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Keywords:

  • JAK2V617F;
  • thrombosis;
  • splanchnic vein thrombosis;
  • polycythaemia rubra vera;
  • essential thrombocythaemia

Summary

  1. Top of page
  2. Summary
  3. JAK2V617F and myeloproliferative disorders
  4. Predicting thrombotic events in MPD
  5. JAK2V617F and thrombosis in MPD
  6. JAK2V617F in the absence of known MPD
  7. Conclusion
  8. References

Since the discovery of the JAK2V617F mutation, the clinical and pathological consequences of this acquired defect have been extensively investigated to determine whether its presence characterises a distinct subgroup of myeloproliferative disorders (MPD). MPD management remains highly dependent on the patient’s thrombotic risk. Whether the presence of the JAK2V617F mutation modifies the thrombotic risk is currently contentious, although there is increasing clinical evidence to suggest that the mutation may be variably associated with thrombosis. These observations are further supported by laboratory parameters which suggest that the JAK2V617F mutation may confer increased activation of leucocytes and platelets in MPD. The role of screening for the JAK2V617F mutation in patients presenting with thrombosis without overt MPD is unclear, but appears justified in cases of idiopathic splanchnic vein thrombosis.


JAK2V617F and myeloproliferative disorders

  1. Top of page
  2. Summary
  3. JAK2V617F and myeloproliferative disorders
  4. Predicting thrombotic events in MPD
  5. JAK2V617F and thrombosis in MPD
  6. JAK2V617F in the absence of known MPD
  7. Conclusion
  8. References

Essential thrombocythaemia (ET) and polycythaemia vera (PV) are the commonest Philadelphia-negative myeloproliferative disorders (MPD). Their importance lies in their predisposition to thrombosis, particularly arterial thrombosis, and the somewhat rarer tendency to transform to acute leukaemia and myelofibrosis. Current management recommendations emphasise the importance of prognostic stratification, especially with regard to thrombotic risk, in determining the start and nature of therapy.

In early 2005, five groups reported the presence of an acquired point mutation, 1849G>T, in the Janus kinase 2 (JAK2) gene (JAK2) in the majority of patients with Ph-negative MPD (Baxter et al, 2005; James et al, 2005; Kralovics et al, 2005a; Levine et al, 2005; Zhao et al, 2005). JAK2, a cytoplasmic tyrosine kinase, is critically important in relaying signalling between activated type 1 cytokine receptors, such as the erythropoietin receptor, and intracellular proliferation mechanisms. The G1849T substitution results in a valine to phenylalanine change at position 617 (V617F) of the JAK2 molecule, within its auto-inhibitory pseudo kinase domain; this results in cytokine-independent activation, uncontrolled downstream signalling, and eventually to unregulated cell proliferation. Stronger JAK2 activation can also occur secondary to a range of exon 12 mutations which have been described in less than 5% of PV patients (Scott et al, 2007).

JAK2V617F is detectable in 90–95% of cases of PV, but only 50–60% of ET cases, prompting speculation as to whether the JAK2V617F-negative ET cases represent a distinct pathological entity, with a different natural history, than the JAK2V617F-positive ones. It has been shown that the latter typically have a higher mean haemoglobin concentration at presentation, a higher leucocyte count, but a lower platelet count and ferritin concentration, leading to suggestions that JAK2V617F-positive ET is a forme fruste of PV, or is at least located along a continuum, at one end of which lies PV, and, at the other, JAK2V617F-negative ET (Campbell et al, 2005). As PV is accepted to have a higher thrombotic risk than ET, the possibility that patients who were JAK2V617F-positive might be at higher risk of thrombotic complications than those who were not, was soon raised.

The identification of a biological marker for ‘high-risk’ patients with MPD would be of great clinical utility: ET and PV comprise a heterogeneous group of chronic diseases, and their treatment may last for decades and, in some cases, cause side-effects more significant than the complications of the disorders themselves. The search for such a marker is not novel, and in 1999 it was established that patients with ET whose myeloid cells were clonal (using X-chromosome inactivation pattern analysis, XCIP) were at greater risk of thrombotic complications than those whose neutrophils were not (Harrison et al, 1999). However, XCIP analysis is age and sex-limited, relatively time-consuming, operator-dependent, and is not widely available outside research laboratories. By contrast, JAK2 mutation screening is now readily available to most hospitals. Consequently, the suggestion that patients with the JAK2V617F mutation, whose presence may imply clonal haematopoiesis, could also be at higher risk of thrombotic complications, is both an attractive and plausible one.

