1. GENETICS & biology OF MYELOPROLIFERATIVE NEOPLASMS
Jason Gotlib, MD, MS
Associate Professor of Medicine (Hematology) Stanford Cancer Institute
MPN Advocacy and Education International
Patient Symposium, San Mateo, CA
May 22, 2014

2. 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

3. 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.

4. Normal Human Blood Development (Hematopoiesis) MPNs, and Stem Cells
Progenitor Cell
CD34+CD38+
Mature
MPN
Cells
CD34-
modified from Tan et al., 2006

5. 2005: identification of jak2 v617F
Lancet
Nature
Cancer
Cell
NEJM

6. JAK2 V617F Mutation Frequency
Polycythemia Vera Essential Thrombocythemia Primary Myelofibrosis
95-98% % %
Exon 12 JAK2 ~2%
JAK2 gene

7. JAK-STAT Signaling
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
EPO or TPO
Receptor
Shuai, K. & Liu,B. (2003) Nature Reviews Immunology 3:900

8. JAK2 V617F Mutation Acquired Arises in blood stem cells
EPO or TPO
Receptor
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

9. Mutations in other genes besides JAK2 cause
activated JAK-STAT signaling in MPNs
LNK
(PV, ET, MF)
<5%
MPL
1-5% ET
5-10% PMF
CBL
~6%
(PMF)
Oh and Gotlib, Exp Rev Hematol, 2010

10. ASH 2013: CALR mutations in non-mutated JAK2 ET and MF patients
‘Triple negative’
10%
MPL Mutant 5%
JAK2 exon 12
mutant
50-60%
JAK2V617F
mutant
30-40%
CALR
????
50-60%
JAK2V617F
mutant
97%
JAK2V617F
mutant
Polycythemia
Vera
Essential Thrombocythemia
Primary Myelofibrosis
Kralovics & Green labs, ASH 2013

12. Referred to as ‘indels’
Mutations in the CALR
gene all occur in one
region (exon 9)
Two most common
mutations in the
CALR gene:
Type 1: 52-bp deletion
Type 2: 5-bp insertion
Referred to as ‘indels’
5-bp insertion
52-bp deletion
Klampfl et al,
NEJM 2013

13. (but not previously known to be relevant
Normal Functions of CALR in Cells
CALR
Mutation
Calcium
Regulation in the cell
Protein folding
Cell
adhesion
Immune-mediated
cell death
Programmed
cell removal
Activation of
JAK-STAT
signaling
(but not previously known to be relevant
to this pathway)
CALR Functions

14. Both JAK2- and CALR-mutated MPN patients show a gene
expression signature associated with activated JAK-STAT signaling
Rampal et al,
Blood, 2014

15. Mutations in genes outside of the JAK-STAT pathway in MPN patients
JAK2 V617F
JAK-STAT
Pathway
CBL JAK2 exon 12
MPL CALR
LNK
TET2
ASXL1
IDH1, IDH2
Outside of
JAK-STAT
Pathway
DNMT3A
EZH2
SRSF2

16. Mutation Frequency in Chronic Phase
and Post-MPN AML
Gene
Chronic Phase
Blast Phase / AML
JAK2 V617F
PV: 98%; ET /PMF: %
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
EZH2
5-13% of MPNs
P53
27%
SRSF2
Mutated genes
related to
JAK-STAT
signaling
5-20%
frequency
Mutated genes
outside of the
JAK-STAT
pathway

17. Average number of acquired mutations in:
PV:
ET:
PMF: 13
Klampfl et al,
NEJM 2013

18. Mutations and Impact on Prognosis

19. IPSS Prognostic Scoring Systems for Primary Myelofibrosis DIPSS Plus
PROGNOSTIC FACTORS
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
DIPSS
Plus
Cervantes et al, Blood, 2009
Gangat et al, J Clin Oncol, 2011

20. DIPSS Plus DIPSS Plus # Adverse Points Median Survival Low risk
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)
Gangat et al, J Clin Oncol, 2011

21. “High-Molecular Risk” Markers in PMF:
ASXL1, EZH2, SRSF2, IDH1/2
Overall Survival
Leukemia-free survival
EZH2
ASXL1
Independent
of IPSS or
DIPSS-plus
SRSF2
IDH1/2
Vannucchi et al.
Leukemia 2012.

