1. Fisiopatologia, diagnosi e classificazione delle trombocitemie
Alessandro M. Vannucchi
UF di Ematologia
Università degli Studi, Firenze

2. Kaushansky K. N Engl J Med 2006;354:2034-2045

3. Classification of Thrombocytosis
Primary Thrombocytosis
Inherited, familial
Acquired, clonal thrombocytosis
Chronic myeloproliferative neoplasms
Chronic myelogenous leukemia
Myelodysplastic syndromes (5q- and RARS-T)
Secondary (Reactive) Thrombocytosis
Acute & transient
Sustained
Life-long
Acute blood loss
Recovery from
thombocytopenia
Acute inflammation or
infection
Response to exercise
Iron deficiency
Hemolytic anemia
Chronic inflammatory or
infectious disease
Cancer
Drug reactions
Splenectomy
Splenic agenesia
(based on pathogenetic mechanism)
Schafer AJ, NEJM 2004

4. Fisiopatologia, diagnosi e classificazione delle trombocitosi primarie
Fisiopatologia della trombosi nella ET

5. Fisiopatologia, diagnosi e classificazione delle trombocitosi primarie
Fisiopatologia della trombosi nella ET

6. Meccanismi molecolari: mutazioni di JAK2 e MPL
Stat5
SOCS1
SOCS3
PI3K
AKT
mTOR
FOXO
Ras
Raf
MEK
ERK
Activation of genes
(proliferation/survival/differentiation)
EPOR
MPL
G/GM-CSFR
IL3R
IL5R
GP130
JAK2
V617F
Exon12
W515L/K
Mpl

7. Only the MPL W515 mutations promote a G(1)/S-phase transition
Chaligne, R. Leukemia 2008

8. Chronic Ph1-neg myeloproliferative neoplasmsPV ET
PMF
JAK2 V617F
≈99 %
≈60%
MPL W515L/K
8%* 8%
JAK2
exon 12
≈50% * nd
* Referred to V617F-negative

9. A PV, rather than an ET phenotype, is reproduced in
JAK2 V617F mice
BM cells showed hypersensitivity of CFU-E to EPO, with 25-30% of CFU-E being EPO-independent. The number of BFU-E was much lower in these mice, suggesting that V617F acts in the most-differentiated erythroid progenitors to promote EPO_independent maturation. THE FACT THAT CENTRAL ERYTHROID FEATURE SOF PV ARE RECAPITULATED BY V617F ARGUES THAT IT IS THE PRIMARY AND DIRECT CAUSE OF PV.
The effects on leukocytes were more variable and strain-dependent.
No increase in plt count despite evidence of proviral integration in Mks, but there was a marked defect in plt function
James C, Nature 2005;Wernig G, Blood 2006; Lacout C, Blood 2006; Zaleskas V, PLoS ONE 2006

10. Ratio of mutant JAK2-V617F to wild-type Jak2 determines the MPD phenotypes in transgenic mice
Transgenic line (FF1-Vav) with an expression of JAK2-V617F lower than the endogenous wild-type JAK2 developed a phenotype resembling ET with strongly elevated platelet counts and moderate neutrophilia.
Transgenic JAK2-V617F/MxCre mice had expression of JAK2-V617F approximately equal to wild-type JAK2 and developed a PV-like phenotype with increased hemoglobin, thrombocytosis, and neutrophilia.
Higher levels of JAK2-V617F in mouse bone marrow by retroviral transduction caused a PV-like phenotype without thrombocytosis.
Tiedt, R. et al. Blood 2008;111:

11. JAK2 V617F allele burden in MPNs20 40 60 80
100
JAK2
V617F
allele burden (%) PV ET
PMF
postPV / ET MF
Antonioli E, Haematologica 2007

12. Identification of disease subtypes in ET based on JAK2 V617F mutation
JAK2V617F pos ET resembles PV:
- increased erythropoiesis
- higher leukocyte count
- more venous thromboses
- more frequent PV “transformation”
- lower ferritin (p < 0.01)
- lower MCV (p < )
- lower serum Epo (p < )
JAK2V617F neg ET
- higher platelet counts
Campbell et al Lancet 2005& Blood 2005; Antonioli et al Leukemia 2005;

