Raymond Wong is a consultant at the Department of Medicine & Therapeutics at Prince of Wales Hospital in Hong Kong. He received his medical degree and training in hematology and internal medicine from the Chinese University of Hong Kong. Dr. Wong has published over 70 articles in peer-reviewed journals on hematology and therapeutics. The presentation discusses the revised classification of myeloproliferative neoplasms and prognostication based on mutations in CALR, JAK2, and MPL. CALR mutations are found in the majority of essential thrombocythemia and primary myelofibrosis patients without JAK2 or MPL mutations. Patients with CALR mutations tend to have less severe disease characteristics compared to those with JAK2
1. RAYMONDWONG,MD
HongKong
• Consultant of the Department of Medicine & Therapeutics,
Prince of Wales Hospital
• Dr. Wong received his medical degree from the Chinese
University of Hong Kong and received his training in
Haematology, Internal Medicine as well as Clinical
Pharmacology and Therapeutics at the Prince of Wales
Hospital, Hong Kong. He also obtained his Doctor of Medicine
from the Chinese University of Hong Kong. He joined the
Prince of Wales Hospital Poison Treatment Centre since its
establishment in 2005 which is a tertiary referral centre for
the management of patients with poisoning. Dr. Wong has
published over 70 articles in peer-reviewed journals including
Blood, Circulation, JAMA and the New England Journal of
Medicine in various areas of haematology and therapeutics.
4. 2008 WHO diagnostic criteria for Ph- MPNs
Diagnosis:
Both major + 1 minor
OR
1st major + 2 minor
All 4 majors All 3 major + 2 minor
5. Incidence of JAK2 mutations
JAK2 V617F
JAK2 ex12
?
JAK2 V617F JAK2 V617F
PV ET MF
N Cross. Hematology Am Soc Hemat Educ Program 2011: 208-14
6. Alternative mechanisms of JAK2 activations
97% JAK2 V617F
2% JAK2 ex12
1% ?
60% JAK2 V617F
3% MPL ex10
3% SH2B3 (LNK)
34% ?
60% JAK2 V617F
5% MPL ex10
3% SH2B3 (LNK)
5% CBL
27% ?
PV ET MF
N Cross. Hematology Am Soc Hemat Educ Program 2011: 208-14
7. Calreticulin (CALR)
• In December 2013, two groups
reported the occurrence of novel
calreticulin (CALR) mutations in
JAK2/MPL-unmutated PMF or ET
• CALR is a multi-functional Ca2+-binding
protein chaperone mostly localized in
the endoplasmic reticulum (ER)
• CALR is located on chromosome
19p13.2, contains 9 exons and its
protein three domains
Ellgaard, L. & Helenius, A. Nature Reviews Molecular Cell Biology 4, 181–191 (2003)
10. Somatic Mutations of
Calreticulin in MPNs
• Among patients with nonmutated
JAK2 or MPL, CALR mutations
were detected in
• 67% of those with ET
• 88% of those with PMF
• A total of 36 types of insertions or
deletions were identified: all
cause a frameshift to the same
alternative reading frame and
generate a novel C-terminal
peptide in the mutant calreticulin
Klampfl et al. NEJM 2013
11. CALR, compared with JAK2 mutations:
ET
• ↓ hemoglobin level
• ↓ leukocyte count
• ↑ platelet count
• ↓ risk of thrombosis
• Better survival (P = 0.04)
MF
• ↓ leukocyte count (P = 0.03)
• ↑ platelet count (P<0.001)
• Better survival (P<0.001)
P<0.001
Klampfl et al. NEJM 2013
The clinical course in patients with CALR mutation was more
indolent than that in patients with the JAK2 V617F mutation
17. Somatic CALR Mutationsin MPNs with Non-mutated JAK2
• In ET, an association between CALR mutations and
• higher platelet count
• lower hemoglobin level
• In addition, the study also suggested an increased
incidence of fibrotic transformation in CALR-mutated
ET without apparent survival difference
Nangalia J, et al. NEJM 2013
18. • 168 MPN patients (PV = 36, ET = 114, PMF = 18)
Ann Lab Med 2015;35:22-27
19. CALR Exon 9 Mutations in MPNs
• ET patients with CALR mutation had lower leukocyte counts
and ages compared with JAK2-mutated ET patients
Ann Lab Med 2015;35:22-27
20. • 576 patients with WHO-defined ET
• In JAK2/MPL-unmutated cases, CALR mutational frequency was 49%
Blood. 2014;123(10):1552-1555
CALR+ JAK2 V617F+ MPL W515+ CALR, JAK2, MPL wt
% of patients 15.5% 64.1% 4.3% 16.1%
CALR+ vs JAK2 V617F+ CALR+ vs MPL W515+ CALR+ vs. triple negative
• Male
• Younger age
• Lower leukocyte count
• Lower hemoglobin level
• Higher platelet count
• Male • Male
21. CALR mutations in ET
• CALR-mutated and triple-
negative cases displayed superior
thrombosis-free survival
• CALR mutation had no impact on
survival or transformation to
post-ET myelofibrosis
Blood. 2014;123(10):1552-1555
23. JAK2 or CALR mutation in ET
