Human myeloproliferative neoplasms:

1
Human myeloproliferative neoplasms molecular
mechanisms, diagnosis and classification
Tony Green Cambridge Institute for Medical
Research University of Cambridge and Addenbrookes
Hospital
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1. Normal mammary epithelial development
7. Diagnosis
Nik-Zainal et al Cell 2012
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Myeloproliferative neoplasms
Red cells
Blood stem cell
Progenitors
Platelets

• Arise in blood stem cell
• Increased production of mature cells
• Window on earliest stage of tumorigenesis
• Tractable accessible tissue, chronic diseases,
  clonal analysis

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The BCR-ABL negative myeloproliferative neoplasms
Essential thrombocythaemia (ET)
Acute myeloid leukaemia
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The JAK2 V617F mutation
neg
JAK2
Exon 16 mutations
Exon 12 mutations
V617F mutation
A T G T N T C T
A T G T T T C T
A T G T G T C T
Grans
T cells
Homozygous
Mitotic recombination
James et al Nature 2005 Baxter et al Lancet
2005 Levine et al Cancer Cell 2005 Kralovics
et al NEJM 2005 Scott et al NEJM 2007 Bercovich
et al Lancet et al 2008
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It all starts to make sense
EPOR TPOR
JAK2
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Diagnosis
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Rapid direct clinical impact
2005
2010
Identification of JAK2 mutation PT-1
trials Specialist MPN clinic
Recognition of new disease subtypes
Molecular testing in regional diagnostic service
Therapeutic JAK2 inhibitors
Selected Green lab translational papers since 2005
Harrison et al NEJM 2005 Baxter et al Lancet
2005 Campbell et al Lancet 2005 Scott et al Blood
2006 Campbell et al Blood 2006a
Campbell and Green NEJM 2006 Campbell et al Blood
2006b Scott et al NEJM 2007 Wilkins et al Blood
2008 Beer et al Blood 2008
Zhao et al NEJM 2008 Campbell et al JCO 2009 Beer
et al Blood 2010 Chen et al Cancer Cell
2010 Godfrey et al Blood 2012
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Old criteria for diagnosis of PV
A 1 Red cell mass gt25 above predicted or Hct
gt60 male, gt56 female 2 No cause for 2o
erythrocytosis 3 Palpable splenomegaly
4 Clonality marker B 1 Platelet count gt400
x109/l 2 Neutrophils gt10 x109/L (gt12.5 in
smokers) 3 Splenomegaly on imaging
4 Endogenous erythroid colonies or reduced serum
epo A1 A2 any other A
establishes diagnosis A1 A2 two
of B establish diagnosis
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Diagnostic criteria 20012

• Raised Hb
• 2 JAK2 mutation
• 3 No cause for 20 erythrocytosis
• - normal art O2
• - serum epo
  not high

McMullin et al BCSH guidelines BJH 2005, 2007
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Idiopathic erythrocytosis or PV?
Scott et al NEJM 2007
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PV variant with JAK2 exon 12 mutations
Isolated erythrocytosis
Can be low level in blood
granulocytes
erythroid colony
High Resolution Melt Analysis
Multiple mutations
Scott et al NEJM 2007 Percy et al Haematologica
2007 Boyd et al BJH 2010
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Diagnosis of ET JAK2 or MPL mutation-positive
BCSH guidelines 2009 Sustained platelet count
gt450 Acquired mutation (JAK2 or MPL) No other
myeloid malignancy
WHO 2008 Sustained platelet count gt450 Acquired
mutation (JAK2 or MPL) No other myeloid
malignancy Typical bone marrow appearances
(reticulin grade 2)
BUT in absence of mutation still need to exclude
reactive causes
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Distinguishing different MPNs
ET
PV
PMF
diagnose and
Beer et al Blood 2011
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Distinguishing different MPNs
ET
PV
PMF
diagnose and
Beer et al Blood 2011
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ET is heterogeneous
Key issue can Cologne/WHO histological criteria
identify distinct disease entities
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WHO book is like a country with good aspects
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.. but some aspects less good
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.. but some aspects less good
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Three questions

• Is prefibrotic PMF really distinct
• Can WHO criteria be applied reproducibly
• Is prefibrotic MF a useful concept

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Distinguishing ET from PMF Role of histology
ET
?

