After 10 years of JAK2V617F: Disease biology and current management
strategies in polycythaemia vera

Jacob Grinfeld, Anna L Godfrey

PII: S0268-960X(16)30107-2
DOI: doi:10.1016/j.blre.2016.11.001
Reference: YBLRE 462

To appear in: Blood Reviews

Please cite this article as: Grinfeld Jacob, Godfrey Anna L, After 10 years of
JAK2V617F: Disease biology and current management strategies in polycythaemia vera,
Blood Reviews (2016), doi:10.1016/j.blre.2016.11.001

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After 10 years of JAK2V617F: disease biology and current

management strategies in polycythaemia vera

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Jacob Grinfeld and Anna L Godfrey*

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Jacob Grinfeld MBChB Bsc MRCP FRCPath

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Department of Haematology, Cambridge University Hospitals NHS Foundation Trust
Hills Rd, Cambridge, CB2 0QQ, UK MA
E-mail: jg738@cam.ac.uk
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* Corresponding author:
Anna L Godfrey MA BMBCh PhD MRCP FRCPath
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Department of Haematology, Cambridge University Hospitals NHS Foundation Trust

Hills Rd, Cambridge, CB2 0QQ, UK
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Tel.: +44 1223 217073

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Fax: +44 1223 762670

E-mail: anna.godfrey1@nhs.net
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Abstract

The JAK2V617F mutation accounts for the vast majority of patients with

polycythaemia vera (PV) and around half of those with other Philadelphia-negative

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myeloproliferative neoplasms. Since its discovery in 2005, numerous insights have

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been gained into the pathways by which JAK2V617F causes myeloproliferation,

including activation of JAK-STAT signalling but also through other canonical and

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non-canonical pathways. A variety of mechanisms explain how this one mutation can

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be associated with distinct clinical disorders, demonstrating how constitutional and

acquired factors may interact in the presence of a single mutation to determine

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disease phenotype. Important biological questions remain unanswered in PV, in

particular how JAK2V617F affects stem cell function and what mechanisms drive

myelofibrotic and leukaemic transformation. Whilst current management is largely

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centred on prevention of cardiovascular events, future therapies must aim to target

the JAK2-mutant clone, to reverse the underlying marrow pathology and to address
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the risk of transformation events.

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Keywords:

Polycythaemia vera

Erythrocytosis

Myeloproliferative

JAK2
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1. Introduction

Polycythaemia vera (PV), one of the Philadelphia-negative myeloproliferative

neoplasms (MPNs), is a clonal haematopoietic stem cell disorder characterised by an

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increased red cell mass, associated with proliferation of erythroid, granulocytic and

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megakaryocytic elements of the bone marrow1. In 2005 the identification of the

JAK2V617F mutation elucidated a molecular basis for the disorder in over 95% of

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patients2-5, followed two years later by the identification of JAK2 exon 12 mutations in

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most remaining patients6, 7. The vast majority of patients with PV therefore carry

mutations in a single gene, and a wealth of studies has since illuminated the many
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molecular and cellular consequences of these mutations.

Whilst JAK2 exon 12 mutations are specific to PV, the JAK2V617F mutation is also
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found in other myeloid disorders: 50 to 60% of those with essential

thrombocythaemia (ET) or primary myelofibrosis (PMF) 2-5, about half of patients with
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the MDS/MPN entity “refractory anaemia with ring sideroblasts and marked
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thrombocytosis” (RARS-T)8, and lower frequencies in AML, other myeloproliferative

and myelodysplastic disorders9, 10. ET and PMF in particular share important clinical
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features with PV, including the presence of neutrophilia and/or thrombocytosis in

some patients, hypercellularity of the bone marrow, frequent splenomegaly, and risks

of thrombosis and haemorrhage1. In a minority of patients, PV, ET and PMF can all

transform into acute myeloid leukaemia (AML),11-13 those with ET may develop

transformation to PV14, and both ET and PV may transform into secondary

myelofibrosis11, 13, 14. The identification of JAK2V617F offered some basis for the

clinical similarities between these MPNs, but the molecular mechanisms behind the

phenotypic differences between the JAK2V617F-positive disorders has remained the

subject of intensive investigation.

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The identification of JAK2 mutations in PV led to the introduction of a rapid and non-

invasive diagnostic test, with mutation testing now widely available and incorporated

into national and international clinical guidelines1, 15-17. Moreover the development of

JAK2 inhibitors has led to a much-needed novel therapeutic avenue for patients with

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myelofibrosis18, 19, and most recently these drugs have also been shown to benefit

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certain patients with PV20. However, clinical management of PV remains

predominantly centred on the reduction of vascular complications and symptoms. In

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spite of significant advances that have been made in understanding the disease

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biology of PV, agents that can target the underlying neoplastic clone have remained

elusive and the risk of disease transformation, with its almost inevitably poor
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outcome, remains an important and unmet clinical need.

This review will first examine current understanding of the molecular basis of PV,
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both in terms of the pathological effects of JAK2 mutations and the factors that may

contribute to phenotypic differences between PV and other JAK2-mutated MPNs.

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Diagnostic criteria and their controversies will be considered. Key prognostic factors
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in PV will then be examined, followed by a discussion of current management

strategies and unresolved clinical challenges.

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2. The molecular and cellular effects of JAK2 mutations

JAK2 is one of a family of cytoplasmic tyrosine kinases that mediates signal

transduction from cell surface cytokine receptors, including the erythropoietin and

thrombopoietin receptors21. Binding of ligand to receptor leads to JAK

autophosphorylation, receptor transphosphorylation and creation of binding sites for

signalling molecules such as STAT proteins. STATs are phosphorylated, translocate

to the nucleus and affect target gene transcription. For example, binding of

erythropoietin to its receptor (EpoR) causes phosphorylation of JAK2 and STAT522,

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together with activation of other effectors including MAP kinase and phosphoinositide

3-kinase (PI3K)/Akt pathways23.

2.1. Biochemical effects of JAK2 mutations

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In early studies, the V617F mutation was found to have activating effects on JAK2

signalling activity, cytokine sensitivity and cytokine-dependent survival in cell lines2-5.

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This activation of JAK2 is thought to reflect the location of the mutation within the

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JH2 “pseudokinase” domain of the protein, which normally has auto-inhibitory effects

on the catalytically active JH1 kinase domain24-27. Mechanisms may include the
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interference of the mutation with direct phosphorylation of JH1 by JH228, and

cooperation of V617F with other residues in JH2 to enhance JH1 kinase activity29, 30.

Interestingly, recent work on the JAK1 JH2 crystal structure has also suggested that
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JAK2V617F activation requires a specific conformational change in the SH2-JH2

linker domain31, which is the area targeted by exon 12 mutations. Exon 12 mutations
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appear have similar activating effects to V617F in cell lines, including cytokine
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independence and constitutive activation of JAK2 and its downstream mediators in

the absence of erythropoietin6.

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Several signalling changes appear to mediate the cellular effects of JAK2V617F.

Increased phosphorylation of STAT5 has been demonstrated in numerous

experimental systems, including cultured erythroid cells and bone marrow trephine

biopsies from patients with MPNs, and in JAK2V617F knock-in mouse models2, 4, 5, 32-
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. The particular importance of STAT5 activation in PV is illustrated by the

observation that expression of constitutively active STAT5 in erythroid progenitors

can drive formation of the same erythropoietin-independent endogenous erythroid

colonies that are typical of PV42. Moreover it has been shown more recently that

Stat5 deletion in either a Jak2V617F knock-in mouse43 or retroviral bone marrow

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transplantation model44 abrogates the marked erythrocytosis that is seen in the

presence of Stat5.

Other signalling processes that appear to be activated by JAK2V617F include the

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PI3K/Akt and MAPK/ERK pathways, which have been studied in many experimental

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systems2, 5, 33, 34, 36, 38, 45. There is evidence that the mutation has direct effects on

apoptosis, both through the extrinsic pathway that acts through cell surface death

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receptors such as Fas46, and through the intrinsic pathway, with increased levels of

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the anti-apoptotic protein Bcl-xL and impairment of its normal role in the DNA

damage response47-49. Other mediators implicated in apoptotic dysregulation include

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ID1, Pim1/2 kinases, the BH3-only proteins Bad and Bim and the Bcl2-like protein

Mcl-1, downstream of STAT3, STAT5 and ERK1/2 pathways50-53. Several studies

have also suggested that JAK2V617F may not only impair the normal cellular
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responses to DNA damage but may also directly contribute to increased DNA

damage and to levels of intracellular reactive oxygen species39, 54-56.

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Whilst activation of pathways such as STAT5 are considered canonical effects of

JAK2V617F, several other “non-canonical” effects have also been reported including
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modification of chromatin. JAK2 can enter the nucleus and phosphorylate histone H3

at Tyr41 (Y41), which prevents binding of the heterochromatin protein HP1α57. This

allows JAK2 to have direct effects on the regulation of gene expression, a process

which can occur in combination with, or independently of, STAT proteins58.

JAK2V617F may also modulate chromatin by phosphorylating the arginine

methyltransferase PRMT5 and impairing its histone methylation activity, which may

in turn promote growth and erythroid differentiation of haematopoietic cells59.

There is also evidence that JAK2V617F may not only act through intrinsic cellular

pathways, but may have important effects through the micro-environment. For
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example, the mutation has been reported to increase levels of the cytokine TNFα,

which is increased in the serum of MPN patients and may paradoxically stimulate

growth of JAK2-mutant cells60. An important role for the microenvironment was also

suggested by two reports that macrophage depletion abrogates erythrocytosis in

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mouse models of JAK2V617F-driven polycythaemia61, 62.

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2.2. Cellular effects of JAK2 mutations

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Early studies of the cellular effects of JAK2V617F on haematopoiesis analysed cell

populations at different stages of differentiation from primary patient samples, with or

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without a period of ex vivo culture. These studies were mostly in keeping with an

expansion advantage for JAK2V617F-mutant cells at later stages of myeloerythrod

differentiation, particularly at low erythropoietin levels and in the presence of

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homozygosity for the V617F mutation63-67. The effects of JAK2 mutations on

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haematopoietic cells have been elucidated further using mouse models. Importantly,
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early work demonstrated that expression of JAK2V617F in retroviral bone marrow

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transplantation models could cause a myeloproliferative phenotype2, 5, confirming the

causal role of the mutation in human MPNs. These models had high, dysregulated
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levels of JAK2V617F expression, whilst knock-in models allow observation of the

phenotypes obtained when the gene is expressed at physiological levels, under the

normal regulatory elements of the mouse Jak2 gene. These knock-in models have

supported the conclusions from patient studies, showing that JAK2V617F is

associated with increased numbers of lineage-restricted progenitors, including those

of the megakaryocyte/erythroid and granulocyte/macrophage lineages38-41, 68.

