Jason Gotlib, MD, MSAssociate Professor of Medicine (Hematology)
Stanford Cancer Institute
MPN Advocacy and Education InternationalPatient Symposium, San Mateo, CA
May 22, 2014
GENETICS & BIOLOGY OF MYELOPROLIFERATIVE NEOPLASMS
DISCUSSION POINTS• Refresher on the ‘established genetics’ & biology of MPNs:
• JAK2 V617F, MPL• JAK-STAT signaling
• Introduction to the ‘newer genetics’ • Calreticulin (CALR)
• Other gene mutations outside of the JAK-STAT pathway
• Relevance of mutations to diagnosis, prognosis and treatment of MPNs
William Dameshek, 1951 Blood Editorial“Some Speculations on the Myeloproliferative Syndromes”
“It is possible that these various conditions- ‘myeloproliferative disorders’- are all somewhat variable
manifestations of proliferative activity of the bone marrow cells, perhaps due to a hitherto undiscovered stimulus.
Normal Human Blood Development (Hematopoiesis) MPNs, and Stem Cells
modified from Tan et al., 2006
CD34+CD38+
CD34-
MPNNormal
MPN Stem Cell
MPN Progenitor
Cell
MatureMPNCells
2005: IDENTIFICATION OF JAK2 V617FLancet
CancerCell
Nature
NEJM
Polycythemia Vera Essential Thrombocythemia Primary Myelofibrosis
JAK2 V617F Mutation Frequency
95-98% 50-60% 50-60% Exon 12 JAK2 ~2%
JAK2 gene
• A well characterized signaling pathway involved in normal hematopoiesis, inflammation, and immune function
• Four members of JAK family• JAK1, JAK2, JAK3 and Tyk2• They are tyrosine kinases
• JAK2 specifically mediates growth factor signaling for red blood cells and for platelets
Shuai, K. & Liu,B. (2003) Nature Reviews Immunology 3:900
JAK-STAT Signaling
EPO or TPOReceptor
JAK2V617F
•Acquired
•Arises in blood stem cells
•Results in constitutively (i.e. always)active JAK2 tyrosine kinase
•Causes disease in mice (PV → MF)
Shuai, K. & Liu,B. Nature Reviews Immunology 2003:3:900
JAK2 V617F Mutation
EPO or TPOReceptor
Mutations in other genes besides JAK2 causeactivated JAK-STAT signaling in MPNs
LNK(PV, ET, MF)
<5%
CBL~6%
(PMF) Oh and Gotlib, Exp Rev Hematol, 2010
MPL1-5% ET
5-10% PMF
ASH 2013: CALR MUTATIONS IN NON-MUTATED JAK2 ET AND MF PATIENTS
97%JAK2V617F
mutant
JAK2 exon 12mutant
PolycythemiaVera
50-60%JAK2V617F
mutant
????
