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CLASSIC MYELOPROLIFERATIVE NEOPLASMS |

APRIL 20, 2023

Genetic basis and molecular

profiling in myeloproliferative
neoplasms
Damien Luque Paz, Robert Kralovics, Radek C. Skoda

Blood (2023) 141 (16): 1909–1921.

https://doi.org/10.1182/blood.2022017578 Article history 

Connected Content
A related article has been published: Essential
thrombocythemia: challenges in clinical practice and
future prospects
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A related article has been published: Biology and
therapeutic targeting of molecular mechanisms in MPNs

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Abstract
BCR::ABL1-negative myeloproliferative neoplasms
(MPNs) are clonal diseases originating from a
single hematopoietic stem cell that cause
excessive production of mature blood cells. The 3
subtypes, that is, polycythemia vera (PV), essential
thrombocythemia (ET), and primary myelofibrosis
(PMF), are diagnosed according to the World
Health Organization (WHO) and international
consensus classification (ICC) criteria. Acquired
gain-of-function mutations in 1 of 3 disease driver
genes (JAK2, CALR, and MPL) are the causative
events that can alone initiate and promote MPN
disease without requiring additional cooperating
mutations. JAK2-p.V617F is present in >95% of PV
patients, and also in about half of the patients with
ET or PMF. ET and PMF are also caused by
mutations in CALR or MPL. In ∼10% of MPN
patients, those referred to as being “triple
negative,” none of the known driver gene
mutations can be detected. The common theme
between the 3 driver gene mutations and triple-
negative MPN is that the Janus kinase–signal
transducer and activator of transcription
(JAK/STAT) signaling pathway is constitutively
activated. We review the recent advances in our
understanding of the early events after the
acquisition of a driver gene mutation. The limiting
factor that determines the frequency at which
MPN disease develops with a long latency is not
the acquisition of driver gene mutations, but rather
the expansion of the clone. Factors that control the
conversion from clonal hematopoiesis to MPN
disease include inherited predisposition, presence
of additional mutations, and inflammation. The full
extent of knowledge of the mutational landscape in
individual MPN patients is now increasingly being
used to predict outcome and chose the optimal
therapy.

Subjects: Free Research Articles, Hematopoiesis

and Stem Cells, Myeloid Neoplasia, Review
Articles, Review Series

Introduction
The clonal origin of myeloproliferative neoplasms
(MPNs) had already been recognized in 1976 using
X-chromosome inactivation patterns in the
peripheral blood of female MPN patients,1 and it
was later firmly established using somatic gene
mutations as the clonal markers.2,JAK2-p.V617F is
detectable in purified human hematopoietic stem
cells (HSCs),3 and the stem cell origin of MPNs
was demonstrated experimentally in mouse
models of MPN disease. by transplanting a single
HSC that expresses JAK2-p.V617F into recipient
mice, which then developed MPN disease.4 Recent
studies using whole-genome sequencing (WGS) of
DNA from single hematopoietic colonies
confirmed the single-HSC origin in human MPN
disease and established a timeline for the
preclinical initiation and expansion of the JAK2-
mutant clone (Figure 1).5,6 With the use of next-
generation sequencing (NGS), our knowledge of
the mutational landscape in MPN disease has
greatly improved, and we now have an advanced
understanding of the genetic basis of MPNs. Until
recently, the risk factors used to predict prognosis
in MPN disease were mostly based on clinical
parameters, but information on the mutational
landscape of MPNs is now increasingly being
added to improve and individualize the prognostic
scores.

Figure 1.

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Reconstructing the timeline of clonal evolution in

myeloproliferative neoplasms. Schematic drawing of the
timeline (not to scale) and the di!erent hematopoietic
compartments during the clonal evolution of MPNs
caused by JAK2-p.V617F. Starting in embryogenesis with
the first division of an ancestral HSC, the daughter cells
acquire a number of mutations in their genomes that can
be used as markers to distinguish them from all other cells
that underwent cell division. New mutations are added
during each of the next cell divisions, and by comparing
the sequence similarities and di!erences of individual
HSCs, a phylogenetic tree can be reconstructed. This
analysis relies on taking bone marrow cells after MPN has
been diagnosed and depositing single HSCs or progenitor
cells into wells where they can be expanded in liquid
culture to obtain single cell–derived colonies. DNA from
each of these colonies is then analyzed by WGS. By
comparing the sequence similarities and di!erences of
individual HSCs, a phylogenetic tree can be reconstructed
that originates in a common ancestor HSC that first
divided at the time of gastrulation. The estimate of the
time when JAK2-p.V617F mutation was acquired is
calculated by assuming a constant mutational rate of 19
mutations per HSC per year. This estimate is not
confounded by the increased cell division rate of JAK2-
p.V617F mutant HSCs, because only sequence alterations
that occurred before JAK2-p.V617F was acquired were
used for deriving the estimate.