The effects of JAK2V617F on haematopoietic stem cells (HSCs) have been more

controversial. Early studies used xenotransplantation experiments, in which CD34+

cells from MPN patients were transplanted into NOD/SCID mice. Whilst these cells
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could engraft in the mice, the proportion of JAK2V617F-positive cells was frequently

reduced in the reconstituted marrow compared to the original cells, arguing against

an advantage for JAK2V617F-positive over wild-type HSCs69, 70. In two knock-in

models of murine Jak2V617F, an increase in the stem cell-enriched Lin-Sca-1+Kit+

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(LSK) cell compartment was reported38, 68, whilst in another LSK numbers were

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unchanged40. With respect to HSC function, competitive transplantation experiments

in one of these models suggested a selective advantage for JAK2V617F-mutant cells

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in primary transplantation, associated with an increase in HSC proliferation and

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decrease in apoptosis, but this advantage was not maintained in secondary

transplants68. Similarly, competitive transplantation experiments in a second model

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suggested normal early function but a late advantage in primary recipients40, 71, whilst

subsequent data have suggested a lack of advantage in secondary recipients72. In

the one knock-in model with a human JAK2V617F construct there was a quantitative
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and qualitative HSC defect, with reduced LSK cell numbers and evidence of

increased DNA damage, reduced cell cycling, and impaired function in competitive
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transplantation experiments, which was most marked in cells homozygous for

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JAK2V617F39, 73. Further studies of this latter model suggested that the mutation may

promote differentiation of HSCs at the expense of self-renewal74.

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Interestingly the concept of a JAK2V617F-induced HSC defect is consistent with

other evidence from the human MPNs. For example, allele burdens for JAK2V617F

are low in many patients64 and may remain stable over years75, and it was reported

that JAK2V617F-positive cells did not expand in the recipient of an allogeneic stem

cell transplant from a JAK2V617F-positive donor76. Conversely it was reported that a

JAK2V617I mutation, found in a family with hereditary thrombocytosis in the absence

of additional MPN-associated molecular abnormalities, was associated with

increased numbers of phenotypic HSCs and an advantage in xenotransplantation

assays77. However this mutation also showed qualitative signalling differences to

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V617F and may be associated with a different HSC phenotype, and moreover the

constitutional nature of the V617I variant would obviate the requirement for a

competitive advantage in human disease. The concept that oncogenes may not

impart a stem cell advantage is also consistent with data from other malignancies.

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For example, the BCR-ABL fusion gene has been associated with reduced HSC self-

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renewal in vitro78, in NOD/SCID transplantation experiments79 and in secondary

transplants from a transgenic mouse model80, and oncogene-induced senescence is

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a recognised barrier to malignant progression in certain haematological and non-

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haematological neoplasms81, 82.

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In summary, numerous studies have contributed to a body of knowledge regarding

the biochemical and cellular consequences of JAK2 mutations. However, it has

remained less clear how the various signalling perturbations contribute to the specific
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clinical features of MPNs, and which are the other factors that distinguish between

the different JAK2-mutant MPN phenotypes. For example, STAT5 activation appears
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to play an important role in erythrocytosis, the defining feature of PV, since

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knockdown of Stat5 in JAK2V617F-driven models of erythrocytosis abrogates this

aspect of the phenotype43, 44. By contrast Stat5 deletion did not prevent development
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of myelofibrosis in a Jak2V617F retroviral bone marrow transplantation model44.

Moreover studies of haematopoietic cells from MPN patients found that patterns of

pSTAT3, pSTAT5, pERK1/2 and pAkt correlated better with MPN subtype rather than

with the presence or burden of the JAK2V617F mutation83, highlighting a potential

role for additional molecular mechanisms in these signalling changes.

3. Factors determining phenotype in PV vs other JAK2-mutated MPNs

In 2005, analysis of ET patients in the PT1 trial demonstrated that in comparison to

JAK2V617F-negative patients, JAK2V617F-positive patients showed higher

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haemoglobin levels, neutrophil counts, more bone marrow erythropoiesis and

granulopoiesis, lower platelet counts, mean corpuscular volume (MCV), serum

erythropoietin and ferritin, and more frequent PV transformation84. Subsequent

studies confirmed these associations14, 85-88, suggesting that JAK2V617F-positive ET

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has some characteristics of a mild form of PV, as well as a similar incidence of

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thrombosis89. These clinical similarities between PV and JAK2V617F-positive ET are

mirrored by certain biological similarities (e.g. a low frequency of driver mutations

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additional to JAK2V617F90) and are in keeping with a model in which the two

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disorders form a phenotypic continuum84. However there are also important

differences between PV and JAK2V617F-positive ET, which have prompted

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speculation and investigation of the factors that push an individual’s phenotype

towards one or other disorder. These factors include the presence and size of clones

homozygous for the JAK2V617F mutation; the presence of additional mutations and
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their subclonal hierarchy in relation to JAK2V617F; germline genetic factors;

differences in gene expression; and other constitutional factors including age and
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gender. We discuss these factors in more detail below.

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3.1. The role of JAK2V617F homozygosity in PV phenotype

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A “homozygous” JAK2V617F sequence (>50% mutant) in granulocyte DNA was

originally identified in 25-30% of those with PV, 9-20% with PMF, and 0-3% with ET2-
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. Clinical studies next investigated how JAK2V617F allele burden, a measure of

“gene dosage” for the mutation that is most often measured in granulocyte DNA,

correlated with certain clinical features. Initial studies divided PV patients into

“heterozygous” (<50% mutant allele) and “homozygous” (>50% mutant allele)

groups, whilst subsequent studies analysed mutant allele burden as a continuous

variable. Together these studies indicated that within PV, higher JAK2V617F allele

burdens were associated with higher haemoglobin levels, higher white cell counts
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and lower platelet counts, together with other features suggesting a “more extreme”

PV phenotype (lower MCV, lower serum ferritin and erythropoietin, more

splenomegaly, more pruritus and more need for cytoreductive therapy)13, 63, 64, 91-95.

These data suggested that a higher JAK2V617F allele burden might promote a more

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“PV-like”, rather than “ET-like”, phenotype. Within ET, higher JAK2V617F allele

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burdens were associated with some of the same features as in PV (higher white cell

counts and more splenomegaly), but in contrast to PV there was an association with

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higher platelet counts63, 64, 88. This may reflect a limitation of measuring allele burden

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in bulk granulocyte DNA: the assays did not demonstrate the contribution of

JAK2V617F-heterozygous and homozygous cells to the overall mutant allele burden.

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Whilst these early patient studies showed a correlation between JAK2V617F allele

burden and PV phenotype, mouse models have since been used to establish the
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causal nature of this relationship. One transgenic mouse model was consistent with

the concept that a phenotype of ET or PV could result from lower and higher levels of
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JAK2V617F expression levels, respectively, suggesting a role for gene dosage in

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determining MPN phenotype96. However the same switch from ET to PV was not

seen with increasing gene dosage in other transgenic models97, 98, and all transgenic
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models are complicated by variable transgene activation between cells.

More recently the development of knock-in mouse models has allowed study of

JAK2V617F when expressed at physiological levels under its normal regulatory

elements38-41, 68. Four knock-in models with a heterozygous murine mutant Jak2 allele

developed a phenotype consistent with PV with erythrocytosis, leucocytosis,

splenomegaly, variable thrombocytosis and increased megakaryocyte-erythroid

progenitors38, 40, 41, 68. One of these studies reported generation of a homozygous-

mutant mouse, which showed more pronounced leucocytosis, thrombocytosis and

splenomegaly but reduced or unchanged haemoglobin levels compared to

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heterozygous mice38; similar phenotypes were observed in a hemizygous mouse with

deletion of the wild-type Jak2 allele99. By contrast in a fifth model with a human

JAK2V617F allele, mice heterozygous for the mutation showed a moderate

thrombocytosis with minimal erythrocytosis, normal spleen size and plasma

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erythropoietin levels, whilst homozygosity was associated with marked erythrocytosis

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and reduced platelet counts, significant splenomegaly and suppressed plasma

erythropoietin levels. These features closely parallel those of human ET and PV,

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respectively, and provided direct genetic evidence that homozygosity for human

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JAK2V617F can be associated with a switch from ET-like disease to a PV-like

phenotype73. The reasons behind the phenotypic variation between the different
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knock-in models remain unclear however, and may reflect differences in technical

aspects of the targeting strategies or inherent differences in the mutant human and

mouse proteins.
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Further analysis of the precise role of JAK2V617F homozygosity in human PV has

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utilised genotyping of individual erythroid colonies grown from MPN patient samples.
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This approach circumvents the limitations of allele burden studies in bulk granulocyte

DNA, by investigating the presence and frequency of homozygosity at the level of

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single precursors. An early study of BFU-E colonies grown in high erythropoietin

conditions found that while virtually all PV and ET patients produced both wild-type

and mutant colonies, JAK2V617F-homozygous colonies were detectable in almost all

of the PV patients, but in none of the cohort with ET100. Subsequent studies largely

supported these observations63, 64, but raised the possibility that JAK2V617F-

homozygous erythroid colonies could be found in a small number of patients with

ET63, 64, 101, whilst PV patients lacking JAK2V617F-homozygous colonies were also

described63, 64. A large study of PV and ET patients then analysed the genotypes of

erythroid colonies grown in low erythropoietin conditions, aiming to select for even

the smallest homozygous-mutant clones. This study confirmed that JAK2V617F-

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homozygous precursors could be identified from approximately 80% of PV

patients102. A higher proportion of JAK2V617F-homozygous erythroid precursors,

relative to heterozygous precursors, was associated with more pronounced PV-like

clinical features, again supporting a causal role for homozygosity in this

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phenotype103. However small homozygous-mutant clones could also be identified

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reproducibly in approximately half of ET patients102. Moreover many patients with PV

and ET were shown to have acquired JAK2V617F homozygosity on multiple

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occasions in independent subclones, but PV was distinguished by the expansion of

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one dominant homozygous-mutant subclone.

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Together these data have demonstrated that in the 80% of PV patients with

significant JAK2V617F-homozygous clones, these may have a causal role in the PV

phenotype. However mitotic recombination occurs at the JAK2 locus remarkably

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frequently; this observation remains unexplained, although JAK2V617F itself has

also been associated with increased homologous recombination and DNA replication
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stress54, 56. Moreover acquisition of homozygosity is not sufficient to drive a

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phenotype of PV, but the important step appears to be whether a homozygous-

mutant clone expands sufficiently to impact on the disease phenotype. Further work
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has aimed to investigate what factors may drive the expansion of individual

subclones, together with the additional factors that may influence phenotype in the

presence of a particular balance of JAK2V617F-homozygous and -heterozygous

subclones.

