS. In
almost every case (96% to >99%) at least one mutation is
readily detected. The vast majority of cases bear two or more
mutations, so that gene–gene interactions have to be taken
into account, especially for prognostic evaluation.
The mutational landscape in AML displays a highly specific co- mutation pattern– while some mutations are frequently found co- mutated, others are mutually exclusive. A
good example is the recurrently mutated gene NPM1. Comutations of NRAS are frequently detected in amino acid
residues G12 and G13, but never for Q61. The same holds
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true for the co- mutation pattern of NPM1 and IDH2,
co- mutations are restricted to IDH2- R140 and not observed
for IDH2- R172.
The importance of the genetic context for prognosis is
increasingly recognized. Depending on occurring comutations, an NPM1 mutation can be associated with a
favorable or an inferior prognosis. The presence of concomitant DNMT3A and FLT3- ITD mutations confers an adverse
outcome. Most interestingly, a strong prognostic impact was
only detectable when all three genes were mutated. In contrast, an NPM1 mutation in the presence of mutated
DNMT3A and NRAS- G12 or NRAS- G13 as well as FLT3 wild
type is a positive prognostic indicator.
Two recurrently mutated genes define AML disease categories among the group “AML with defining genetic abnormalities.” Aside from the respective mutation, blast counts
greater than 20% are required in WHO classification to meet
the diagnostic criteria for AML with CEBPA mutation. To
change the blast criterion, there is not yet enough data. In
contrast, AML with NPM1 mutation can be diagnosed independently of the blast count. There are no specific morphological features associated with either of the two molecularly
defined AML subtypes.
AML with NPM1 mutation. Found in 27–35% of all adult
AML cases and in 45–64% of adult cases with a normal
karyotype. Multi- lineage dysplasia occurs in 20–25% of
cases, but does not take precedence over the NPM1 muta-
tion. While the individual prognosis highly depends on
the genomic context, as discussed above, patients with this
subtype generally correspond well to induction therapy
and have a good prognosis.
AML with CEBPA mutation. As is the case for AML with
NPM1 mutation, this subtype is also associated with a
normal karyotype and a favorable outcome. The presence
of multi- lineage dysplasia does not influence prognosis.
FLT3- ITD and GATA2 zinc finger 1 are recurrently found
co- mutated in this AML subtype; however, their prognos-
tic impact is still unclear.
The former provisional entity “AML with mutated
RUNX1” of the 4th edition of the WHO classification (2017)
was determined to lack enough specificity to define a discrete AML type, since it overlaps with a broad range of defining molecular features. Accordingly, there is no separate
entity defined by this aberration in the current WHO classification 2022.
Differences and similarities of the WHO
classification 2022 and the ICC 2022
As previously mentioned, two new classification systems
were published in 2022: The WHO classification and the
ICC. In both new classifications, the paradigm shift from
phenotype to genotype that we have been experiencing for
some time is evident. Although both classifications have
their advantages, the parallel use of two different classifications may pose difficulties for diagnosticians and a solid
overview of the crucial similarities and differences is helpful
(Tables4.4 and4.5).
Concerning MDS, one important difference between the
two systems is the renaming of “myelodysplastic syndromes”
into “myelodysplastic neoplasms” of the WHO to underscore
their neoplastic nature and harmonize terminology with
MPN. Within the ICC, the term “myelodysplastic syndromes” remains. The short-
term “MDS” is currently used in
both classifications. The WHO distinguishes two major subtypes in MDS: “MDS with defining genetic abnormalities”
and “MDS, morphologically defined.” This broad distinction
is not found in the ICC, but in the respective subgroups,
there exists overlap. The group of MDS with defining genetic
alterations includes the well- known subgroup of MDS- 5q
and the two subgroups of MDS- SF3B1 and MDS- biTP53.
The new group of MDS- SF3B1 is also introduced by the ICC.
According to the WHO, the term MDS with minor blasts
and ring sideroblasts can be further used for SF3B1 wildtype cases in which the proportion of ring sideroblasts is
≥15%. Moreover, the subgroup of MDS- biTP53 includes
cases with two or more TP53 mutations or a TP53 mutation
with concomitant TP53 deletion or copy number neutral loss
of heterozygosity.
In AML, too, the WHO distinguishes between two major
groups “Acute myeloid leukemia with defining genetic
abnormalities” and “Acute myeloid leukemia, defined by differentiation.” Equivalently, the ICC is also increasingly
guided by genetic characteristics when dividing AML into
subgroups, but without the use of these two main categories.
An important WHO modification is the adjustment of the
respective blast count (%). As described in the previous sections, in the group of “AML with defining genetic abnormalities,” the blast criterion of 20% now only applies to the entities
AML with BCR::ABL1 fusion (in order to guarantee a possible distinction from CML) and AML with CEBPA mutation
(due to the current data situation). Also in the ICC, the
defining blast percentage was lowered to 10% in some of the
genetically defined AML subtypes. Furthermore, within the
WHO classification, the group of “AML with biallelic CEBPA
mutation” has been changed to “AML with CEBPA mutation,” so that both, monoallelic mutations (which have to lie
in the “basic leucine zipper (bZIP)” region), or biallelic
mutations are included. Within in ICC, AML with CEBPA
bZIP
in- frame mutations is considered to have a favorable prognosis regardless of whether they are biallelic mutations.
Additionally, AML with TP53 mutations is considered to
be a distinct AML subtype with unfavorable prognosis in
theICC. If pure erythroid leukemia with TP53 mutation is
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