• 3 experienced haematopathologists, large
  prospective cohort (PT1 study) , blinded analysis
  of WHO diagnosis and 16 morphological criteria

Campbell et al Lancet 2005
Scott et al Blood 2006
Wilkins et al Blood 2008
Campbell et al JCO 2009
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Conclusions
Even experienced haematopathologists dont agree
on what to call true ET prefibrotic
PMF Two alternative explanations -
Prefibrotic PMF not a distinct entity
- Prefibrotic PMF exists but special training
required - even if true,
questionable utility of criteria
the application of which is so difficult
even for highly
experienced pathologists
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Utility of prefibrotic PMF criteria (WHO 2008)
Thiele et al Blood 2011 Barbui, Thiele et al JCO
2011
YES
Concordance 73-88
Brousseau et al Histopathology 2010
Distinction between ET and prefibrotic PMF is of
questionable clinical relevance Buhr, Kreipe et
al Haematologica 2012 gt 50 no agreement or
unclassifiable - WHO criteria for
discriminating ET from prefibrotic PMF are poorly
to only moderately reproducible
NO
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Barbui, Thiele et al JCO 2011

• Limitation of consensus approach to histology
• MF progression at 15 yrs 10 vs 17
• 
  true ET vs prefib MF
• Leuk progression at 15 yrs 2 vs 11
• - but no mention of therapy
• Even if real difference prefibrotic PMF likely
  to represent later stage of same disease process

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Three questions
? later stage disease

• Is prefibrotic PMF really distinct
• Can WHO criteria be applied
• reproducibly
• Is prefibrotic PMF a useful concept

NO
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Nomenclature of prefibrotic PMF is flawed
Prefibrotic PMF?
True ET
PV
PMF
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Nomenclature of prefibrotic PMF is flawed
Prefibrotic PMF?
ET
PV
PMF
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Nomenclature of prefibrotic PMF is flawed
5-30
ET
PV
PMF
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Nomenclature of prefibrotic PMF is flawed
Prepolycythaemic PV?
ET
PV
PMF
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Concept of prefibrotic PMF is also potentially
dangerous
For individual patient management -
inappropriate therapy (eg BMT) for low risk
patients For the MPN field - patient
cohorts will not be comparable
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Three questions
? later stage disease

• Is prefibrotic PMF really distinct
• Can WHO criteria be applied
• reproducibly
• Is prefibrotic PMF a useful concept

NO
Flawed Dangerous
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Distinguishing ET from PMF
Thrombocytosis with isolated increased
reticulin

• Relatively common 15-20
• Unclassifiable under WHO
• Benign prognosis

Implications patient predisposed to robust
fibrosis but lacks 2nd
hits needed for evolution of clinical disease

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Distinguishing ET from PMFPMF as presentation in
accelerated phase of pre-existing MPN

• PMF and MF transformation indistinguishable
• PMF patients may have prior thrombocytosis
• PMF exhibits features of late stage disease
• - high clonal burden
• more cytogenetic abnormalities
• - increased progression to AML