13. Induction of thrombocytosis and myelofibrosis by MPLW515L
MPLWT
MPLW515L

14. Clinical correlates of MPL W515 mutations in ET
mutant
wild-type P
value
Median age, years
58 (22-84)
52 (8-97)
0.08
White blood cell count (x109/L)
8.8 ± 3.0
9.3 ± 2.7
0.6
Hemoglobin (g/L)
134 ± 13
140 ± 11
0.02
Hematocrit (%)
40.6 ± 3.8
42.0 ± 4.1
0.04
Platelet count (x109/L)
960 ± 327
860 ± 309
Splenomegaly, no-(%)
6 (19%)
178 (21%)
0.9
Major thromb, no-(%)
10 (32%)
172 (19%)
0.1
Microvessel dis no-(%)
19 (61%)
276 (30%)
0.0005
Major hemorrh no-(%)
3 (10%)
35 (3.6%)
994 ET pts
3.0%
418
V617Fneg
8.1%
12 (40%) W515K
18 (60%) W515L
In 8 pts both MPLW515 and JAK2V617F
AM Vannucchi et al., Blood, 2008

15. Essential Thrombocythemia
WHO 2008 DIAGNOSTIC CRITERIA
Essential Thrombocythemia
Major
criteria
1. Sustained platelet count ≥ 450 x 109/L
2. Bone marrow biopsy showing proliferation mainly of the megakaryocytic lineage with increased numbers of enlarged mature megakaryocytes. No significant increase or left shift of neutrophil granulopoiesis or erythropoiesis
3. Not meeting WHO criteria for CML, PV, PMF, MDS or other myeloid neoplasm
4. Demonstration of JAK2V617F or other clonal marker
or no evidence of reactive thrombocytosis
Minor
Diagnostic combinations
All four criteria must be met
*, Tefferi A, 2007

16. ST ET PMF PV RARS-T 5q- CML 100% 60% 1- 4% 40% 60% 5- 8% 40%
60% 1- 4% 40%
PMF
60% 5- 8% 40% PV
95%
RARS-T
50%
50%
5q-
 5%
95%
CML
100%
Vannucchi AM, ASH2007

17. Fisiopatologia, diagnosi e classificazione delle trombocitosi primarie
Fisiopatologia della trombosi nella ET

18. Thrombosis-free survival in MPN
Crude-analysis
Years
Probability
A GIMEMA retrospective study on 1,224 patients 67
397
339
275
235
206
168
139
120 95 82 PV
Pts at risk
140
637
549
479
421
365
316
262
220
191
169 ET 28
190
123 94 78 68 60 48 40 36 30 MF
Years
Risultati di uno studio retropse4ttivo su oltre 1000 pz con MPD, multicentrioc, italiano, che è una fotografia dello stato attuale con le terapie attuali
Vannucchi AM, Blood 2007 & unpubl.

19. Recurrent Thrombosis in PV or ET
Time after first thrombosis (months)
5.6%pt-yr
3.4%pt-yr
2.2%pt-yr
Cumulative probability (%)
49.9%
ET (n=259) PV (n=235)
n=494
A retrospective GIMEMA study on 494 pts with first thrombosis
La probabilita’ cumulativa di ricorrenza era del 17.7% a 2 anni, 30.8% a 5 anni, de.l 49.9% a 10 anni
De Stefano V, Haematologica 2007

20. Cardiovascular risk factors (*)
Risk Stratification in ET
Risk category
Age >60 or
history of thrombosis
Cardiovascular risk factors (*)
Low NO
Intermediate
YES
High (**)
----
Although their contribution to thrombotic risk is controversial, it is apporpiate counselinmg patients as concerns style of life, particularly smoking, and eventually correcting associated abnormalities such as hypertension or diabetes
(*) Hyperthension, Hypercholesterolemia, Diabetes, Smoking; Familial thrombophilia.
Their contribution to thrombotic risk is controversial