Predicting thrombotic events in MPD

  1. Top of page
  2. Summary
  3. JAK2V617F and myeloproliferative disorders
  4. Predicting thrombotic events in MPD
  5. JAK2V617F and thrombosis in MPD
  6. JAK2V617F in the absence of known MPD
  7. Conclusion
  8. References

Thrombotic risk in MPD

Thrombosis remains the leading cause for morbidity and mortality in MPD representing the initial presentation for 12–39% of patients subsequently diagnosed with PV and 11–25% for those given a diagnosis of ET. Details regarding the incidence of thrombosis in MPD are well outlined in a prior review (Elliott & Tefferi, 2005). Existing management strategies in MPD remain dominated by prognostic stratification in terms of thrombotic risk (Table I). Well described risk factors for thrombosis identified in patients with ET include increasing age, a prior thrombotic event, the presence of clonality and invitro occurrence of spontaneous megakaryocyte colony formation (Cortelazzo et al, 1990; Tefferi & Hoagland, 1994; Landolfi et al, 1997; Ravandi-Kashani & Schafer, 1997; Harrison et al, 1999; Niittyvuopio et al, 2004). The recognition of advanced age (>60 years) as an important pro-thrombotic risk factor has been further confirmed by two recent single institution studies in ET (Wolanskyj et al, 2006; Carobbio et al, 2007), although in the later study the association was significant in relation to arterial but not venous events. Similarly in PV, data from the ECLAP (European Collaboration on Low-dose Aspirin in Polycythaemia Vera) study confirmed the strong association between increasing age and thrombosis risk (Landolfi et al, 2007), a finding also observed in a recent study of 494 ET/PV patients in which 34% experienced recurrent thrombosis (De Stefano et al, 2008). The role of conventional cardiovascular risk factors remains debatable. Interestingly, the study by De Stefano et al (2008) and the aforementioned study by Wolanskyj et al (2006) failed to show the importance of cardiovascular risk factors in predicting thrombotic risk. This is in contrast to the ECLAP group, which established the importance of cardiovascular risk in PV (Finazzi, 2004; Marchioli et al, 2005). Such discrepancies highlight that the weighting given to these conventional cardiovascular risk factors in assigning overall thrombotic risk in MPD remains controversial. The contribution to thrombotic risk of co-existent inherited thrombophilic states in MPD is unresolved, as investigations have produced conflicting results primarily limited to small retrospective studies (Harrison, 2005; Griesshammer, 2006).

Table I.   Established and novel thrombotic risk factors in MPD.
Established risk factorsControversial risk factorsPotential novel risk factors
Age > 60 yearsConventional cardiovascular risk factors (smoking, hypertension, diabetes, dyslipidaemia)Leucocytosis
Previous thrombosisJAK2V617F mutation

Haematocrit

In PV the degree of erythrocytosis is well correlated with the risk of thrombosis (Pearson & Wetherley-Mein, 1978). However, although the haematocrit is the major determinant of whole blood viscosity in vitro, under physiological conditions in vivo, blood flow is also influenced by prevailing high shear rates, red cell axial migration, cellular activation and adaptive vessel diameter changes (Pearson, 1997). So, whilst a high haematocrit may be the main pathogenic mechanism of thrombosis in PV, it is not the only one, with platelets, white cells, plasma proteins and the activity of the endothelium creating a complex viscosity profile. This is an important concept to grasp when considering the pathogenesis of thrombosis in MPD and is illustrated clinically by the observation that, despite comparable haematocrits, patients with secondary erythrocytosis have far fewer thrombotic complications than those with PV (Finazzi et al, 2006). A raised haematocrit appears to alter platelet behaviour secondary to modified flow dynamics. During normal flow conditions, axial migration of red cells displaces platelets to the mural plasma zone, exposing them to maximal vessel wall shearing forces (Beck & Eckstein, 1980). With increasing haematocrits, the zone of flowing platelets is effectively squashed by the large numbers of red cells, narrowing the platelet mural flow zone and increasing the shear forces acting on the platelets. This encourages greater interaction between fellow platelets and platelets and the endothelium and is greatest at high wall shear rates (eg. arterioles and capillaries). This may explain the predominance of arterial events over venous events in MPD (Landolfi et al, 1995). In addition to the rheological effects, the red cells in PV may be dysfunctional with increased adhesiveness (Wautier et al, 2007) and features that contribute metabolically to platelet activation (Santos et al, 1997; Valles et al, 2002).