• Compared with JAK2-mutated cases, CALR-mutated patients
were:
• younger
• ↓ leukocyte count
• ↓ hemoglobin
• ↑ platelet count
Blood. 2014;123(10):1544-1551
24. JAK2 or CALR mutation in ET
CALR- vs JAK2-mutated patients
• No difference in OS, risk of
leukemic or fibrotic
transformation
• Better thrombosis-free survival
25. CALRexon 9 frameshiftmutationsin patientswith
thrombocytosis(not confirmedET)
• In 289 patients referred for evaluation of persistent thrombocytosis
Chi J, et al. Leukemia 2014
26. CALRvs JAK2vs MPL-mutatedor triple-negativeMF
• 254 MF patients who were cytogenetically characterized and screened for
several MPN-characteristic mutations including ASXL1, EZH2, IDH and
spliceosome mutations (SF3B1, SRSF2 and U2AF1)
• In JAK2/MPL-unmutated cases, CALR mutational frequency was 74%
• Patients with CALR mutations:
• younger
• higher platelet count
• lower DIPSS-plus score
• less likely to be anemic, require transfusions or display leukocytosis
Tefferi A, et al. Leukemia (2014) 28, 1472–1477
CALR+ JAK2 V617F+ MPL W515+ CALR, JAK2, MPL wt
% of patients 25% 58% 8% 9%
27. CALR vs JAK2 vs MPL-mutatedor triple-negativeMF
• CALR mutations had a favorable survival
independent of DIPSS-plus risk and
ASXL1 mutation status
• Triple-negative patients displayed
inferior leukemia-free survival
• “CALR–ASXL1+” and “triple-negative”
mutation profiles are prognostically
detrimental
Tefferi A, et al. Leukemia (2014) 28, 1472–1477
28. CALR-mutated patients (vs. JAK2)
Essential Thrombocythemia
• younger age
• male sex
• higher platelet count,
• lower hemoglobin level
• lower leukocyte count
• lower incidence of
thrombotic events
Primary Myelofibrosis
• younger
• higher platelet count
• better risk profile
• higher hemoglobin
• lower leukocyte count
• less spliceosome mutations
Tefferi A, et al. AJH 2015
29. Frequencyof JAK2, CALR, andMPLmutationsin MPNs
96%
3%
55% 65%
4%
8%
15-24% 25-35%
Nanglia J and Green R, Hematology 2014; Tefferi A and Barbui T, AJH 2015
30. Diagnostic algorithm for BCR-ABL1-negative MPNs
• Genotyping for CALR could be a useful diagnostic tool for JAK2-or MPL-
negative ET or PMF patients in conjunction with BM morphology
Tefferi A, et al. AJH 2015
31. ProposedRevised ET Diagnostic Criteria
2008 (Current) Proposed Revision
Tefferi A, et al. Leukemia 2014
All 4 major or first 3 major + 1 minorAll 4 major criteria
32. ProposedRevised MF Diagnostic Criteria
2008 (Current) Proposed Revision
Tefferi A, et al. Leukemia 2014
All 4 major or first 2 major + all minorAll 3 major criteria + 2 minor criteria
33. “MaskedPV”
• JAK2-mutated patients who display PV-characteristic BM
morphology but display hemoglobin levels 16-18.5 g/dl for men
and 15-16.5 g/dl for women
Barbui T, et al. AJH 2013
34. A. Thrombosis-free survival
B. Myelofibrosis / leukemia-
free survival
C. overall survival
in masked and overt PV
“MaskedPV”
35. ProposedRevised PV Diagnostic Criteria
2008 (Current) Proposed Revision
Tefferi A, et al. Leukemia 2014
Both major + 1 minor
1st major + 2 minor
All 3 major OR first 2 major + minor
36. Summary
• The molecular diagnostic gap in JAK2/MPL-unmutated ET/PMF
is now partially addressed by the recent discovery of
calreticulin (CALR) mutations in the majority of such cases
• Genotyping for CALR could be a useful diagnostic tool for JAK2-
or MPL-negative ET or PMF patients. BM marrow morphology
remains the central diagnostic platform
• CALR mutation may be a distinct disease group, with different
clinical and hematological characteristics than that of JAK2-
mutated patients
• Inclusion of CALR mutations in the WHO classification system
for ET/PMF has been proposed
CALR mutations were not seen in 382 cases of PV but were detected in 25% of patients with ET (n=311) and 35% of those with PMF (n=203)
Two variants constituted more than 80% of the CALR mutations seen: type 1 variant (p.L367fs*46) resulted from 52 bp deletion and was more frequent in PMF, and type 2 variant (p.K385fs*47) resulted from 5-bp TTGTC insertion.
Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a
majority of patients with myeloproliferative neoplasms with nonmutated JAK2
Where do CALR mutations fit in the above scheme? CALR mutations are frequent in JAK2/MPL-unmutated ET/PMF (estimated at 49% in strictly WHO-defined ET and 74% in WHO confirmed PMF) and thus provide a much needed clonal marker in such cases.
Genotyping for CALR could be a useful diagnostic tool for JAK2-or MPL-negative ET or PMF patients.