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Molecular classification of ET and PMF
Chronic phase
Heterogeneous (mostly Pl)
JAK2
MPL
Campbell and Green NEJM 2006
Beer et al Blood 2011
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Distinguishing different MPNs
ET
PV
PMF
diagnose and
Beer et al Blood 2011
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Distinguishing ET from PV
809 ET patients in PT-1 trial
JAK2-positive ET is forme fruste of PV
Campbell et al Lancet 2005
Scott et al Blood 2006
Harrison et al NEJM 2005
Campbell et al Lancet 2005
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Suppression of Epo in JAK2-pos ET
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JAK2 WT
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JAK2 V617F
30
25
Serum Epo (mU/mL)
20
plt0.0001
15
10
5
0
11
12
13
14
15
16
Haemoglobin (g/dL)
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Distinguishing ET from PV
Normal
ET
N
14 16 18 20
Hb
Inherent problem in using continuous variable (eg
Hct or RCM) to make a binary distinction
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One mutation but two diseases
Polycythaemia vera
Essential thrombocythaemia
Hypothesis homozygosity for JAK2 mutation
causes PV phenotype
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JAK2V617F knockin mouse homozygosity causes
phenotypic switch from ET to PV
Haematocrit
Platelets
Haematocrit ()
Platelets (x103)/µl
WT Het Hom
WT Het Hom
Li et al Blood 2010 Juan Li, David Kent
unpublished
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ET
Heterozygous JAK2V617F
9p LOH
PV
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Homozygous mutant BFU-E present in many patients
with ET
PV 80
ET 52
Higher number of homozygous colonies in PV
patients compared to ET - homozygous clone
has selective advantage in PV but not ET -
recurrent acquisition of homozygosity
Godfrey et al Blood 2012
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Recurrent acquisition of 9p LOH in patient with PV
A B C
Heterozygous LOH
Tel
Mb from telomere
JAK2
A
C
B
170 180
170 180
170 180
125 130 135
125 130 135
125 130 135
Cen
Number of colonies
44 5 4
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Summary

• Recurrent acquisition of homozygosity
• - in 5/8 PV patients and 2/2 ET
  patients tested
• - resolution limited (2.3 to 14.2 Mb)
  so number of distinct
• clones may be underestimate
• PV distinguished from ET by presence of dominant
  homozygous clone 10 fold larger than minor
  clones
• - ? additional lesions
• Multiple clones arise in HSPC compartment
• - persist over time
• - detectable in sorted CMPs

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ET
Heterozygous JAK2V617F
Recurrent 9p LOH
PV
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Differential STAT1 signaling in heterozygous
colonies from patients with ET and PV
Arrays
Pathway
Key regulator
DAPI
pSTAT1
Actin
ET
PV
Chen et al Cancer Cell 2010
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Essential thrombocythaemia
Polycythaemia vera
JAK2 V617F
JAK2 V617F
STAT1 defect
JAK2 homozyg
Clonal expansion
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Conclusions and questions

• Unexpected complexity in early phase of simple
  malignancy
• Questions
• - how does clone expand given HSC defect
• - what drives recurrent mitotic
  recombination
• - what drives expansion of dominant
  homozygous clone in PV
• - cause and effects of pSTAT1 defect in
  heterozygous PV cells
• Exomes coming

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Green lab Maria Ahn Athar Aziz Philip
Beer Edwin Chen Jyoti Evans Anna Godfrey Tina
Hamilton David Kent Juan Li Steve
Loughran Charlie Massie June Park Dean
Pask Yvonne Silber Rachel Sneade Addenbrookes/BRC
Mike Scott Joanna Baxter/Anthony Bench MPD
clinic, TRL team NCRI MPN Study Group PT-1 trial
team
Acknowledgements
Sanger Institute Peter Campbell, Mike
Stratton Andy Futreal, Elli Papaemmanuil Cambridg
e University Anne Ferguson-Smith Carol
Edwards Nick Cross Amy Jones Claire
Harrison Mary-Frances McMullin Adam
Mead Sten-Eirik Jacobsen Alessandro
Vannucchi Eva Hellstom Ghulam Mufti Jean
Jacques Kiladjian
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The myeloproliferative neoplasms
Primary Myelofibrosis
Essential thrombo- cythaemia (ET)
Acute myeloid leukaemia
Thrombosis
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JAK2 mutations
neg
FERM
SH2
JH2
Kinase
exon 14 V617F 95 PV 50 ET 50 IMF
exon 16 R683 Acute Lymphoblastic Leukaemia
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Difference between mutation pos ET and PV
Acquired mutations
Gender
Genetic modifiers
Depleted iron stores
Erythrocytosis
JAK2 Mutation
Normal Hb
Campbell et al Lancet 2005
Scott et al NEJM 2007
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Nuclear JAK2 signaling directly regulates
chromatin structure