Thrombocytosis

In contrast to haematocrit, even extreme elevations in platelet counts do not significantly contribute to whole blood viscosity. Although intuitively we consider thrombocytosis to increase the risk of thrombosis, most prospective and retrospective studies to date have failed to demonstrate a clear correlation between platelet counts and the incidence of thrombosis in MPD (Cortelazzo et al, 1990; Colombi et al, 1991; Wehmeier et al, 1991; Tefferi & Hoagland, 1994). This includes the PVSG-01 (polycythaemia vera study group) trial (the largest prospective cohort of PV patients) in which platelet counts closest to the thrombotic event did not predict its occurrence (Berk et al, 1986). Further, in the ECLAP study (1638 PV patients), there was no correlation between platelet count and the 226 recorded thrombotic events (Di Nisio et al, 2007). Even in ET, no direct relationship between high platelet count and thrombosis has been demonstrated (Lengfelder et al, 1998). The high- risk arm of the MRC-PT1 (Medical Research Council Primary Thrombocythaemia) trial (Harrison et al, 2005) which compared cytoreduction with hydroxycarbamide (hydroxyurea) versus anagrelide (in addition to low dose aspirin) found that the ET patients receiving hydroxycarbamide were less likely to experience an arterial thrombotic event. Yet both groups achieved similar control of their platelet counts. Perhaps the improved outcome reflects an overall effect of myelosupression per se affecting other factors including white cell count and haematocrit.

There is, however, some evidence to support a link between thrombocytosis and thrombosis. Thrombosis has been correlated with increased platelet turnover in patients with thrombocytosis as measured by circulating reticulated platelets (Rinder et al, 1998). Regarding MPD, a small study by Cortelazzo et al (1995) involving 114 ET patients showed that reducing the platelet count with hydroxycarbamide significantly reduced the incidence of thrombotic events. Also, a study of 155 patients with idiopathic myelofibrosis and 31 thrombotic events, identified a platelet count >450 × 109/l as a predictor for major cardiovascular risk, with five-year thrombosis-free survival calculated as 80·6% and 96·2% depending on whether they were below or above this cut-off, respectively (Cervantes et al, 2006). Finally, in a small study of 33 PV patients, higher platelet counts (P = 0·0349) were observed in a subgroup of six patients who developed thrombosis (Ohyashiki et al, 2007a). Although limited by study size, the authors speculated that their observations support the notion that uncontrolled thrombocytosis is a risk factor for thrombosis.

Platelet dysfunction

Platelet aggregometry and more recently, flow cytometric techniques have revealed a myriad of structural and functional platelet abnormalities in MPD. Platelet adrenergic receptors crucial for efficient haemostasis have been shown to be decreased or impaired (glycoprotein (GP)1b, GpIIb/IIIa, GPVI) resulting in reduced platelet responsiveness (Harrison, 2005; Falanga et al, 2007). Increased GpIV, loss of prostaglandin D2 receptors and abnormalities in the thrombopoietin receptor (cMPL) have also been described (Harrison, 2005). Other findings have included observations of enhanced platelet activation with spontaneous platelet aggregation and circulating platelet aggregates; increased platelet microparticles; acquired storage pool disease; and abnormal arachidonic acid metabolism (Schafer, 1984, 2006; Elliott & Tefferi, 2005). Aside from a possible association between thrombosis and both increased GPIV (Jensen et al, 2000), and increased platelet derived microparticles (Villmow et al, 2002) studies have failed to show a consistent correlation between any of the described platelet abnormalities and thrombosis. Perhaps the type of platelet dysfunction observed can evolve with the natural history of the disease, making it difficult to observe an association between thrombosis and any one particular platelet aberration, or perhaps several simultaneous platelet abnormalities may work in concert to counterbalance any haemostatic effect in MPD.