22. “High-Molecular Risk” Markers in PMF:
0, 1, or >2 mutations 1 1
>2
>2
Guglielmelli et al, Leukemia, 2014

23. Impact of CALR Mutations
on Outcomes in ET / PMF
Klampfl et al,
NEJM 2013

24. Type 1 vs Type 2 CALR mutations
may have different effects on prognosis
Tefferi et al, Leukemia, 2014

25. Two Faces
of ET
Chao, Gotlib, Blood, 2014

26. 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

27. Are TET2 mutations the “pre-JAK2” mutation?
JAK2 mutant +
TET2 normal CD34+
TET2 mutant CD34+
High % engraftment
Low % engraftment
JAK2 mutant +
TET2 mutant colonies
JAK2 normal
No JAK2 mutant
TET2 normal colonies!!
These data suggest that TET2 mutations
preceded acquisition of JAK2 mutations in MPN patients.
*Delhommeau et al NEJM 2009

28. TET2 mutations in normal elderly individuals with clonal blood formation
A proportion of patients with
clonal blood formation and no
clinically apparent hematological
disorder have TET2 mutations.
In some cases, the acquisition of
the TET2 mutation actually
preceded development of
JAK2 mutant MPN. .
Beerman et al. Curr Opin Immunology 2010
Busque et al. Nat Genetics 2013

29. 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

30. 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, 2006
Lacout, et al, Blood, 2006
Wernig et al, Blood, 2006
Zaleskas et al, PLoS ONE, 2006

31. Predisposition genes identified in collaboration with 23andMe
Model of MPN
Development
? Environmental
Factors
Inherited
Variations in
DNA that Predispose
To MPN
Acquired
MPN
Mutations
Chronic
Phase
MPN
JAK2 V617F
Blast
Phase
JAK2 46/1
LNK
ASXL1
TERT
EZH2
LNK
TET2
TET2
MPL
IDH 1/2
Predisposition genes identified in collaboration with 23andMe
SRSF2
P53

32. Diagnosis and Treatment
Genetic Mutations in
Diagnosis and Treatment

33. Summary: Role of JAK2 and other mutations in the diagnosis of MPNs
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

34. Summary: Mutations and Treatment 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?

35. Do mutations in MPN affect
response to therapy?
Examined impact of mutations on outcome in MF patients treated on COMFORT-II trial.
Frequency
JAK2
75%
ASXL1
33%
TET2
11%
MPL 7%
EZH2
CBL 4%
SRSF2 3%
SH2B3 1%
IDH1/2
73 patients best
Available therapy **
** = High molecular risk (HMR) category
219
patients
146 patients
Ruxolitinib
Survival at 114 weeks
Best available tx
Ruxolitinib
Low Molecular Risk
71% pts alive
85% pts alive
High Molecular Risk
58% pts alive
79% pts alive
Guglielmelli et al, Blood, 2014

36. Acknowledgements Our Patients Stanford Division of Hematology
Andrea Linder Jim Zehnder
Cheryl Langford Jason Merker
Cecelia Perkins Andy Fire
Jenny Ma Biquan Luo
Cristina Williams Krishna Roskin
Wan-Jen Hong Mark Chao
Colleagues
Ruben Mesa
Ross Levine
Claire Harrison
Animesh Pardanani
Ayalew Tefferi
23andMe
David Hinds
and team
Our Patients
Stanford Division of Hematology
MPN Advocacy and Education International
Charles and Ann Johnson Foundation