3.2. Other mutations and PV phenotype

Since the identification of JAK2V617F, a range of other mutations have been

identified in MPNs. Mutations in CALR, MPL and LNK are found predominantly in

JAK2-unmutated MPNs and seem likely to be have parallel effects on cellular

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signalling that drive the specific MPN phenotype. By contrast many of the other

mutations (Table 1) are found in other myeloid malignancies as well as MPNs and

seem likely to contribute to disease through less specific effects on HSC survival or

self-renewal104. Amongst the MPNs these mutations tend to be commonest in

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myelofibrosis and/or blast-phase disease with low frequencies in PV, but nonetheless

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they are candidates for driving expansion of JAK2-mutant subclones in PV and ET.

For example, TET2 and DNMT3A mutations have been identified patients with PV

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who lack JAK2V617F-homozygous precursors105, 106. These patients tend to have

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particularly large heterozygous-mutant subclones, perhaps accounting for the

development of sufficient erythrocytosis to meet a diagnosis of PV rather than ET63,

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102
. It is likely that these additional mutations may contribute to the phenomenon of

“clonal dominance”, a description used for patients with a similar JAK2V617F allele

burden in CD34+ progenitor cells compared to neutrophils64. This feature is

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particularly seen in myelofibrosis but is also commoner in patients with PV compared

to those with ET, the latter tending to show lower JAK2V617F allele burdens in
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CD34+ progenitor cells compared to more mature granulocytes107.

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A recent finding is that it is not only the combination of mutations that is important in
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determining subclonal balance and phenotype, but also the order of their acquisition.

In a series of patients carrying both JAK2 and TET2 mutations, acquisition of mutant

JAK2 prior to TET2 was more likely to be associated with a phenotype of PV than

ET, and as expected this correlated with a higher proportion of JAK2V617F-

homozygous colonies108. The double-mutant clone was larger in these patients at the

level of haematopoietic stem and progenitor cells (HSPCs), in contrast to the “TET2-

first” group in which TET2-single-mutant HSPCs predominated, and “JAK2-first”

patients also had a higher risk of thrombosis. Certain other mutations may not only

promote the expansion of JAK2-mutant clones but may also contribute directly to PV

phenotype. An example is that of truncating NF-E2 mutations that were identified in a

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small number of patients with JAK2V617F-positive PV, as well as some with

myelofibrosis109. These mutations are associated with a proliferative advantage in

haematopoietic colony assays and cell lines. Moreover a murine bone marrow

transplantation model showed erythrocytosis, thrombocytosis and neutrophilia in the

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presence of an NF-E2 mutation, but the combination with JAK2V617F led to an even

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more pronounced erythrocytosis than JAK2V617F alone, suggesting a cooperative

effect on phenotype.

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The factors contributing to clonal balance are frequently unclear however. There is

evidence to suggest that in some patients, additional mutations may not account for
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differential subclonal expansion. In two PV patients with multiple JAK2V617F-

homozygous subclones, an additional “driver” mutation was identified by exome

sequencing in the dominant homozygous-mutant subclone110. However these

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mutations were not specific to the dominant subclone and were each present in at

least one minor homozygous-mutant subclone. These findings raise the possibility
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that in some patients, stochastic mechanisms may account for the relative sizes of
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JAK2-mutant clones and therefore for differences in phenotype.

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3.3. Germline factors in the phenotype of JAK2-mutated MPNs

Germline JAK2 mutations affecting V617 (V617I)77 and other residues outside the

pseudokinase domain111, 112 have recently been reported to cause hereditary

thrombocytosis, and all have been suggested to have weaker consequences for

JAK2 function than V617F77, 111, 112. Equivalent mutations have not been identified in

patients with acquired ET or PV, however.

Germline factors are important in genetic predisposition to acquired MPNs: a specific

constitutional JAK2 haplotype, designated 46/1, is strongly associated with the

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development of JAK2V617F-positive MPNs113, 114, as well as with JAK2 exon 12-

mutated and MPL-mutated disease115-117. These findings raised the possibilities that

either the specific haplotype is associated with an increased risk of developing a

pathogenic JAK2 mutation, or that the acquisition of a mutation is more likely to lead

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to clinical disease in the presence of the 46/1 haplotype118. A small study of PV

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patients suggested that homozygosity for the 46/1 haplotype was associated with a

greater likelihood of increasing allele burden over time119, raising the possibility that

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this genotype may promote the development or expansion of JAK2V617F-

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homozygous clones. A study in the Chinese population also suggested that the 46/1

haplotype is associated with a higher haemoglobin and white cell count at diagnosis
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in ET and PMF, and with higher platelet counts in PMF120, although other studies

have not found associations with clinical features118.

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More recently, extensive genome-wide association studies have identified a number

of other novel single-nucleotide polymorphisms (SNPs) associated with MPNs. A

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germline variant in the TERT gene was associated with MPNs but not with disease
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phenotype or with the presence of the JAK2V617F mutation121, 122, and recently

additional variants related to the TET2 and SH2B3 gene loci have been described123.
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A particularly interesting observation was that a SNP located between the HBS1L

and MYB genes showed an association with CALR- and MPL-mutant MPNs, but

within JAK2-mutated patients showed a significantly different allele frequency

between ET and PV122. The risk allele was found to specifically increase the risk of

ET but not PV and was associated with significantly lower MYB expression in

haematopoietic colonies; moreover MYB-knockdown mice show a transplantable ET-

like disease124. These findings, together with other studies125-127, suggest a potentially

important role of germline factors in determining disease phenotype within JAK2-

mutant MPNs.
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3.4. Differential gene expression in JAK2-mutant PV and ET

A number of studies have investigated differences in gene expression between the

different MPNs. One novel approach was to use clonally-derived erythroid cells from

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patients with PV and ET, in each case comparing JAK2V617F-heterozygous mutant

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cells to a wild-type sample from the same patient and thus controlling for inter-

individual variation in gene expression128. In ET but not in PV, JAK2-mutant cells

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showed upregulation of interferon target genes compared to wild-type cells,

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associated with increased activation of pSTAT1, which was absent in PV patients.

Functional experiments suggested that differential activation of STAT1 could

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contribute to altered megakaryocytic and erythroid differentiation. These findings are

also consistent with data from a JAK2V617F transgenic mouse model, in which loss

of Stat1 resulted in more marked erythrocytosis and reduction of thrombocytosis

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compared to normal Stat1 levels129. Interestingly this study found that serum
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interferon-gamma levels were increased in the mice with thrombocytosis and also in
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patients with ET, with lower levels in those with PV. These findings raise the
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possibility that varying levels of interferon-gamma might account for differences in

signaling seen between PV and ET patients. However the molecular explanation for
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these data in patients remains elusive and it is unclear if this phenomenon may relate

to acquired or constitutional differences in interferon biology.

Within PV, a microarray-based study of CD34+ peripheral blood cells suggested that

gene expression could be used to differentiate between two groups of PV patients

who also showed differences in disease duration, haemoglobin level,

thromboembolic events, splenomegaly, exposure to chemotherapy, leukaemic

transformation and survival130. These findings were independent of JAK2V617F allele

burden and raise the possibility that differences in gene expression between patients

with PV may contribute to phenotypic heterogeneity, although the genetic or

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epigenetic mechanisms are uncertain. Deregulation of microRNA expression has

also been implicated in the biology of PV131 and a small study suggested that

perturbations in microRNA expression may also differ between PV and ET

patients132.

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In summary, a number of factors are likely to play a role in determining a precise

phenotype PV or ET in the presence of a JAK2 mutation. There are likely to be many

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other germline genetic influences. For example, we have studied two patients with

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beta-thalassaemia trait in whom colony assays have revealed large JAK2V617F-

homozygous clones, typical of PV (unpublished data). However in both cases a

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phenotype of ET is evident clinically, a phenomenon that seems likely to reflect a

constraining effect of the beta-globin abnormalities on the development of

erythrocytosis. Age may influence the phenotype obtained and gender may be
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particularly important, possibly through modulating the phenotypic effects of large

JAK2V617F-homozygous clones103, 133. It has also been reported that gender may be
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associated with additional phenotypic features in PV including age of presentation,

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presence of splenomegaly and thrombotic events133, 134. Genome-wide association

studies in normal individuals have identified a number of genetic loci associated with
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full blood count parameters135-138, confirming for example that variants in genes

related to iron metabolism may influence haemoglobin levels139, 140. Given that

haemoglobin and platelet levels are polygenic traits in the normal population, they

are likely to be similarly influenced by genetic determinants in patients with MPNs.

The timing of presentation to medical care and institution of therapy may also be

important, since many PV patients show a thrombocytosis during the evolution of

their disease and could gain a diagnostic label of ET if they receive cytoreduction

before the full PV phenotype is allowed to develop141. Together all of these data

support a model in which JAK2-mutant PV and ET form a biological continuum, with

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the precise phenotype determined by a range of discrete and continuous variables

(Figure 1).

3.5. JAK2 exon 12 mutations in PV

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Over 20 different mutations in exon 12 of JAK2 have been reported in PV but have

not been described in ET. Exon 12 mutations tend to be associated with a particular

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clinical phenotype: compared to PV patients with JAK2V617F, patients are younger,

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with higher haemoglobin concentrations, lower white cell and platelet counts, and

isolated bone marrow erythroid hyperplasia without granulocytic or megakaryocytic

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morphological abnormalities6, 142. In a series of 106 patients with exon 12 mutations,

64% presented with isolated erythrocytosis, with 15% having additional leucocytosis,

12% additional thrombocytosis and 9% both, with a similar incidence of thrombosis,

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myelofibrosis, acute leukaemia and death to those with JAK2V617F142. By contrast to

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JAK2V617F-positive PV, patients tend to carry predominantly heterozygous-mutant

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clones, with small homozygous-mutant clones detectable in 40-50% of patients

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(similar to ET)102.
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The mechanism for the marked, and relatively isolated, erythrocytosis seen in

patients with JAK2 exon 12 mutations is not entirely clear. Some in vitro studies6, but

not others143, have suggested that these mutations might be associated with more

marked activation of JAK2 than V617F. Expression profiling has demonstrated that

STAT1 activation is preserved in the presence of exon 12 mutations and that

attenuated STAT1 signaling is therefore not the explanation for erythrocytosis in

these patients144.

3.6. Factors contributing to disease transformation from PV: myelofibrosis and acute

myeloid leukaemia
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Whilst JAK2V617F is also found in 50 to 60% of those with PMF, it appears likely

that additional factors contribute to this more complex, heterogeneous phenotype.