The contribution that platelets play in thrombotic risk remains unclear, but this should not undermine several important facts that implicate platelets in the pathogenesis of thrombosis. For one, the efficacy of low dose Aspirin in reducing the risk of cardiovascular events in PV is well demonstrated in the ECLAP study (Di Nisio et al, 2007). Secondly, erythromelalgia is very effectively treated by Aspirin (Michiels et al, 2006), and biopsies derived from erythromelalgic skin reveal platelet rich thrombi that stain only very weakly for fibrin (van Genderen & Michiels, 1997). Finally, there is the observation that cytoreduction and the (presumed) contribution of a reduction in platelet count reduces the incidence of thrombosis in ET (Cortelazzo et al, 1995; Spivak, 2002; Harrison et al, 2005).

Leucocytes

More recent attention has shifted to the role of leucocytes in contributing to the pathogenesis of thrombosis. Activated neutrophils and monocytes can promote thrombosis by releasing granules and forming aggregates with platelets, which, in turn, can trigger monocyte tissue factor expression, pro-inflammatory cytokines and mediators of cell damage. Several studies have shown enhanced neutrophil activation, increased formation of neutrophil-platelet aggregates, and increased markers of endothelial damage in PV and ET patients compared to controls (Falanga et al, 2000; Jensen et al, 2000). Greater mean percentages of neutrophil-aggregates have been described in patients with a history of microvascular or thrombotic events (Jensen et al, 2001). Other markers of platelet-leucocyte activation (p-selectin, monocyte CD11b and monocyte membrane tissue factor) have been shown to be higher in ET patients with a history of thrombosis compared to patients without thrombosis (Falanga et al, 2007). The same group has shown that the formation of leucocyte-platelet aggregates may be attenuated by aspirin therapy (Falanga et al, 2005). Hydroxycarbamide has also been reported to reduce tissue factor expression on activated white cells (Maugeri et al, 2006).

Recent analysis of the ECLAP study data revealed that PV patients with a white cell count greater than 15 × 109/l were at a higher risk of thrombosis than those patients with white cell counts below 10 × 109/l (HR1·71, 95%CI, 1·10–2·65; P = 0·017) (Landolfi et al, 2007). Similarly, in ET an increased leucocyte count (>8·7 × 109/l) at diagnosis has been associated with thrombosis during follow up (RR2·3, 95%CI 1·4–3·9, P = 0·0001) (Carobbio et al, 2007). This association appeared to be attenuated by myelosuppressive therapy, suggesting an alternative explanation for the efficacy of hydroxycarbamide and aspirin therapy in MPDs, more akin to the efficacy noted in sickle cell disease, where leucocytosis also correlates with thrombotic events (Stuart & Nagel, 2004).

JAK2V617F and thrombosis in MPD

  1. Top of page
  2. Summary
  3. JAK2V617F and myeloproliferative disorders
  4. Predicting thrombotic events in MPD
  5. JAK2V617F and thrombosis in MPD
  6. JAK2V617F in the absence of known MPD
  7. Conclusion
  8. References

Activated haemostasis and JAK2V617F

In the era of a better molecular understanding of MPDs, much attention has focused on whether the presence of JAK2V617F has any bearing on thrombotic risk. Several hypothetical mechanisms exist. It is now widely recognised that the JAK2 phenotype of ET results in a higher haemoglobin and higher white cell count, factors which both increase the risk of thrombosis through concepts of rheology and platelet activation. However, there may be more specific actions. It has recently been observed that JAK2V617F may modify red cell adhesion molecules and promote increased adhesiveness favouring thrombosis (Wautier et al, 2007). The direct effect of the JAK2-activating mutation may be involved in impaired expression of cMPL signal transduction for TPO-induced platelet priming, leading to chronic platelet hyper-responsiveness (Kubota et al, 2004). JAK2 may also affect platelet activation by modifying cMPL cell surface localisation and stability (Royer et al, 2005). Abnormal expression of bcl-x, an inhibitor of apoptosis, and overexpression of certain genes (CD177 and transcription factor NF-E2) are also thought to be due to activation of the JAK/STAT pathway, a consequence of the JAK2 mutation (Kralovics et al, 2005b) and may indirectly affect coagulation.

