1. Which mutations matter in myelofibrosis?
Alessandro M. Vannucchi
Laboratorio Congiunto MMPC
Department of Experimental and Clinical Medicine
University of Florence, Italy

2. PMF-associated Mutations: Current Status
JAK2 V617F
MPL W515
Unmutated
CALR
Klampfl T et al. NEJM 2013; 369: ; Nangalia J et al. NEJM 2013;369:

3. JAK2 V617F Mutation and Prognosis in PMF
Overall Survival
Blast Transformation
P=0.198 WT
V617F WT
V617F
P=0.839
HR: 1.05 (95% CI, )
A JAK2 V617F mutated genotype did not impact significantly on prognosis compared with JAK2 wild type (but might be different if we consider CALR mutated and triple-negative patients separate)
N=483
Vannucchi AM et al, Leukemia 2013;27:1861-9

4. JAK2 V617F Allele Burden and Blast Transformation
IPSS prognostic score
4.33 ( )
0.007
JAK2 V617F wt or Homo vs. Hetero
2.43 (1.44-4)
0.02
Barosi G. et al. PlosOne 2013; 3:e59791

5. Prognostic Impact of CALR Mutations in PMF
Median 8.2 years
P<0.0001
MPL mut
Median 4.3
years
Triple negative
Median 2.5 years
JAK2 mut
Median 4.3 years
CALR mutated patients have better prognosis than other mutation categories
Klampfl T et al. NEJM 2013;369: ; Tefferi A et al. Leukemia Feb 5, online.

6. CALR mutations :Type 1 & Type 2
Klampfl T et al. NEJM 2013; 369: ; Tefferi A et al., Leukemia 2014; Feb 26 Epub

7. CALR Type 1 vs Type 2 Mutations in PMF
Type 1 CALR mut
Median years
Median years
JAK2 V617F mut
Median 4.0 years
P<0.0001
Tefferi A et al., Leukemia 2014; Feb 26 Epub

8. Mutation Landscape* in PMF
79.1% had at least one mutation
154 pts (32.5%) had > 2 mutations
31 pts (6.4%) had > 3 mutations
* Partial view
Vannucchi AM et al, Leukemia 2013;27(9):1861-9

9. High Molecular Risk Prognostic Category
harboring >1 mutation in any one of ASXL1, EZH2, SRSF2, IDH1/2
Overall Survival
Blast Transformation
A HMR status is associated with reduced OS and increased risk of blast transformation in PMF patients independent of IPSS/DIPPS-plus
Vannucchi AM et al, Leukemia 2013;27:1861-9

10. HMR: How many patients would be reclassified?
IPSS Risk Categories
ASXL1
N. (%)
EZH2
SRSF2
N.(%)
IDHs
N (%) Of HMR patients
LOW
24/162
(14.8%)
6/165
(3.6%)
7/151
(4.6%)
2/157
(1.3%)
35/166
(21.1%)
INT- 1
28/142
(19.7%)
6/143
(4.2%)
6/136
(4.4%)
6/142
34 /146
(23.4%)
INT- 2
23/100
(23.0%)
4/99
(4.0%)
9/97
(9.3%)
2/96
(2.1%)
31 /104
(29.8%)
HIGH
27/65
(41.5%)
8/66
(12.1%)
16/63
(25.4%)
1/60
(1.7%)
39/68
(57.3%)

11. Patients Distribution by Number of HMR Mutated Genes (no mut, 1 mut, > 2mut)
LMR
LMR
Test Cohort
N= 537
Validation Cohort
N= 292
Refers to mutations in any one of ASXL1, EZH2, SRSF2, IDH1 & 2
Guglielmelli P, et al. Leukemia 2014, Febr 19 Epub

12. Overall Survival by Number of Mutated Genes
P <
No HMR mutation
Median years
N=370
N=127
1 mutation
Median 7.0 years
HR 1.8 ( )
N=40
>2 mutations
Median 2.6 years
HR 3.8 ( )
In a Cox multivariable analysis adjusted for IPSS, the HR for OS for 2 or more mutations was 2.4 (95% CI= )
Guglielmelli P, et al. Leukemia 2014, Febr 19 Epub

13. The Impact of the Number of HMR Mutated Genes on Survival of IPSS-Stratified Patients
LOW + INT-1
INT-2 + HIGH
No mutation
Median 20.2 years
No mutation
Median 5.7 years
1 mutation
Median 4.4 years
HR 1.4 ( )
>2 mutations
Median 2.2 years
HR 2.2 ( )
>2 mutations
Median 3.2 years
HR 4.0 ( )
1 mutation
Median 11.3 years
HR 1.4 ( )
Guglielmelli P, et al. Leukemia 2014, Febr 19 Epub

14. Leukemia Free Survival by Number of
HMR Mutated Genes
P <
No mutation
Median 26.7 years
1 mutation
Median 11.1years
HR 2.6 ( )
>2 mutations
Median 6.6years
HR 6.2 ( )
N=370
N=127
N=40
The negative impact of harboring >2 mutated genes on the time to progression to blast transformation was maintained in the lower (HR 1.8; 95% CI ; P=0.061) and higher (HR 2.5; 95% CI , P=0.02) IPSS risk categories.
Guglielmelli P, et al. Leukemia 2014

15. Prognostic Relevance of the Number
of Mutated Genes
Lundberg P et al. Blood 2013; Jan 29

16. CALR and ASXL1 Mutations-Based Prognostic Risk Stratification
Mayo Clinic, n=277
Florence, n=293
Overall survival
Tefferi A et al, Leukemia 2014, Feb 5,online.