Transformations of ET and PV to secondary myelofibrosis are well recognised and

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patients diagnosed with “primary” myelofibrosis may show evidence of a prior

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undiagnosed chronic-phase MPN145, supporting the concept of myelofibrosis as an

accelerated phase of disease. It is clear that additional genetic lesions are important.

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Exome sequencing studies have demonstrated a higher average number of somatic

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mutations in patients with myelofibrosis compared to those with PV or ET90, and

consistent with this, most of the other recurrent mutations identified in chronic-phase
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MPNs are also commonest in PMF (Table 1). Moreover the spectrum of genetic

lesions in PMF shows increasing overlap both with secondary AML and with poor

prognostic subgroups of other myeloid malignancies. Many of the genes affected are
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predicted to alter DNA and/or histone modifications through a variety of mechanisms,

or to affect the RNA splicing machinery.

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The role of JAK2V617F in progression of chronic-phase PV to AML is particularly

complex. In individuals in whom JAK2V617F is detectable in the leukaemic blasts,

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the mutation may have contributed to disease evolution by causing increased DNA

damage54 and an impaired apoptotic response to DNA damage49. Other patients with

JAK2V617F-positive chronic MPNs may however develop JAK2V617F-negative

AML146, 147. A variety of molecular lesions have been implicated to have a specific

role in leukaemic transformation, including mutations in TP53, RUNX1, c-CBL,

IKZF1, NRAS and KRAS104, 147, 148.

4. Diagnostic criteria for PV

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Clinical assessment of a patient with suspected PV should aim to identify possible

alternative causes of erythrocytosis. Examples include secondary erythrocytosis

associated with respiratory disease, renal disease or obstructive sleep apnoea,

relative erythrocytosis that reflects a contracted plasma volume, or congenital causes

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of erythrocytosis. Investigations are described in guidelines such as those published

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by the British Committee for Standards in Haematology (BCSH)15, 149, but an

essential laboratory investigation is screening for JAK2V617F (and, if necessary,

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exon 12 mutations), for which guidelines are also available150.

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Where an MPN is suspected, criteria have been published by the World Health
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Organisation (WHO) or BCSH to define those patients who should be diagnosed with

PV (Table 2)1, 15, 151. An essential component in both cases is the presence of an

erythrocytosis, but the parameters and thresholds used to define this key criterion
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remain controversial152. The WHO criteria have been recently updated and now
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suggest the use of absolute haemoglobin level, haematocrit, or red cell mass151,
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whilst the BCSH criteria depend on either haematocrit or red cell mass15. Although
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scintigraphic measurement of red cell mass may be considered the gold standard

investigation, it is not universally available and is not absolutely required in either set
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of criteria. However, haemoglobin or haematocrit levels do not provide a perfect

surrogate for red cell mass and their use in diagnostic thresholds for PV have been

questioned153, 154. This issue relates to unpredictable changes in plasma volume in

PV, especially in the presence of splenomegaly155-157, and it has been suggested that

more systematic measurement of red cell mass would be helpful158. Iron deficiency

may further confound the clinical presentation. The term “masked PV” has been used

for those patients whose bone marrow morphology is in keeping with a diagnosis of

PV but who do not meet haemoglobin- or haematocrit-based thresholds for

erythrocytosis159. These patients may represent an important group since they were

reported to have higher rates of myelofibrotic transformation, acute leukaemia and

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thrombosis with inferior survival, compared to those with overt PV159-161, although this

was not confirmed in all studies162. Interestingly it was found that the use of BCSH

criteria resulted in a smaller proportion of patients falling into this category compared

to the 2008 WHO criteria (which depended on absolute haemoglobin level or a

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documented rise in haemoglobin, but not haematocrit)160, 163. These data support the

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use of haematocrit rather than haemoglobin as an indicator of erythrocytosis.

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In clinical practice, the issue of defining erythrocytosis is most relevant in

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distinguishing PV from ET in the presence of a JAK2 mutation. Both WHO and BCSH

criteria for ET require the exclusion of PV and therefore, to at least some extent,
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depend on the same thresholds used to define erythrocytosis. A study of patients

with PV (overt or masked) and ET suggested that the most optimal cut-offs to

distinguish between JAK2-mutated ET and PV are a haemoglobin level of 165 g/l in

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males and 160 g/l in females, or haematocrit of 49% in males and 48% in females164,
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which is reflected in the 2016 WHO criteria (Table 2)151. However these criteria have
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not been validated prospectively.

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The BCSH and WHO criteria also differ in the importance given to bone marrow
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morphology. Although the classical histological features of PV are identifiable in most

patients (Figure 2), the use of histology to determine diagnosis within the MPNs, as

recommended by the WHO, remains controversial. Indeed BCSH criteria allow a

diagnosis of PV to be made in the presence of erythrocytosis and a JAK2 mutation

without a bone marrow biopsy. In one study of 272 bone marrow biopsies, blinded

histological evaluation by seven haematopathologists resulted in a diagnostic

consensus in only 53% of patients, and even where a histological consensus was

obtained, the concordance rate with the clinician’s diagnosis was only 71%165. There

are therefore ongoing uncertainties about the reliability of histology in discriminating

between PV and JAK2-mutated ET in routine clinical practice. WHO criteria also

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retain serum erythropoietin level as a minor criterion for the diagnosis of PV,

although this has been shown to add little to the accuracy of PV diagnosis in the era

of JAK2 mutation testing166.

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Although PV and JAK2-mutated ET are discriminated predominantly on the basis of

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pragmatic thresholds for red cell mass, haemoglobin or haematocrit levels, it is clear

that these thresholds are somewhat arbitrary and some patients with JAK2V617F-

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positive ET will be more “similar” to PV than others. Moreover this dichotomous

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classification does not fully capture the phenotypic heterogeneity within and overlap

between the two groups of patients. As discussed above, JAK2V617F-positive PV

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and ET can be considered as two ends of a biological continuum84 in which specific

phenotypic features (blood count parameters, and perhaps others such as spleen

size and thrombotic risk) may be influenced by the balance between clones of
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different JAK2 genotype together with genetic and environmental modifiers (Figure

1). This concept of a disease spectrum rather than a dichotomy raises questions as
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to whether it is appropriate to divide treatment studies and guidelines according to

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current diagnostic criteria, in which JAK2V617F-positive and -negative ET are

considered together, and quite separate to PV16, 149. Interestingly one report
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suggested that higher rates of thrombosis in masked PV might reflect a lower

intensity of treatment as compared to overt PV161. This raises the possibility that a

more unified diagnostic (and management) approach to those with JAK2-mutated

MPNs (PV or ET) might improve outcomes, but this has not yet been tested in clinical

trials.

5. Prognosis in PV

5.1. Factors predictive of overall survival and vascular events

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Patients with PV have a reduced life expectancy in comparison to an age-matched

healthy population and compared to patients with ET. A study of 396 consecutive PV

patients demonstrated a 15-year survival of 65%, compared to 73% in ET167, while a

multi-centre study of 1545 PV patients showed a median survival of 14.1-18.9

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years168. Age and thrombotic history have been identified as independent poor

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prognostic factors for overall survival in several studies168, 169 and leucocytosis,

abnormal karyotype and a leucoerythroblastic blood smear have also been shown to

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confer an adverse prognosis168.

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Venous and arterial thromboses are the leading causes of mortality in patients with
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PV and are the presenting feature in 20%170. After diagnosis, the rate of thrombotic

events has been estimated as 3.4-5.5 per 100 patients per year, with age at

diagnosis and history of prior thrombotic episodes being most strongly predictive of
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further events169, 170. The European Collaboration on Low-Dose Aspirin in

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Polycythemia Vera (ECLAP) study also found that a smoking history carried a small
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but significant risk for thrombosis (hazard ratio 1.9), but there is little convincing
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evidence that other factors traditionally associated with cardiovascular risk, such as

hypertension, hyperlipidaemia or diabetes mellitus, play a significant role in the

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context of PV171.

A correlation between haematocrit and risk of thrombosis was first described in a

retrospective report in 1978172, leading to the recommendation that the haematocrit

should be maintained below 0.45. As well as being associated with increased blood

viscosity, a raised haematocrit may also contribute to increased platelet activation

due to displacement towards the vessel wall, and may be associated with red cell

aggregates173. Polycythemia Study Group (PVSG)-01 and 05, a large randomised

controlled study initiated in 1967, allowed further indirect assessment of the impact of

haematocrit on thrombotic risk, since a fall in thrombotic events in the venesection-

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only arm was observed once the target haematocrit was reduced from 0.52 to

0.45174, 175. However the use of the haematocrit as a therapeutic target was only

validated formally in the recent CytoPV study176. This prospective study randomised

365 patients with PV to intensive (target haematocrit <0.45) or less intensive (target

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haematocrit 0.45-0.50) treatment, with the choice of therapy (venesection,

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cytoreduction or both) at the investigator’s discretion. With a median follow-up of 31

months, 9.8% of patients in the low-intensity arm met the primary endpoint of death

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from cardiovascular or major thrombotic events compared to 2.7% in the intensive

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treatment arm. Although this lends more definitive support to a haematocrit target of

<0.45, it remains unclear whether an even lower threshold might offer a further
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reduction in thrombotic events. A number of centres advocate different targets for

males and females – i.e. <0.45 and <0.42, respectively - based on differences in

steady-state physiological levels155, 177. This is reasonable but currently there is no

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prospective or retrospective evidence to support the use of different targets.

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Interpretation of the CytoPV study is confounded by the fact that patients in the
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lower-intensity arm tended to have higher white cell counts than those in the

intensive arm, most likely reflecting differences in the use of cytoreductive drugs.
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This is important because leucocytosis may itself be a risk factor for thrombosis. In

ET, a leucocyte count >15x109/l was identified as an independent predictor both of

overall survival and thrombosis in a study of 322 patients178, and the prospective PT1

study also found a linear relationship between leucocyte count on follow-up and the

estimated hazard ratio for thrombosis179. Proposed mechanisms for this association

include the activation of platelets by granule-derived proteases, and direct

interactions via CD11b-CD42b or -CD63b173, 180. Data are more limited in the context

of PV however. Analysis of the prospective ECLAP cohort did demonstrate an

increased thrombotic risk in patients with a leucocytosis (HR 1.71), mainly reflecting

an increased risk of myocardial infarction171, although no association was seen in a

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retrospective analysis that included 153 PV patients181.