Recently, Robertson et al (2007) reported significantly elevated soluble p- selectin levels in MPD patients with the JAK2 mutation compared to wild type. P-selectin mediates platelet binding to leucocytes, which induces highly procoagulant microparticles and platelet-leucocyte aggregates that promote thrombosis. Similar results have been observed using a whole blood flow cytometric method (as opposed to enzyme-linked immunosorbent assay, ELISA), which revealed significantly higher p-selectin expression both at baseline (P = 0·006) and following arachidonic acid activation (P = 0·03) for JAK2V617F positive ET cases versus the wild type (Arellano-Rodrigo et al, 2006). A separate study, with a slightly larger patient group, evaluated platelet p-selectin (CD62p) also by flow cytometry and reported that whilst they observed an over-expression of p-selectin in platelets from ET patients, they did not find any difference in CD62p expression between the JAK2 mutation carriers compared to the JAK2 wild type subjects (Falanga et al, 2007).

However, this group did identify a number of other haemostatic variables that appeared to be associated with the JAK2V617F mutation. One of these was platelet membrane-bound tissue factor (TF). Compared to JAK2 wild type cases, JAK2V617F positive cases had significantly (P < 0·01) higher levels of TF-positive platelets by flow cytometric analysis. This was associated with a concomitant increase in the total TF antigen content in washed platelets, as measured by ELISA. The increased platelet surface TF in the JAK2 mutation carriers remained significant even after multivariate analysis taking age, sex and treatment into account. The exact origin of platelet expressed tissue factor is unresolved and may be from contact with leucocytes or microparticles or may have been formed in the platelets themselves (Siddiqui et al, 2002; Camera et al, 2003). Nevertheless, the overriding point is that the JAK2V617F mutation appears to have an increased presence of aberrant TF, a definitive feature of a prothrombotic phenotype.

Falanga et al (2007) also confirmed previous findings of increased levels of circulating platelet/leucocyte aggregates in ET patients, but in addition demonstrated that these aggregates are significantly greater in JAK2V617F mutation carriers compared to the wild-type cases of ET. The investigators also observed significantly increased surface expression of neutrophil CD14 and leucocyte alkaline phosphatase (LAP) in JAK2V617Fversus wild type cases. A Similar study in 49 ET patients also found higher levels of platelet–neutrophil and platelet monocyte complexes in JAK2 mutation carriers compared to wild type counterparts, but in this instance the difference did not reach statistical significance (Arellano-Rodrigo et al, 2006). Similar parameters have been studied in patients with primary myelofibrosis (PMF), where although platelet-leucocyte aggregates were increased regardless of JAK2 phenotype, there was no difference regarding JAK2 status (Alvarez-Larran et al, 2008). However, these investigators did show that compared to the JAK2 wild type, patients with the JAK2V617F mutation had significantly higher monocyte and neutrophil CD11b expression, both at baseline and following activation. Here, as is observed in many MPD studies, mutation positive patients also had higher leucocyte counts, but in this study the analysis failed to demonstrate that the higher CD11b was independent of the leucocyte count. The authors speculated that the increased CD11b expression would enhance neutrophil adhesion to megakaryocytes, and facilitate emperipolesis, a phenomenon often implicated in myelofibrotic development.

Other markers of haemostatic activation, including prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin (TAT) complexes have previously been shown to be increased in MPD compared to controls (Falanga et al, 2000). Recently investigators have addressed these (and other markers) in relation to JAK2 status. Robertson et al (2007) did not find any association of hypercoagulation markers TAT and F1 + 2 or D-dimer with JAK2 status, or even the presence of MPD, but their control group were all patients with uncontrolled hypertension and at increased risk of vascular events. Conversely, in 75 ET patients, F1 + 2, TAT, D-dimer, plasminogen activator inhibitor (PAI-1), tissue plasminogen activator (t-PA), thrombomodulin (TM) and neutrophil elastase were analysed (Falanga et al, 2007). All haemostatic markers were higher in the ET patients, with only TAT complex levels not reaching significance. However, with regards to the JAK2 mutation, only soluble TM levels were significantly higher when compared to wild type ET patients (P = 0·020). In PMF, D-dimer, F1 + 2 and TM have been investigated and high levels of the latter two measures have been reported in association with the disease, but in a subgroup of JAK2V617F positive patients, only F1 + 2 levels were observed to be significantly higher compared to wild type PMF patients (Alvarez-Larran et al, 2008).