Essential ThrombocythemiaPrimary Myelofibrosis
50-60%JAK2V617F
mutant
30-40%CALRmutant
Kralovics & Green labs, ASH 2013
‘Triple negative’10%
MPL Mutant5%
52-bp deletion5-bp insertion
Klampfl et al,NEJM 2013
Mutations in the CALRgene all occur in one
region (exon 9)
Two most common mutations in the
CALR gene:
Type 1: 52-bp deletionType 2: 5-bp insertion
Referred to as ‘indels’
CALR FUNCTIONS
Calcium Regulation in
the cell
Protein folding
Cell adhesion
Immune-mediatedcell death
Programmedcell removal
CALRMutation
Normal Functions of CALR in Cells
Activation ofJAK-STAT signaling
(but not previously known to be relevant
to this pathway)
Both JAK2- and CALR-mutated MPN patients show a gene expression signature associated with activated JAK-STAT signaling
Rampal et al,Blood, 2014
JAK2 V617F
CBL JAK2 exon 12MPL CALR LNK
TET2ASXL1
IDH1, IDH2
DNMT3AEZH2
SRSF2
Mutations in genes outside of the JAK-STAT pathway in MPN patients
JAK-STAT Pathway
Outside ofJAK-STAT Pathway
Gene Chronic Phase Blast Phase / AMLJAK2 V617F PV: 98%; ET /PMF: 50-60%
Exon 12 JAK2 PV: ~1-2%
CALR ET/PMF: ~30-40%
MPL ET: 1-5%; PMF: 5-10%
LNK PV, ET, PMF: <5% ~10%
CBL PMF: 6%
TET2 PV: 7-16%, ET: 4-11%, PMF: 8-17%
ASXL1 PV: 2-5%; ET: 5-8%; PMF: 7-17% 19%
DNMT3A PV: 7%, ET: 3%, PMF: 7-15% 17%
IDH 1/2 PMF: 4% 21%
IKZF1 19%
EZH2 5-13% of MPNs
P53 27%
SRSF2 19%
5-20%frequency
Mutation Frequency in Chronic Phase and Post-MPN AML
Mutated genesrelated toJAK-STATsignaling
Mutated genesoutside of the
JAK-STATpathway
Average number of acquired mutations in:PV: 6.5ET: 6.5PMF: 13
Klampfl et al,NEJM 2013
Mutations and Impact on Prognosis
• Age >65• Hb < 10 g/dL• WBC > 25,000/mm3
• Constitutional symptoms• Peripheral blood blasts >1%
• RBC transfusion dependence• Platelet count < 100,000/mm3
• Unfavorable cytogenetics
IPSS
DIPSSPlus
Prognostic Scoring Systems for Primary Myelofibrosis
PROGNOSTIC FACTORS
Cervantes et al, Blood, 2009Gangat et al, J Clin Oncol, 2011
DIPSS Plus # Adverse Points Median Survival
Low risk 0 185 months (15.4 yrs)
Intermediate-1 risk 1 78 months (6.5 yrs)
Intermediate-2 risk 2-3 35 months (2.9 yrs)
High risk 4-6 16 months (1.3 yrs)
DIPSS Plus
Gangat et al, J Clin Oncol, 2011
“High-Molecular Risk” Markers in PMF:ASXL1, EZH2, SRSF2, IDH1/2
Overall Survival Leukemia-free survival
EZH2
ASXL1
SRSF2
IDH1/2
Independentof IPSS or
DIPSS-plus
Vannucchi et al. Leukemia 2012.
“High-Molecular Risk” Markers in PMF:0, 1, or >2 mutations
0
1>2
0
1>2
Guglielmelli et al, Leukemia, 2014
Impact of CALR Mutationson Outcomes in ET / PMF
Klampfl et al,NEJM 2013
Type 1 vs Type 2 CALR mutations may have different effects on prognosis
Tefferi et al, Leukemia, 2014
Chao, Gotlib, Blood, 2014
Two Faces of ET
How does one mutation cause 3 diseases?(1) JAK2 Dependent Effects
JAK2V617F homozygosity Polycythemia Vera JAK2V617F heterozygosity Essential Thrombocytosis
(2) JAK2-Independent Effects - Co-occurring mutations
(3) Genetic background of the patient
- Variations in the DNA that one is born with that may predispose to greater susceptibility to MPN later in life
ARE TET2 MUTATIONS THE “PRE-JAK2” MUTATION?
JAK2 mutant + TET2 mutant colonies
JAK2 normalTET2 mutant colonies
No JAK2 mutantTET2 normal colonies!!
JAK2 mutant + TET2 normal CD34+
JAK2 mutant + TET2 mutant CD34+
High % engraftment
Low % engraftment
*Delhommeau et al NEJM 2009
These data suggest that TET2 mutationspreceded acquisition of JAK2 mutations in MPN patients.
TET2 MUTATIONS IN NORMAL ELDERLY INDIVIDUALS WITH CLONAL BLOOD FORMATION
A proportion of patients withclonal blood formation and noclinically apparent hematologicaldisorder have TET2 mutations.