There have been mixed reports regarding the significance of JAK2V617F

homozygosity in determining thrombotic risk both in PV and ET. Tefferi et al95

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compared rates of thrombosis between 13 JAK2V617F-“homozygous” and 45

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JAK2V617F-“heterozygous” patients and found no difference, with a later larger

study reaching a similar conclusion13. However these were both retrospective studies

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that distinguished between homozygous and heterozygous patients using semi-

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quantitative techniques. Subsequently JAK2V617F allele burden was assessed using

quantitative real-time PCR in diagnostic samples from a cohort of 173 PV patients,

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with a median follow-up of 24 months91. The 18% of patients with mutant allele

burdens of >75% had greater rates of cardiovascular events at the time of, and

following, diagnosis, as well as greater numbers of thrombotic events post-diagnosis.

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Allele burden remained the only independent variable predictive of cardiovascular

events in a multivariate analysis that included age, prior thrombosis and leucocytosis.
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In summary, age and thrombotic history remain established risk factors for

thrombosis in PV, whilst leucocytosis and JAK2V617F allele burden may have
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significance and warrant further investigation in prospective studies. Interestingly

there has been no evidence, either in ET or PV, that the degree of thrombocytosis

correlates with thrombotic risk. In fact, in ET a U-shaped relationship has been

demonstrated between platelet count and the risk of haemorrhage179, which is likely

to be explained by the development of an acquired von Willebrand syndrome at high

platelet counts (generally >1000x109/L)182. In PV, analysis of the ECLAP cohort

demonstrated a prior bleeding history as the only independent predictor for

haemorrhage169, while data from the German Study Alliance Leukaemia MPN

registry, which includes 142 patients with PV, identified prior thrombosis,

splenomegaly and heparin use as risk factors for bleeding in univariate analyses183.
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5.3. Factors predictive of transformation to myelofibrosis or acute leukaemia

Factors that have been associated with increased risk of myelofibrotic transformation

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in PV include the presence of increased bone marrow fibrosis at diagnosis184 and a

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diagnostic white cell count >15x109/l185. JAK2V617F allele burden was associated

with risk of disease transformation in a prospective study of 338 patients with PV93;

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2% of patients transformed to secondary myelofibrosis, all of whom had allele

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burdens >50%, and this association remained significant on multivariate analysis.

Although the latter study did not identify an association between allele burden and
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progression to AML, the frequency of this endpoint was low (3%). The incidence of

leukaemic transformation varies widely between studies, and has been reported as

high as up to >30% in trials with follow-up of greater than 20 years186. Studies with
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higher rates of transformation to AML have implicated leucocytosis, age and

abnormal karyotype as additional risk factors168, 187. There is also a strong association
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with the use of certain cytoreductive agents (see section 6.4).

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Although a number of somatic mutations have been associated with progression to

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myelofibrosis or acute leukaemia (see section 3.6), it is less clear whether the

mutational profile at diagnosis can predict transformation events or other outcome

measures. Although a number of studies have addressed this question in primary

myelofibrosis, only one study to date has comprehensively investigated other MPN

subtypes. Lundberg et al188 screened for mutations in 104 genes in a 197 patient

cohort that included 94 patients with PV. Of these, only mutations of TP53 and TET2

were associated with increased rates of leukaemic transformation.

6. Management of PV
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The aims of treatment in PV are to reduce disease-related symptoms and the risk of

venous and arterial thrombosis, while minimising treatment-related toxicity,

haemorrhagic risk and, in particular, the risk of progression to post-PV myelofibrosis

and acute leukaemia. As such management largely adopts a risk-adapted approach,

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targeting cytoreductive treatment towards those at higher thrombotic or

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haemorrhagic risk. Although, with the exception of smoking, there is no association

between established cardiovascular risk factors and thrombotic risk in the context of

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PV, it is generally accepted that these should be targeted regardless of other specific

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management steps.

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6.1. Use of anti-platelet agents

Since platelet activation seems to be a central mechanism in MPN-associated

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thrombosis173, 189 and there is evidence of increased platelet thromboxane

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biosynthesis in PV190, there has been interest in the use of anti-platelet agents, in
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particular aspirin, for thromboprophylaxis in MPNs. High-dose aspirin (900mg) or

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dipyridamole (225mg) was used in the venesection arm of the PVSG-05 trial (which

was a comparator for pipobroman), but this was discontinued early due to excessive
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gastrointestinal bleeds and deaths from haemorrhage175. This initially led to the

avoidance of aspirin in patients with MPNs, but subsequently the use of low-dose

aspirin was assessed in the ECLAP trial191. This multicentre, double-blind trial

randomised 518 high- or low-risk PV patients without a specific indication for aspirin

to receive daily aspirin 100mg or placebo, with a mean follow-up period of three

years. The composite primary end-point of non-fatal myocardial infarction, non-fatal

stroke, pulmonary embolism, major venous thrombosis, or death from cardiovascular

causes was met in 7.9% of the placebo-treated group and 3.2% of the aspirin treated

group (p=0.03), with overall incidences of thrombotic events of 15.5% and 6.7%

respectively (p=0.003). No significant differences were seen in the incidence of

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major, or minor bleeding. It is worth noting that this study was terminated early due to

poor recruitment and only one third of the target number of patients were actually

recruited; as such it may have been under-powered to detect significant differences

in bleeding complications192. Nonetheless this study established a role for low-dose

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aspirin in primary prevention of thrombotic events in PV and did not demonstrate a

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significant increase in haemorrhagic events, leading to the widespread use of aspirin

across all risk groups in PV, as well as in ET16. This approach is further supported by

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a recent retrospective study in low-risk ET that showed a reduction in venous events

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in JAK2-mutated patients who received antiplatelets, compared to observation alone

(although an increase in bleeding, without a reduction in thrombosis, was seen in

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CALR-mutated ET patients and it is now unclear that universal aspirin use remains

appropriate in this latter group)193. A Cochrane meta-analysis194 included both the

ECLAP study and an earlier study by Gruppo Italiano Studio Policitemia that
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assessed the safety of low-dose aspirin (40mg), and included thrombotic/embolic

events as secondary endpoints195. The reduction in thrombosis-related mortality did

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not reach significance in this analysis, but there was no significant increase in
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haemorrhagic complications. This review also concluded that aspirin is safe to use in

PV. However, these and other authors have highlighted the need for further research
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into which subgroups might benefit most or might be at greater risk of bleeding

complications, as well as into alternatives, for example thienopyridine anti-platelet

agents such as clopidogrel, which might be used in patients intolerant of or resistant

to aspirin192.

6.2. Venesection

As discussed above, a number of studies have indicated that thrombotic risk is

significantly reduced when the haematocrit is maintained below 0.45 and that

venesection of 250-500ml per session can be sufficient to achieve this target. This is
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achieved both by immediate reduction in haematocrit by the removal of red cells with

compensatory expansion of the plasma volume, but also by the longer term induction

of an iron-deficient state that constrains erythropoiesis. Furthermore, venesection

alone compared favourably compared to radiophosphorus (32P) and chlorambucil in

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the PVSG trials, which randomised 431 patients to these three treatments with

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median survivals of 13.9, 11.8 and 8.9 years respectively. However, higher rates of

early thrombotic events were observed in the venesection-only arm, particularly in

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patients over 70196. This has led to the recommendation that venesection (rather than

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cytoreduction) may be adequate to control haematocrit in low-risk patients but raised

the question of whether cytoreductive agents have a particularly important role in the
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high-risk group. Conversely the fact that a haematocrit target of 0.52 was initially

used in this trial, and subsequently reduced to 0.45, does makes its interpretation

problematic. Venesection is generally well tolerated and avoids potential toxicities

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associated with cytoreductive drugs but necessitates regular outpatient visits (in

some cases it may need to be performed daily until the haematocrit is range). Other
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potential problems include difficult venous access, anxiety regarding the use of
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needles, and symptoms resulting from iron deficiency (such as fatigue, cognitive

impairment or restless legs) or cardiovascular instability/vasovagal events (where

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isovolaemic venesection may be more appropriate).

6.3. Historical perspective: Cytoreduction using radiophosphorus or alkylating agents

32
P has been used for cytoreduction since the 1940s and has the advantage that only

intermittent treatments are required, but has long been associated with increased risk

of leukaemic transformation. A number of early trials therefore addressed whether

other myelosuppressive, predominantly alkylating, agents could provide an

alternative. However, even greater rates of leukaemic transformation were seen with

the use of chlorambucil in the PVSG trials (6% with 32P and 11% with chlorambucil,
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compared to 1% in the venesection arm)196. The European Organisation for

Research on Treatment of Cancer (EORTC) group randomised 293 patients to 32P or

busulfan197. No differences were seen in rates of leukaemic transformation (1-2%

with median follow-up of 8 years), but the busulfan-treated group had longer

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durations of remission and lower rates of thrombosis and non-haematological

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malignancies.

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Pipobroman, a piperazine derivative structurally similar to many alkylating agents,

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has also been assessed as a first line agent in a number of trials and demonstrated

rates of haematological remission of >92%, but rates of leukaemic transformation by

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10 years of 5-14%187, 198, 199. In the French Polycythaemia Study Group (FPSG) study

292 patients aged under 65 years were randomised to hydroxycarbamide or

pipobroman186, 200. Although there was no observed difference in thrombotic or

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haemorrhagic events, rates of transformation to AML or myelodysplasia were

significantly higher in patients treated with pipobroman (52% compared to 24% by 20

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years). This was associated with a median overall survival of 15.4 years in the
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pipobroman arm compared to 20.3 years in the hydroxycarbamide arm186.

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The rates of leukaemic transformation in alkylator- or 32P-treated or untreated

patients are inconsistent between trials, which may reflect differences in patient

selection or treatment intensity. These associations were assessed more

systematically in the larger ECLAP trial, where the leukaemia transformation rate

was 1.8 per 100 patients per year in 32P/alkylator-treated patients compared to 0.29

per 100 patients per year in patients treated with venesection alone, or interferon-

alpha201. Similarly Tefferi et al reported an overall incidence of AML of 2.3% by 10

years, with significantly higher rates seen in patients treated with single agent 32P,

pipobroman, chlorambucil or combined pipobroman and busulfan therapy168.