Genetic basis and clonal evolution

of MPNs
A large number of genes carrying mutations in
hematopoietic cells from MPN patients have been
identified by targeted NGS and whole exome
sequencing (WES; Table 1). No general consensus
has been reached regarding the nomenclature that
should be used to classify these mutations,
although the distinction between “driver” vs
“passenger” mutations is widely used. In the
context of MPNs, we propose to further distinguish
between “disease driver” mutations and “clonal
driver” mutations.

Table 1.
Recurrent somatic gene mutations in
myeloproliferative neoplasms (MPNs) and
secondary acute myeloid leukemia (AML)

Gene Function Location Frequency, %

and type
of PV ET
mutation

Disease
driver
mutations

JAK2 JAK-STAT V617F or 98% 55%

signaling exon 12

MPL TPO receptor Exon 10 0% 5%-7%

JAK-STAT
signaling

CALR Chaperone Frameshift 0% 25%-30%

protein CALR- in exon 9
mut binds and
activates
MPL

Clonal
driver
mutations

TET2 Epigenetic All exons 10%-20% 3%-10%

regulation

DNMT3A Epigenetic R882 and 5%-10% 1%-5%

regulation all exons

IDH1 Epigenetic R132 1%-2% 1%-2%

regulation

IDH2 Epigenetic R140 or 1%-2% 1%-2%

regulation R172

ASXL1 Epigenetic Mostly 2%-7% 5%-10%

regulation nonsense
/
frameshift
in the last
exon

EZH2 Epigenetic All exons 1%-2% 1%-2%

regulation

NRAS ERK/MAPK G12, G13, <2% <2%

signaling or Q61

KRAS ERK/MAPK G12, G13, <2% <2%

signaling or Q61

SH2B3 JAK signaling Exon 2 2%-9% 1%-3%

regulation

CBL JAK signaling Exons 8 <2% <2%

regulation and 9

SRSF2 mRNA P95 <2% <2%

splicing

SF3B1 mRNA Exon 14- 2%-3% 2%-5%

splicing 16

U2AF1 mRNA S34 or <2% <2%

splicing Q157

NFE2 Transcriptional All exons 3%,6% 1%-7%

factor

RUNX1 Transcriptional All exons <2% <2%

factor

TP53 Transcriptional All exons <2%

factor

Only gene mutations occurring

with a frequency of >2% are listed.
CALR-mut, calreticulin mutant;
ERK, extracellular signal-regulated
kinase; ET, essential
thrombocythemia; HMR, high
molecular risk category for PMF;
ICC, international consensus
classification; JAK, Janus kinase;
MAPK, mitogen-activated protein
kinase; MPL, thrombopoeitin
receptor; PMF, primary
myelofibrosis; PV, polycythemia
vera; TPO, thrombopoietin; WHO,
World Health Organization.

“Disease driver” mutations recapitulate the human

MPN phenotype when introduced into model
organisms such as mice. A large proportion of
MPN patients carry mutations solely in 1 of the 3
disease driver genes JAK2, CALR, or MPL,7-15
without additional clonal driver mutations
detectable by targeted NGS.16,17 This finding was
also confirmed by WES and WGS, which found
that some sequence alterations can be detected,
but these were not in any of the genes known to
have a functional impact on hematopoiesis.14,15

“Clonal driver” mutations do not cause the MPN

phenotype when expressed in mouse models, but
they modify the phenotype when they are
combined with one of the “disease driver”
mutations. Some of these “clonal driver” mutations
alter blood counts, increase HSC fitness or
proliferation rate, and induce pre-malignant
changes in hematopoiesis not directly related to
MPN. The “clonal driver” genes that are most
frequently mutated in MPNs include TET2,
DNMT3A, ASXL1, and EZH2. These gene mutations
are not MPN-specific and can be found across all
myeloid malignancies. The MPN “disease driver”
mutations, in particular JAK2-p.V617F, can also be
“clonal drivers” in the context of clonal
hematopoiesis, without resulting in MPNs.

