Jason Gotlib, MD, MS

Associate 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 12

MPL CALR LNK

TET2ASXL1

IDH1, IDH2

DNMT3A

EZH2

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 / AML

JAK2 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