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6.4. Hydroxycarbamide use in PV and leukaemogenic potential

Given the associations between leukaemic transformation and alkylating agents, the

use of hydroxycarbamide, a ribonucleoside reductase inhibitor that interferes with

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DNA replication and may also act as a nitric oxide donor, has been explored as an

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alternative. An early phase 2 study demonstrated its tolerability and efficacy in a

cohort of 118 PV patients202. There has been a relative paucity of phase 3 data

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examining the use of hydroxycarbamide in PV, although the FPSG trial described

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above showed comparable efficacy in preventing vascular events as compared to

pipobroman200. However subsequent data from randomised trials in ET have been

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informative in establishing hydroxycarbamide as a safe and effective first-line agent

for the prevention of vascular events in high-risk MPNs. Cortelazzo et al randomised

114 high-risk ET patients to hydroxycarbamide or no myelosuppressive treatment

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with a median follow-up of 27 months, and reported vascular events in 24% of the

untreated arm but only 3.6% of the hydroxycarbamide-treated arm203. In the high risk
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arm of the PT1 trial, 806 ET patients were randomised to aspirin plus anagrelide, an
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inhibitor of cyclic AMP phosphodiesterase that blocks megakaryocytic differentiation

and proliferation, or hydroxycarbamide plus aspirin11. Patients in the anagrelide arm

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were more likely to reach the composite primary end-point of arterial or venous

thrombosis, serious haemorrhage or death from thrombotic or haemorrhagic causes,

and showed higher rates of myelofibrotic transformation, arterial thrombosis and

serious haemorrhage.

The risk of leukaemia associated with hydroxycarbamide therapy has been more

controversial. From trials such as the FPSG <65 study it is clear that the risk of AML

is lower with hydroxycarbamide than with alkylating agents, but even in this study a

surprisingly high AML/MDS transformation rate of 24.2% was seen in the

hydroxycarbamide arm at 20 years (16.6% in patients who had only received

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hydroxycarbamide and no other therapy)186. Nonetheless AML is part of the natural

history of PV and a major issue has been the lack of trials in PV comparing

hydroxycarbamide to venesection alone, or to other “non-leukaemogenic agents”.

Analysis of the ECLAP cohort showed no difference in the rate of transformation in

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venesected/interferon-treated patients compared to those treated with

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hydroxycarbamide201. This was also the case in a Swedish case-control study that

included 162 patients with transformation204 and another cohort of 1545 PV

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patients168. Further evidence against a significant leukaemogenic effect of

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hydroxycarbamide comes from an analysis of hprt locus mutations and “illegitimate”

VDJ recombination events in untreated patients, hydroxycarbamide-treated MPN

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patients and hydroxycarbamide-treated patients with sickle cell anaemia205. No

difference was seen in mutation rates between MPN patients treated with prolonged

hydroxycarbamide exposure compared to those with short exposure or untreated

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controls. However, it is worth noting that assessment of VDJ recombination is only of

relevance in T cells, and whether the conclusions of this analysis can be extended to
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myeloid or haematopoietic stem/progenitor cells is unclear.

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Although the available data do not conclusively support a direct leukaemogenic effect
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of hydroxycarbamide monotherapy, combination therapy with other agents may

increase the risk of AML. In the FPSG trial of patients older than 65 years, patients

were randomised to 32P or 32P with hydroxycarbamide maintenance206. A higher

actuarial risk of leukaemic transformation was observed in the latter arm, despite the

potential to reduce the radiation dose with hydroxycarbamide maintenance. In the

long-term follow-up of the Italian ET study described above, higher rates of

leukaemic transformation and of other secondary malignancies were also seen when

hydroxycarbamide was used sequentially following busulfan, but were much rarer in

those treated with hydroxycarbamide alone207.

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In summary, hydroxycarbamide is an effective treatment to achieve haematological

responses and reduce vascular events in MPNs. There is evidence that the

leukaemic risk associated with hydroxycarbamide is much lower than that seen with

many other myelosuppressive agents, specifically alkylating agents and 32P, but this

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risk may be increased if hydroxycarbamide is used in combination or sequentially

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with these agents. The European Leukaemia Network (ELN) have generated

response criteria for the treatment of PV208 (Table 3), which depend largely on

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features of PV biology rather than the development of complications. Approximately

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11% of patients fail to achieve an adequate response with hydroxycarbamide, and a

further 14% will discontinue due to intolerance, such as development of

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mucocutaneous ulceration, skin cancers, pneumonitis or gastrointestinal side

effects209. Resistance to hydroxycarbamide, as defined according to ELN criteria210

(Table 4), has been shown to identify a subset of patients with increased risk of
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death and transformation to acute leukaemia or myelofibrosis in one retrospective

study209. In particular, the lack of resolution in leucocytosis was associated with

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worse overall survival, and lack of platelet control with risk of thrombosis and
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bleeding. Perhaps surprisingly, control of haematocrit did not correlate with

thrombotic risk. Hydroxycarbamide-resistant patients therefore represent a difficult

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group of patients to manage and this has driven the search for other effective non-

leukaemogenic agents.

6.5. Interferon-alpha and anagrelide in PV

Recombinant interferon alpha is an attractive agent for first-line treatment in PV. It

has no known leukaemogenic potential, can achieve good haematocrit and platelet

count control, and has been shown to selectively inhibit the growth of the malignant

clone leading to a reduction of JAK2V617F burden or reversion of cytogenetic

abnormalities, and restoration of polyclonal haematopoiesis211, 212. Furthermore, it

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can antagonise platelet-derived growth factor and impair fibroblast differentiation,

providing possible theoretical mechanisms for an inhibition of myelofibrotic

transformation, supported by some evidence in early myelofibrosis213. To date

however, trials evaluating interferon have predominantly been small, single-arm

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studies and there have been no randomised controlled trials allowing comparison

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with other agents such as hydroxycarbamide. A literature review of trials performed

in PV has provided evidence for the efficacy and tolerability of interferon for a total of

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349 patients across 16 studies214, although a formal meta-analysis was not possible

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due to differences in the drug (interferon alpha 2a/2b, pegylated/unpegylated) and

response criteria used. This analysis demonstrated that on average 60% of patients
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were able to discontinue venesection, although this was as high as 97% in some

studies, and patients generally suffered low rates of thrombosis. Treatment was

limited by discontinuation rates of 30-40%, with the main side effects including
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fatigue, headache, pyrexia, myalgia and neuropsychiatric symptoms. This tolerability

profile, together with the need for subcutaneous administration, are the main factors
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limiting use of interferon in practice.

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Pegylated interferons are those that are bound by urethane or amide bonds to a
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polyethylene glycol molecule. This property prolongs their half-lives so that weekly

rather than 24-48 hourly dosing is possible, and the reduced peak dose may

potentially reduce toxicity. Three studies have assessed response rates and

tolerability using pegylated interferons in PV. Kuriakose et al assessed 76 patients

with PV, 28 of whom were treated with recombinant interferon-alpha-2b, 18 with

pegylated interferon-alpha-2a (Pegasys), 8 with hydroxycarbamide and 19 with other

agents (imatinib, dasatinib, 32P and busulfan)215. Haematological responses were

seen in 89.3%, 88.9% and 59.3% in the interferon-alpha-2b, pegylated interferon and

non-interferon groups respectively. Discontinuation rates were 16.1% in the non-

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pegylated interferon arm, compared to 5.6% with pegylated treatment. Low rates of

molecular response were observed across all arms (15.2% of interferon-treated

patients having a partial molecular response) and were independent of

haematological response. By contrast, Pegasys treatment in two other studies of 40

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and 37 PV patients was associated with molecular responses in 54% and 90% of

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patients respectively and complete molecular responses (i.e. undetectable

JAK2V617F) in 14% and 24%, with haematological responses in 80% and 100%212,

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216
. Five patients in the former study had a sustained complete molecular and

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haematological response after discontinuing Pegasys, and no thrombotic events

were observed in the study. Discontinuation rates were 10% and 24.3% respectively,
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suggesting pegylated inteferons may be a viable alternative to other interferon

preparations or to hydroxycarbamide as a first-line treatment.

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Phase 3 clinical trials comparing interferons with hydroxycarbamide are ongoing and

may further inform the choice of first-line agents in future. These include the PROUD-
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PV study (NCT01949805), in which patients were randomised to either

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hydroxycarbamide or pegylated interferon alpha-2b (PEG-intron), as well as DALIAH

(NCT01387763) and NCT01259856, which compare hydroxycarbamide to Pegasys

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and/or PEG-intron. Further molecular characterisation may also help target the use

of interferons: there is evidence that sensitivity to interferon may correlate with the

presence of JAK2V617F-homozygous clones217 and, conversely, that clones carrying

non-JAK2 mutations (e.g. TET2) may be more resistant to interferon218, 219.

Anagrelide selectively targets platelet production and therefore its role in PV is

limited. However, it has demonstrated responses when used in combination with

hydroxycarbamide in a cohort of hydroxycarbamide-resistant patients, suggesting

combination therapy may be a possibility as a second-line option220. Anagrelide has

the advantage of being non-leukaemogenic but its use is further limited by the
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increased potential for myelofibrotic transformation11 and its side effect profile which

includes tachycardias, fluid retention and headaches.

6.6. JAK2 inhibitors in PV

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Ruxolitinib is a JAK1/JAK2 inhibitor currently approved for clinical use in

myelofibrosis and in patients with PV resistant or intolerant of hydroxycarbamide. A

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Phase 2 trial first assessed its use in a cohort of 34 PV patients who were resistant

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or intolerant to hydroxycarbamide, with a median follow-up of 152 weeks221.

Haematological response (by ELN criteria) was achieved in 97% of patients with 61%
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achieving a durable response (no requirement for venesection) for 144 weeks. 70%

of patients with splenomegaly showed responses in spleen size within 24 weeks.

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This was followed by the Phase 3 RESPONSE trial comparing ruxolitinib to best

available therapy in 222 PV patients with both hydroxycarbamide

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resistance/intolerance and splenomegaly20. The primary composite end-point of

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haematocrit control and reduction in spleen size of at least 35% was met in 20.9% of

the ruxolitinib arm and 0.9% of the standard therapy arm, with 23.6% and 8.9% of
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patients, respectively, achieving a complete response by ELN criteria. Ruxolitinib

was particularly effective in improving constitutional symptoms, as was seen in

previous trials in myelofibrosis18, 19. Although the trial was not sufficiently powered to

detect a difference, there was a low rate of thrombosis in the ruxolitinib arm (1

patient, compared to 6 in the standard therapy arm). Overall ruxolitinib had a good

safety profile: anaemia was seen in 43.6% of patients and thrombocytopenia in

24.5% but grade III/IV toxicity was only seen in 1.8% and 5.4% respectively,

compared to 0% and 3.6% in the standard treatment arm. The most commonly

reported side effects were headache, fatigue, diarrhoea, muscle spasms and

dizziness and the rate of discontinuation due to toxicity was low (3.6%). These
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studies therefore indicate a role for ruxolitinib in achieving haematological control in

hydroxycarbamide-resistant/intolerant patients with PV and also suggest that

ruxolitinib may offer additional advantages in terms of reduction of splenomegaly and

symptom control. However, the generalisability of the results is limited by the highly

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selected patient group; in particular the presence of significant splenomegaly. The

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RESPONSE-2 and MAJIC studies are comparing ruxolitinib to best available therapy

in hydroxycarbamide-resistant or -intolerant patients without a requirement for

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splenomegaly and their results should therefore be more generalisable to routine

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practice.