21. Thrombocytosis and Thrombosis: an Uneven Relationship - 1
Finding
Clonal
Thrombocytosis
Secondary
Digital or cerebrovascular ischemia
Characteristic No
Large vessel arterial or venous
thrombosis
Increased risk
Bleeding complications
EVEN BEYIND mpdS
Schafer AJ, NEJM 2004

22. Thrombocytosis and Thrombosis: an Uneven Relationship - 2
ECLAP cohort
In a series of 99 young (16-59) ET pts, receiving or not prophylactic cyroreductive therapy, no difference in thrombosis (nor hemorrhages) was noted
Berk PD et al, 1995; Di Nisio M et al, BJH 2006; Tefferi A et al, Blood 2006

23. Thrombocytosis and Thrombosis: an Uneven Relationship - 3
Platelet count* (x109/L)
Hazard Ratio (95% CI) p-value
650 to 1,000
More than 1,000
Total thrombosis
1) Unadjusted
0.7
( )
0.5
( )
P= 0.03
2) Variables adjusted
0.6
( )
( )
P=0.01
3) +JAK2V617F adjusted
( )
Carobbio A, Blood 2008

24. Thrombocytosis and Thrombosis: an Uneven Relationship - 4
MRC PT-1 Trial
Harrison C et al, NEJM 2005

25. Leukocytosis and Thrombosis in ET
Wolanskyi A, Mayo Clin Proc, 2006
WBC (x109/L)
HR (95% CI)
P (*)
>15.0
1.74 ( )
0.01
Carobbio A, Blood 2007
WBC (x109/L)
HR (95% CI)
P (*)
<7.2 1
2.4 ( )
0.04
2.7 ( )
0.02
>10.4
3.0 ( )
0.008
Carobbio 439 pts: count at diagnosis precits for thrombosis in the follow-up; Wolanskyj 322
Unlike Carobbio, Tefferi et al bllod (2007, letter) found an association of leukocytosis with both Arterial and Venous events at diagnosis, BUT NOT in follow-up unlike Carobbio. However, he did not perform multivariable time-dependent analysis.
(*) Multivariable analysis

26. Leukocytosis and “conventional” risk factors in ET
Low-risk
High-risk
P=.003
Cumulative probability of thrombosis in the follow-up
Carobbio A et al, Blood 2007; JCO 2008

27. Rate of CV events (% pts/year)
Interaction of leukocytosis and thrombocytosis on thrombotic risk in ET
WBC
Platelet count
Rate of CV events (% pts/year)
JAK2V617F mutated (%) RR
(P=)
<11,000
>1,000
1.59 26 1
<1,000
2.26 56
1.92
(0.034)
>11,000
2.88 61
2.38
(0.026)
2.95 77
2.43
(0.017)
Carobbio A, Blood 2008

28. V617F Status and Thrombosis in ET
Venous thrombosis
Arterial thrombosis
Thrombosis at presentation
2.09 [1.44, 3.05]
1.96 [1.43, 2.67]
1.88 [1.38, 2.56]
Ziakas PD et al, Haematologica 2008

29. V617F Burden and Thrombosis in ET
(95%CI) P WT 1
Hetero
1.49
( )
0.13
Homo
3.97
( )
0.013 *
adjusted for age, sex, previous thrombosis and leukocytosis
Vannucchi AM et al, Blood 2007

30. Interactions between JAK2 V617F genotype and inherited thrombophilia
132 ET pts, 45 pts (34%) with major thrombosis.
83 pts (62.8%) V617F+, 8 pts (6%) were Homo RR
JAK2 V617F+
2.1 ( )
Homo
3.7 ( )
Hetero
1.9 ( )
Homo vs Hetero
1.9 ( ) RR
JAK2+ Thr’ph’+
vs WT Thr’ph’-
4.4 ( )
JAK2+ Thr’ph’-
2.1 ( )
vs JAK2+Thr’ph’-
2.1 ( )
De Stefano V, EHA 2008

31. Acknowledgments Tiziano Barbui Chairman of the GIMEMA-MPD WP
and all the colleagues of the WP
Paola Guglielmelli, Elisabetta Antonioli,
Lisa Pieri, Alberto Bosi
Dip. Ematologia, Firenze