In some cases, the acquisition ofthe TET2 mutation actually preceded development of JAK2 mutant MPN.
.
Beerman et al. Curr Opin Immunology 2010 Busque et al. Nat Genetics 2013
How does one mutation cause 3 diseases?
(1) JAK2 Dependent Effects JAK2V617F homozygosity Polycythemia Vera JAK2V617F heterozygosity Essential Thrombocytosis
(2) JAK2-Independent Effects - Co-occurring mutations
(3) Genetic background of the patient- Variations in the DNA that one is born with that may
predispose to greater susceptibility to MPN later in life or type of MPN
JAK2 V617F: One Mutation, Three Diseases: Effect of genetic background: mice example
Balb/c mice:
High red blood cell count, high white blood cell count, and myelofibrosis
C57Bl/6 mice:
High red blood cell count, normal-mildly increased white blood cell count, and fibrosis only in the spleen (not marrow)
Bumm , et al, Cancer Res, 2006Lacout, et al, Blood, 2006Wernig et al, Blood, 2006Zaleskas et al, PLoS ONE, 2006
Acquired MPN
Mutations
Inherited Variations in
DNA that PredisposeTo MPN
? EnvironmentalFactors
JAK2 46/1
TERT
TET2
JAK2 V617F
LNKEZH2
TET2MPL
ASXL1
ChronicPhaseMPN
BlastPhase
IDH 1/2
P53SRSF2
Model of MPNDevelopment
LNK
Predisposition genes identified in collaboration with 23andMe
Genetic Mutations in Diagnosis and Treatment
• JAK2 V617F, MPL, or CALR mutations establish the presence of a primary bone marrow disorder, almost always an MPN, instead of a reactive condition (e.g. infection, inflammation)
• However, the diagnosis of an MPN requires a combination of clinical, laboratory, histopathology, and mutation testing
• The majority of patients with ET and MF with non-mutated JAK2 or MPL have CALR mutations
Summary: Role of JAK2 and other mutations in the diagnosis of MPNs
In 2014:
• Treatment decisions about PV, ET, or PMF are not based on JAK2 mutation status• IPSS/DIPSS-Plus are used to risk stratify patients into low, intermediate-1/2,
and high risk groups
• JAK inhibitors demonstrate activity in myelofibrosis patients with normal or mutant JAK2
• Gene panels are becoming available in labs to evaluate for mutations in 20+ genes • May be useful:
1) in triple negative patients 2) to assess for poor-risk molecular markers: ASXL1, EZH2, SRSF2, IDH 1/2 Poor risk markers: earlier referral to transplant for intermediate-1 risk patients?
Summary: Mutations and Treatment of MPNs
Do mutations in MPN affect response to therapy?
• Examined impact of mutations on outcome in MF patients treated on COMFORT-II trial.
146 patients Ruxolitinib
73 patients best Available therapy219
patients
FrequencyJAK2 75%
ASXL1 33%TET2 11%MPL 7%EZH2 7%CBL 4%
SRSF2 3%SH2B3 1%IDH1/2 1%
Best available tx RuxolitinibLow Molecular Risk 71% pts alive 85% pts aliveHigh Molecular Risk 58% pts alive 79% pts alive
Survival at 114 weeks
**
**
**
**
** = High molecular risk (HMR) category
Guglielmelli et al, Blood, 2014
Our Patients
Stanford Division of Hematology
MPN Advocacy and Education International
Charles and Ann Johnson Foundation
AcknowledgementsStanford
Andrea Linder Jim Zehnder Cheryl Langford Jason MerkerCecelia Perkins Andy FireJenny Ma Biquan LuoCristina Williams Krishna RoskinWan-Jen Hong Mark Chao
ColleaguesRuben MesaRoss Levine
Claire HarrisonAnimesh Pardanani
Ayalew Tefferi
23andMe
David Hinds and team