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6.7. Histone deacetylase inhibitors and other agents in PV

Histone deacetylase (HDAC) inhibitors have been shown to have anti-tumour activity
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in AML and multiple myeloma, and also have anti-angiogenic properties. A phase 2

study assessed the use of vorinostat in a 63 patient cohort that included 44 patients
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with PV. Although patients achieved complete resolution of pruritus (present initially
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in 19% of patients), improvement in splenomegaly with resolution in 23% of patients

and a decrease in JAK2V617F burden in 65% of patients, 44% discontinued

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treatment during the 24-week trial period due to adverse events that included severe

fatigue, diarrhoea, vomiting and weight loss222. Givinostat, which has been shown in

vitro to selectively inhibit JAK-STAT signaling and proliferation of JAK2-mutant

primary cells and cell lines223, was tolerated better in a pilot study of 12 PV, 1 ET and

16 myelofibrosis patients224. Although patients also experienced diarrhoea (62%),

nausea (10%), fatigue (7%) and abdominal pain (17%), these were Grade I-II in

almost all cases and only one patient discontinued treatment because of drug-related

adverse events. Seven of the thirteen PV or ET patients showed a clinical response

by ELN criteria. This study was followed by a trial of 50 or 100mg givinostat in

combination with hydroxycarbamide in a cohort of 44 patients who were non-

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responsive to maximal doses of hydroxycarbamide225. The combination was

generally well tolerated, with only 18% discontinuing treatment within 24 weeks.

Responses were seen in 55% of patients on 50mg and 50% on 100mg givinostat,

with resolution of pruritus in 64% and 67%, respectively. These studies therefore

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suggest a potential role for HDAC inhibitors in disease control and symptomatic

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management of hydroxycarbamide-resistant patients.

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There are a number of other agents currently under investigation for

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hydroxycarbamide-resistant patients. These include inhibitors of signalling down-

stream of JAK2 (such as as TGR-1202, a PI3K-delta inhibitor). CEP-701 (lestaurtinib,

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a FLT3 inhibitor), RG7388 (a selective inhibitor of p53-MDM2 binding), AUY922 (an

inhibitor of heat-shock protein 90) and Imelestat (a telomerase inhibitor that has

shown some efficacy in ET and myelofibrosis226, 227).

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6.8. Symptom burden and control in PV

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Beyond the morbidity and mortality related to PV-related complications such as

thromboses, haemorrhage and disease transformation, PV itself carries a significant

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symptomatic burden. This can be formally quantified using the MPN symptom

assessment form (MPN-SAF), where severity of a number of symptoms are scored

from 1-10, and from which a total symptom score (TSS, out of 100) can be calculated

as a global measure of morbidity228. This score was found to correlate well with other

measures of quality of life, with PV patients in a large international study having a

mean TSS of 21.3, compared to 18.7 for those with ET and 25.7 for those with

myelofibrosis229. Fatigue showed the greatest severity in patients with PV (mean

4.4/10, present in 88%), followed by itch (62%), concentration problems (65%), early

satiety (64%) and inactivity (61%), and the incidence and severity of all of these

symptoms was higher for patients with PV than those with ET.
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The RESPONSE trial reported significant improvements in constitutional symptoms

with ruxolitinib compared to standard therapy, particularly those related to cytokines

such as pruritus, sweats, fatigue and muscle aches20. The effects on symptoms and

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quality of life were described further in a post-hoc analysis that used a number of

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tools including the MPN-SAF and EORTC QLQ-C30 quality of life questionnaire230.

Ruxolitinib treatment was associated with a fall in TSS and with improvements in

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other global measures of quality of life, social, physical and emotional functioning,

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while patients in the standard treatment arm showed a deterioration on average in all

these measures. More specifically the EORTC QLQ-C30 demonstrated

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improvements in fatigue, pain, anorexia, dyspnoea, diarrhoea and constipation in the

ruxolitinib arm but not the standard treatment arm, while insomnia improved in both

treatment arms. The symptomatic benefit of ruxolitinib was further assessed in the
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RELIEF study, a double blind, double-dummy switch study which randomised

symptomatic patients who were otherwise well controlled on hydroxycarbamide, with

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no venesection requirement or splenomegaly, to either ruxolitinib and

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hydroxycarbamide-placebo, or hydroxycarbamide at the same dose/schedule and

ruxolitinib-placebo231. Both ruxolitinib- and hydroxycarbamide-treated patients

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showed mean improvements in cytokine-related total symptom scores, although a

significant difference between these arms was not observed.

Pruritus is particularly an issue in PV and is seen in 62% of patients compared to

46% of those with ET and 50% with MF229. It can be associated with discomfort,

problems sleeping, social embarrassment, mood changes, intolerance of bathing,

depression and even suicidal ideation232. Antihistamines are widely prescribed but

the literature is inconsistent on whether they are beneficial. Phototherapy has been

used in small numbers of patients to good effect, and there is also some evidence for

the use of selective serotonin reuptake inhibitor anti-depressant agents (reviewed in

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Saini et al232). A review in 2000 of 11 reports of use of interferon alpha found that

81% of the 80 PV patients evaluated showed improvement in pruritus on interferon

treatment, as well as improvements in splenomegaly in 77%233. Most recently there is

not only evidence that ruxolitinib can significantly improve the pruritus associated

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with PV20, 230, but also that HDAC inhibitors may prove to be useful in this setting225.

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6.9. Management of special situations in PV: pregnancy and splanchnic thrombosis

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Recommendations on the management of PV in pregnancy are limited by its low

incidence (median age at presentation is 60 and there is a male predominance) and

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the fact that most reports include only small numbers of patients and are

retrospective. The experience in ET shows that myeloproliferative conditions carry a

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significant risk to the mother and fetus. A literature review of 291 pregnancies in 190
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women with ET revealed a first trimester miscarriage rate of 39% and a live birth rate

of only 61%234, while the rates of maternal thrombosis and haemorrhage were 3%
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and 2%, respectively. In PV a review of 36 pregnancies in 18 patients reported a

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neonatal survival rate of only 50%235. Three mothers suffered thrombotic

complications, one a large post-partum haemorrhage, four developed pre-eclampsia

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and there was one maternal death.

On the basis of this limited experience, the following recommendations have been

suggested234, 235. Patients should continue on aspirin 50-100mg and thromboembolic

deterrent (TED) stockings should be worn throughout pregnancy and for six weeks

post-partum. 4 weekly full blood counts should be performed until 24 weeks gestation

and 2 weekly thereafter to ensure adequate haematocrit control, and iron

supplementation should be avoided. Patients over 35 years old, with previous

pregnancy complications or microcirculatory events, additional thrombophilic factors

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(such as a positive lupus anticoagulant) or platelet count over 1000x109/l should be

considered high risk and therefore should be considered for additional low molecular

weight heparin (LMWH) or cytoreduction. Cytoreduction, in particular, would be

indicated if there is inadequate control of the haematocrit or a rising platelet count,

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while the use of LMWH is supported by experience of patients with multiple other

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thrombotic risk factors in pregnancy, outside of the context of PV236. Those with a

previous thromboembolic or haemorrhagic event are considered at highest risk, in

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which case both low molecular weight heparin and cytoreduction would be indicated.

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No studies have addressed the optimal haematocrit in pregnancy. Given the

expansion in plasma volume in this state, resulting in a significantly lower

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haematocrit for a given red cell mass, a target of <0.36 has been recommended by

some155.
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Where indicated, prophylactic doses of subcutaneous LMWH are used (for example,

5000 units dalteparin or 40 mg enoxaparin daily) and monitoring of anti-Xa levels are
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recommended every 2-3 months. Ultrasound scanning is recommended at weeks 12,

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20, 26, 30 and 36 and uterine artery Doppler at 20 and 24 weeks, and evidence of

placental insufficiency would be an indication to escalate LMWH treatment or to

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consider cytoreduction. There is evidence, predominantly from animal studies, of

teratogenicity with hydroxycarbamide use, and anagrelide can potentially cross the

placenta. Therefore interferon alpha is the agent of choice for cytoreduction. If aspirin

is discontinued for spinal or epidural analgesia, this period should be covered with

prophylactic LMWH, with the last dose at least 12 hours before. Aspirin and LMWH

should be continued for at least 6 weeks post-partum for all risk groups, as the post-

partum period carries a high thrombotic risk for the mother.

The proportion of patients with splanchnic vein thrombosis (SVT) found to have an

underlying myeloproliferative neoplasm can be as high as 70% in some series, and

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this complication is seen in up to 5% of patients with PV237. The diagnosis of PV may

be masked by plasma volume expansion or bleeding/iron deficiency in those with

portal hypertension, and therefore needs to be considered even in those with a

normal haematocrit at diagnosis. Molecular studies are essential and red cell mass

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studies may be helpful in the situation. Currently there are no specific

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recommendations for the management of SVT, beyond those for the management of

venous thrombosis in general. Immediate anticoagulation with therapeutic LMWH is

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required and usually followed with a vitamin K antagonist. The duration of treatment

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required is unclear, but in patients with spontaneous SVT an argument can be made

for indefinite anticoagulation, given that SVT is potentially life-threatening and PV

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remains an ongoing risk factor. The utility of novel oral anticoagulant agents in this

context is unclear and there is a risk of interactions with JAK inhibitors. The

occurrence of thrombosis is an indicator of high-risk PV and warrants cytoreduction

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but data are lacking regarding blood count targets; a more stringent haematocrit

target of <0.42 has been adopted in some centres and may be more appropriate
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given both the high thrombotic risk in these patients, and the fact that splanchnic vein
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thromboses may be associated with expansion of plasma volume238.

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6.10. Summary of recommendations for the management of PV

Together the available evidence indicates that the use of low-dose aspirin and

achievement of a haematocrit target of <0.45 (and potentially even lower in some

contexts) are effective strategies for reducing thrombotic risk in patients with PV.

These measures are therefore recommended for all patients, regardless of the

presence of additional risk factors. Venesection is a safe and generally well-tolerated

therapy for haematocrit control.

Cytoreduction is indicated for high-risk patients – those over the age of 60 and/or
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those with a history of previous thrombosis – or for patients intolerant of or

inadequately controlled on venesection, those with intractable symptoms such as

pruritus, evidence of progressive myeloproliferation such as worsening splenomegaly

or leucocytosis (itself a risk factor for thrombosis) or with progressive thrombocytosis

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(and therefore at increased risk of haemorrhage)239. Patients with SVT require

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cytoreduction as well as anticoagulation and it is uncertain whether more stringent

treatment targets would result in better outcomes in this group. There is insufficient

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evidence to determine whether cytoreductive agents are beneficial in patients at

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intermediate risk – i.e. those under 60 with additional risk factors such as diabetes

mellitus – but the management of these patients should include modification of risk
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factors and a lower threshold for considering cytoreductive agents.