Finally, “passenger” mutations are sequence

alterations without a functional consequence. They
can be useful as markers to determine and follow
the subclonal structure in MPN. In many cases,
whether a gene mutation alters function is di!icult
to predict, because many mutations have not yet
been analyzed functionally. Such mutations are
considered “variants of unknown significance”.
Recurrence of such mutations can be used as a
criterion in favor of a clonal advantage, but
functional assays, such as those in cell lines, will
be required in order to assign variants of unknown
significance to either the “passenger” or “clonal
driver” category.

Given that MPNs originate from a single HSC, early

events after the acquisition of a driver gene
mutation that lead to the expansion of the clone
have become the focus of intense research. This
focus is particularly relevant for JAK2-p.V617F,
because JAK2 is also one of the genes most
frequently mutated in healthy individuals with
clonal hematopoiesis. Clonal hematopoiesis in the
context of MPNs can be defined as the
overproportional presence of blood cells that
originate from a single HSC. Detecting clonality
relies on finding markers that allow these clonal
cells to be distinguished from the progeny of other
HSCs.

In a seminal study, DNA from single hematopoietic

colonies derived from bone marrow of a healthy
donor was analyzed by WGS to reconstruct the
phylogeny of HSCs.18 The method relies on the fact
that during each cell division, the 2 daughter cells
acquire a number of mutations in their genomes,
owing to the imperfect fidelity of DNA replication
and DNA repair. The vast majority of these
sequence alterations are functionally silent, but
they can be used as markers that distinguish the
daughter cells from all other cells that underwent
cell division. With a su!icient number of sequence
alterations, markers can be found that distinguish
very early HSC ancestors, from which a large
proportion of adult HSCs are derived. By WGS of
140 single cell–derived colonies, all blood cells
were mapped to originate from a common
ancestor at the time of gastrulation (Figure 1).18
Markers that identify very early cell divisions, when
applied to adult hematopoiesis, would define large
“clones” in normal hematopoiesis, and would
falsely suggest clonality, because the markers are
shared by a large number of adult HSCs. The most
useful markers of clonality in hematopoiesis are
gene alterations that distinguish between HSCs at
the time when the clonal driver gene mutation was
acquired—that is, the best clonality marker is the
actual “clonal driver” mutation itself. A second
aspect to consider when assessing clonality is the
threshold percentage of contribution of a single
HSC to peripheral blood that is su!icient to be
considered “overproportional.” Unfortunately, the
number of di!erent HSCs that at any given time
are contributing to peripheral blood cells under
healthy steady state conditions is not known. Age-
dependent constriction of the HSC pool was
recently described, and this could decrease the
threshold for “clonality.”19

Clonal hematopoiesis of
indeterminate potential and the
evolution of MPNs
The term “clonal hematopoiesis of indeterminate
potential” (CHIP) was created to denote a
potentially “pre-malignant” state in individuals
without a hematologic malignancy, and the most
frequently found markers in CHIP, not surprisingly,
also represent mutations in known cancer-
associated genes, in particular the epigenetic
regulators DNMT3A, TET2, and ASXL1, and at lower
frequencies also JAK2. Recurrent somatic
mutations in TET2 were the first to be described in
elderly individuals with clonal hematopoies.20 The
threshold for CHIP in most studies was set at
variant allele fraction (VAF) >2% (ie, >4% of
peripheral blood cells carry this heterozygous
mutation). The measured %VAF is also influenced
by the source of cells used for the analysis. DNA
from unfractionated total white blood cells, which
represent a mixture of B and T-cells with a long
half-life, will decrease the VAF of most CHIP-
markers, compared with granulocytes that have a
short half-life and high turnover.