17. CALR and ASXL1 Mutations-Based Risk Stratification
According to IPPS Categories
IPSS LOW + INT1
IPSS INT2 + HIGH
CALR- ASXL1+
Median 6.03 years
HR 18.7 ( )
Median 3.1 years
HR 3.9 ( )
CALR- ASXL1-
CALR+ ASXL1+
Median n.r.
HR 5.4 ( )
Median 3.7 years
HR 3.0 ( )
CALR+ ASXL1-
P<0.001
P=0.07
CALR/ASXL1 mutations-based prognostic model was DIPSS-plus independent (p<0.0001)
Tefferi A et al, Leukemia 2014, Feb 5,online.

18. Patients’ Mutation Profile in COMFORT-II Trial
150 pts (90.4%) presented with at least one mutation
There was no difference in the frequency of individual mutations between ruxolitinib and BAT arms
Overall, 46 patients in the ruxo arm (38.3%) and 20 patients (43.5%) in the BAT arm were classified as "high molecular risk" (HMR)
Mutations in %
JAK2
75.5
ASXL1
32.5
TET2
10.7
MPL
7.7
EZH2
7.2
CBL
4.4
SRSF2
3.0
KRAS
1.4
SH2B3
1.3
IDH1
0.7
SOCS1
SOCS2
IDH2
>4 0
No.
mutations
BAT, best available therapy
Guglielmelli P, et al. Blood 2014

19. HMR
LMR
Spleen volume reduction
of >35% from baseline
48 weeks
24 weeks
Mean spleen volume % change
from baseline
-23.5
-30.6
-29.0
-29.9
Proportion of patients (%)
Spleen Response by Molecular Status in Patients Receiving Ruxolitinib in COMFORT-II
HMR status did not impact on symptomatic improvement
HMR status did not increase the risk of developing anemia or thrombocytopenia under ruxolitinib treatment
Guglielmelli P, et al. Blood 2014, Jan 23

20. Symptomatic Improvement by Molecular Status in Patients Receiving Ruxolitinib in COMFORT-II
HMR
LMR
Proportion of patients (%)
48 weeks
24 weeks
None of the individual mutations with a frequency >2% correlated with symptomatic improvement
Guglielmelli P, et al. Blood 2014, Jan 23

21. Hematologic Toxicity by Molecular Status in Patients Receiving Ruxolitinib in COMFORT-II
HMR
LMR
Proportion of patients (%)
Anemia
Thrombocythopenia
Mutations of genes of the JAK/STAT signalling pathway (JAK2, MPL, SH3B2, CBL, SOCSs) did not correlate with development of anemia or thrombocytopenia at 48 wk of treatment
Guglielmelli P, et al. Blood 2014, Jan 23

22. Survival Estimates in Patients Stratified by Treatment and Molecular Score
Ruxolitinib arm§
BAT arm§
HMR
LMR
0.79
0.85
0.58
0.71
In multivariate analysis of overall survival by treatment and molecular risk, the HR for treatment (ruxolitinib vs BAT) was 0.57 (95%CI= ) and for LMR vs HMR the HR was 0.62 (95%CI= )
§Median follow up= 151 weeks; Kaplan Meier estimates at 144 weeks
Guglielmelli P, et al. Blood 2014, Jan 23

23. Therapeutic Efficacy of JAK2 Inhibitor Fedratinib
in CALR Mutated PMF Patients
Passamonti F et al, NEJM 2014; 370:1168-9

24. Which Mutations Matter in Myelofibrosis (take home message)
A mutated CALR status delineates a better outcome in PMF patients compared with JAK2/MPL mutated and “triple negative”
A IPSS/DIPSS-plus independent “High-Molecular Risk” (HMR) condition is defined by the presence of any one mutated gene among EZH2, ASXL1, SRSF2 and IDH1/2
ASXL1 and CALR contribute to refine prognostic stratification
The number of (HMR-) mutated genes is an additional determinant of survival
Clinical response to JAK2 inhibitors is independent of HMR category (ruxolitinib) and CALR mutations (fedratinib)

25. Which Mutations Matter in Myelofibrosis (work to do for the future)
How to integrate all these information in an unified molecular/clinical scoring system

26. Acknowledgments Nick Cross, Amy Jones Salisbury & Southampton, UK
Tony Green, Jyoti Nangalia
Cambridge, UK
Robert Kralovics, Thorsten Klampfl
CERMM, Wien, Austria
Ayalew Tefferi, Animesh Pardanani
Mayo Clinic, Rochester, US
William Vainchenker, Jean Luc Villeval
Inst G. Roussy, Paris, France
Niccolò Bartalucci
Costanza Bogani
Laura Calabresi
Tiziana Fanelli
Rajmonda Fjerza
Ilaria M. Farina
Paola Guglielmelli
Carmela Mannarelli
Serena Martinelli
Lucia Merli
Annalisa Pacilli
Alessandro Pancrazzi
Chiara Paoli
Lisa Pieri
Giada Rotunno
Gianni Barosi
Mario Cazzola
Rossella
Manfredini
Alessandro Rambaldi,
Tiziano Barbui
Daniela Cilloni
Elisabetta Dejana