First-line cytoreduction should be either with recombinant interferon alpha or

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hydroxycarbamide. Interferon alpha has the advantages that it is not associated with

leukaemic transformation, has been shown to reduce the JAK2 mutant allele burden
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and potentially reduce disease progression in myelofibrosis, and can be effective in

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reducing pruritus. However its significant side effects limit its use in older patients

and it is contraindicated in those with a history of autoimmune or psychiatric

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conditions. Pegylated interferons may be better tolerated and require less frequent

dosing. Hydroxycarbamide does not share these advantages and there still is a lack

of certainty about potential leukaemogenicity, but it is generally better tolerated.

Therefore it is generally the drug of choice in older patients (over 50 years) and

should be used with caution in patients with previous exposure to leukaemogenic

agents. Patients failing either first-line agent can be changed to the remaining agent

if tolerated. Further options at this point would include JAK inhibitor therapy if

available, busulfan or pipobroman (which carry an increased risk of disease

transformation and should be reserved for patients over 75 years), anagrelide (for

thrombocytosis) or a trial of other experimental agents.

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7. Conclusions

Since the identification of the JAK2V617F mutation in the majority of patients with

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PV, significant progress has been made in elucidating the mechanisms by which this

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genetic abnormality causes disease. The identification of canonical and non-

canonical downstream pathways has demonstrated the diverse molecular and

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cellular effects of this single mutation on haematopoietic precursors and how they

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contribute to MPN phenotype, particularly that of erythrocytosis. It remains less clear

however how JAK2V617F affects haematopoietic stem cell function and whether
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other advantageous genetic lesions are generally involved in the persistence of

chronic-phase disease.
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The question of how the single JAK2V617F mutation can be associated with multiple

different clinical phenotypes has also been widely investigated. Myelofibrosis is

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characterised by a wider spectrum and heavier burden of other mutations, and

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appears to represent a more accelerated phase of disease. By contrast JAK2V617F-

positive ET and PV have characteristics of a biological continuum and there is

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evidence that a number of continuous and discrete variables may determine the

exact clinical phenotype and diagnosis. This clinical spectrum of JAK2V617F-positive

ET/PV and its differences from JAK2-negative ET are not yet fully reflected in

international diagnostic criteria, management guidelines or clinical trials.

Together the biological data indicate that disease phenotype and persistence in

JAK2-mutated PV reflect a broad diversity of molecular perturbations. Moreover the

mechanisms that determine the most clinically damaging events – those of disease

transformation – are even more complex and are likely to depend on additional,

heterogeneous genetic lesions. This complexity accounts for the relative lack of
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progress in development of therapies that affect the underlying disease biology and

in particular the size of the JAK2-mutant clone and risk of disease transformation. It

is clear that inhibition of JAK2 in PV and related disorders does not result in the

same clonal response that is seen with inhibitors of BCR-ABL in chronic myeloid

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leukaemia. Although there are promising suggestions that newer agents such as

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pegylated interferons may specifically target the disease clone, the challenge is now

to elucidate which characteristics of a patient and their disease account for and

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predict response to these treatments. Moreover no agent has yet been shown to

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prevent or delay disease progression. In the meantime, management of PV remains

centred on prevention of cardiovascular complications and studies continue to clarify

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the roles of specific cytoreductive agents and their respective treatment targets.

Nonetheless important areas such as the use of additional thrombotic risk markers,

the impact of novel agents (e.g. JAK2 inhibitors) on thrombosis, and management of
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JAK2V617F-associated splanchnic vein thrombosis, require further study in

prospective trials.
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Practice Points

• Optimisation of cardiovascular risk factors and aspirin 75-100mg daily (unless

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contraindicated) should be employed for all patients.

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A target haematocrit of <0.45 should be used for all risk groups. Some

centres advocate lower targets in females and in certain high-risk situations.

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Venesection alone may be sufficient to achieve this and is preferable in low-

risk patients. Given the evidence for a biological continuum between JAK2-

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mutated ET and PV and the possibility of masked PV, there is an argument
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for maintaining haematocrit <0.45 in all JAK2-mutated patients.

• Consider interferon-alpha for low-risk patients if intolerant of venesection or

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symptomatic, particularly with pruritus.

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• Cytoreduction with interferon-alpha or hydroxycarbamide is indicated in high-

risk patients (age >60 and/or previous thrombosis) and should be considered
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in cases with progressive myeloproliferation (rising white cell count,

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splenomegaly), uncontrolled thrombocytosis or contraindications to

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venesection.

• In patients with an inadequate response, alternative first-line agents can be

attempted and patients should be considered for clinical trials. In older

patients (e.g. >75 years) busulfan or pipobroman may be considered.

• Individualised molecular characterisation (such as JAK2V617F allele burden

and presence of additional somatic mutations e.g. TET2 and TP53) may in

future offer improved risk stratification and inform the choice of therapeutic

agent.
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Research Agenda

• Effects of JAK2V617F on haematopoietic stem cells and mechanisms by

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which disease clones persist in chronic-phase PV

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Prospective evaluation of leucocytosis and JAK2V617F allele burden as

possible additional markers of thrombotic risk in PV

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• Role of additional genetic lesions in disease transformation and in identifying

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patients as candidates for novel therapies

• Effects of interferon-alpha, JAK2 inhibitors and other novel therapies on

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thrombotic risk, JAK2 mutant burden and risk of disease transformation

• Relationship between PV and JAK2V617F-positive ET, as compared to

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JAK2-negative ET, in clinical practice, especially the role of haematocrit in

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thrombotic risk in JAK2V617F-positive ET

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• Treatment targets in very high risk patients, especially those with splanchnic
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vein thrombosis and for other subgroups, e.g. males vs. females, pregnancy.

• The use of novel agents or first-line JAK inhibitors in high-risk patients.

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Conflict of interest statement

JG has no conflicts of interest to declare. ALG has received speaker honoraria from

Novartis and Shire.

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Figure Legends

Figure 1. Factors that may determine a phenotype of PV or ET in the presence

of a JAK2V617F mutation. Numerous factors may modulate the relative sizes of

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JAK2V617F-heterozygous and JAK2V617F-homozygous subclones, as well as the

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phenotypic effects of these clones.

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Figure 2. Bone marrow histology in PV. Low power (left) and high power (right)

images of a bone marrow trephine (H&E stain), showing typical histological features
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of PV including marked hypercellularity, trilineage hyperplasia (“panmyelosis”) and

megakaryocyte pleomorphism and clustering.

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Figure 2

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Table 1. Genes other than JAK2, MPL and CALR that show recurrent mutations

in human MPNs, and their frequencies in PV and other disorders.

Note that the list is not exhaustive and exome sequencing studies have shown

additional genes to be mutated in small proportions of patients90. *JAK2-negative

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idiopathic erythrocytosis.

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Blast
Gene PV (%) ET (%) MF (%)

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phase (%)
LNK240-242 25* 0-7 0-6 10
105, 243, 244
TET2 10-16 <5 7-17 17-32

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245-247
DNMT3A 3-7 <5 2-15 14-17
247-250
ASXL1 2-7 <5 13-32 18-33
251-254
SRSF2 <5 <5 6-17 19-33
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249, 255, 256
EZH2 <5 <5 7-13 <5
147, 247, 257-259
CBL <5 <5 0-8 6-9
260, 261
IDH1/2 <5 <5 <5 9-22
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247, 252, 262

SF3B1 <5 <5 <5 <5
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Table 2. Diagnostic criteria for polycythaemia vera.

M = male, F = female, RCM = red cell mass

World Health Organisation, 2016 revision151 British Committee for Standards in

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Haematology, 2007

Diagnosis of PV requires: Diagnosis of PV requires A1 + A2

All 3 major criteria, or Additional criteria used for JAK2-negative PV

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Major (1) + (2), + minor criterion

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Major (1): A1:
Hb > 165 g/l (M) or 160 g/l (F) or Hct > 0.52 (M) or > 0.48 (F) or
Hct > 0.49 (M) or > 0.48 (F) or Raised RCM (> 25% above predicted)
RCM > 25% above mean normal predicted

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Major (2): BM biopsy showing hypercellularity for A2: Mutation in JAK2
age with trilineage growth (panmyelosis),
including pleomorphic, mature megakaryocytes
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Major (3): Presence of JAK2V617F or JAK2 exon
12 mutation

Minor: Subnormal serum erythropoietin level

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Table 3. European Leukaemia Network criteria for response in polycythaemia

vera (adapted from Barosi et al, 2013208).

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Criteria for complete response (A,B,C AND D are ALL required):
A: Resolution of disease-related signs for at least 12 weeks, including
hepatosplenomegaly and improvement in MPN-SAF TSS of at least 10 points

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B: Haematocrit maintained below 0.45 (without phlebotomy), platelet count below
400x109/l and white cell count below 10x109/l for at least 12 weeks.

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C: No evidence of disease progression or thrombotic or haemorrhagic events.
D: Histological remission, defined as normocellularity, absence of trilineage
hyperplasia, and reticulin fibrosis <2

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Criteria for partial response:
A, B, AND C (as defined above) without:
D: Histological remission, defined as resolution of trilineage hyperplasia
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Criteria for no response:
Failure to meet complete or partial response criteria.
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Table 4. Criteria for the diagnosis of clinical resistance or intolerance to

hydroxycarbamide (adapted from Barosi et al, 2010210).

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A: Need for phlebotomy to maintain haematocrit <0.45 after 3 months of at least 2
g/day of Hydroxycarbamide
B: Platelet count >400x109/l AND white blood cell count >10x109/l after 3 months of

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at least 2 g/day of Hydroxycarbamide
C: Failure to reduce massive splenomegaly (i.e. >10cm below costal margin) by

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greater than 50%, or to reduce splenomegaly-associated symptoms after 3 months
of at least 2 g/day of Hydroxycarbamide
D: Absolute neutrophil count <1.0x109/l OR platelet count <100x109/l or
haemoglobin <100 g/l at the lowest dose of Hydroxycarbamide required to achieve

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a complete or partial clinico-haematological response (defined in Table 3).
E: Presence of leg ulcers or other unacceptable Hydroxycarbamide-related non-
haematological toxicities, such as mucocutaneous manifestations,
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gastrointestinal symptoms, pneumonitis or fever at any dose of Hydroxycarbamide
Any one of A, B, C, D or E is sufficient for the diagnosis of hydroxycarbamide
resistance or intolerance.
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