In the context of JAK2-p.V617F, several studies

used highly sensitive polymerase chain reaction
(PCR) methods and expanded the definition of
CHIP to include individuals with JAK2-p.V617F VAF
as low as VAF 0.01%,21 which means that 1 in 5000
cells was positive for the JAK2-p.V617F. With this
expanded definition of CHIP, the authors
characterized a cohort of 19 958 probands and
found 613 that carried JAK2-p.V617F (3.1%) and 32
that carried CALR mutations (0.2%). With a
threshold of 1% VAF, the prevalence of JAK2-
p.V617F was still 0.46%.21 Within these 613 patients,
16 previously unrecognized cases of MPN were
found, but the vast majority of individuals with
JAK2-p.V617F CHIP did not fulfill the diagnostic
criteria for MPN. The cumulative incidence of
evolution from JAK2-p.V617F CHIP to MPN remains
to be determined, but VAF JAK2-p.V617F >2%, or
an increase in VAF during follow-up, was
associated with a high rate of conversion from
CHIP to MPN.22,23 Some patients with very low VAF
in purified granulocytes can still display MPN
phenotypes, due to selective expansion of the
JAK2-mutant clone in late stages of erythropoiesis
and thrombopoiesis.24

The time required for the conversion from CHIP to

MPN was estimated, in a study that analyzed
blood donors who progressed to MPN, and from
whom earlier blood samples were available for
analysis, to be 5 to 15 years.25 WGS of single cell
colonies was also applied to reconstruct the
phylogeny and timing of acquisition of the JAK2-
p.V617F mutation in MPNs.5,6 Both studies
concluded that the JAK2-p.V617F mutation
occurred several decades before the diagnosis of
MPN. Thus, the conversion from acquisition of
JAK2-p.V617F to manifestation of MPN appears to
take several decades, and from CHIP to MPN
takes 5 to 15 years. Therefore, the time from
acquisition of JAK2-p.V617F until it is detectable as
CHIP is also likely to be in the range of decades
(Figure 1).26

In the context of MPN, a noteworthy point is that

the prevalence of JAK2-p.V617F CHIP is 15-fold
higher than that of mutated CALR. This di!erence
could be due to more frequent acquisition of JAK2-
p.V617F. Alternatively, CALR mutations, by
conferring a higher rate of clonal expansion
compared to JAK2-p.V617F, may shorten the
transition from acquisition of the mutation through
the CHIP phase to MPN, resulting in a lower
prevalence of CHIP.27 Reconstructing the
phylogeny of CALR mutations by WGS will likely
clarify these issues. The likelihood that an HSC
carrying a driver gene mutation will escape
immune surveillance, expand, and produce
peripheral blood cells detectable as CHIP could
also contribute to the observed di!erences. As
many healthy individuals have been found to carry
memory T-cells that recognize the mutated CALR-
neoantigen without any evidence for CALR-
mutated CHIP,28 the lower prevalence of CALR-
mutated CHIP could be due to a more e!ective
elimination of CALR-mutated cells at the pre-CHIP
phase by the immune system in an HLA-restricted
manner. In contrast, mutant CALR protein was also
shown to decrease major histocompatibility
complex-I expression and cause defects in peptide
loading,29,30 providing a possible explanation for
why immune checkpoint blockade and mutant
CALR-derived peptide vaccination failed to
generate clinical responses in MPN patients.31,32
Peptides containing the JAK2-p.V617F mutation
also are unlikely to form high-a!inity interactions
with any HLA allotype,33 and activation of
JAK/STAT signaling by JAK2-p.V617F increases
programmed death ligand 1 (PD-L1) expression.34,35
Therefore, cells expressing JAK2-p.V617F are
expected to evade immune surveillance, which
could explain why JAK2-p.V617F CHIP is so
frequent. Conversely, JAK2-p.V617F CHIP only
rarely progresses to MPN, suggesting that some
mechanisms are active mainly at this later
conversion stage (Figure 2).

Figure 2.

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Evolution of MPNs originating from a single HSC that

acquired a disease driver mutation. Model summarizing
the events from the acquisition of JAK2-p.V617F until the
development of MPN disease. The early events occur
inside the bone marrow. A single HSC with JAK2-p.V617F
can divide to yield 2 HSC daughter cells that carry the
mutation, which leads to persistence, and later limited
expansion of the mutated HSCs. Alternatively, the mutated
HSC can di!erentiate into committed progenitors that
produce a wave of mutant hematopoietic cells, but
eventually are exhausted, due to loss of stemness. The
mutant HSCs can also be eliminated at this early stage by
cells of the immune system. During this phase, cells
carrying JAK2-p.V617F are not yet detectable in peripheral
blood (“pre-CHIP phase”). After expansion of the mutated
HSCs and with a latency of years or decades, mature
hematopoietic cells carrying JAK2-p.V617F are produced
that become detectable in peripheral blood as “clonal
 Genetic basis and clonal evolution of MPNs
hematopoiesis of undetermined potential (CHIP).” In only