Despite the variable conclusions with regards to which disordered haemostatic parameter is associated with JAK2V617F, this should not overshadow the overwhelming evidence supporting the concept of increased neutrophil and platelet activation in ET. Further, there is growing evidence to support the suggestion that the JAK2V617F phenotype displays a greater degree of spontaneous platelet and leucocyte activation compared to the wild type. Large prospective studies are needed to address if the JAK2V617F mutation is indeed related to increased activation of platelets and leucocytes and that these parameters play a vital role in the pathophysiology of thrombosis in MPD.

JAK2V617F status and thrombotic risk – clinical evidence

Fourteen published studies have examined the interaction between JAK2V617F mutation status and thrombotic risk in patients with MPD (Table II). The largest prospective study to address the issue followed 776 patients with ET enrolled into the MRC PT1 trial, and two smaller studies (Campbell et al, 2005). 414 patients were V617F-positive (53%), and these were found to have a significantly higher risk of venous thromboembolism (VTE) in the year prior to diagnosis (11 vs. 2 events, P = 0·04) and a non-significant trend towards VTE after trial entry (12 vs. 4 events, P = 0·06) compared to V617F-negative patients. There was no difference in the rate of arterial events. V617F-positive patients were also significantly older than V617F-negative ones, and had significantly higher haemoglobin concentrations and neutrophil counts. Analysis of response to treatment showed that amongst V617F-positive patients, those randomised to receive anagrelide had a significantly higher arterial thrombotic rate than those who received hydroxycarbamide (19 vs. 5 events, P = 0·03), but this difference was not seen in V617F-negative patients. With respect to VTE, there was no apparent interplay between mutation status, treatment, and thrombotic risk.

Table II.   Studies addressing the association between JAK2V617F and thrombosis.
StudynPatient group%JAK2 V617F+ Median f/up (months)Association between JAK2 V671F and thrombosis on univariate analysis?DetailsPotential confounding factors associated with JAK2 V617F statusIndependent association between JAK2 V617F and thrombosis on multivariate analysis?Comments
  1. ‘Thrombosis’ includes both venous and arterial events unless otherwise stated. ‘Overall’ refers to thrombosis at presentation or follow-up.

  2. *< 0·05.

  3. < 0·005.

  4. ‡Number of evaluable patients in whom JAK2 V617F status was known and clinical data was available.

  5. OR, odds ratio; CI, confidence intervals; ND, not done; PV, polycythaemia vera; ET, essential thrombocytosis; IMF, idiopathic myelofibrosis; VTE, venous thromboembolism; Hb, haemoglobin; WCC, white blood cell count.

Kralovics et al, 2005a244128 PV, 93 ET, 23 IMF48 YesHigher overall rate of thrombosis in V617F+ group (26% vs. 15%)*Age, disease durationNDNo separate analysis for ET subgroup
Wolanskyj et al, 2005150ET49137NoNo difference in rate of thrombosis at any time (45% vs. 38%) or after diagnosis (33% vs. 29%).Age Hb, WCCNoNot powered to detect small differences in thrombotic rate (eg. 5% and 15%)
Campbell et al, 2005776ET53 YesHigher rate of VTE prior to diagnosis in V617F+ group (11 vs. 2 events)*. Trend towards higher rate of VTE after diagnosis (12 vs. 4 events). No difference in arterial events.Age, Hb, neutrophil countNDPatients mostly derived from MRC PT-1 trial
Cheung et al, 2006 60ET48 YesHigher overall rate of thrombosis in V617F+ group (62% vs. 26%)†.HbND 
Tefferi et al, 2006 63PV92 33NoNo difference in overall rate of thrombosis. No 
Heller et al, 2006 50ET48 79YesHigher overall rate of thrombosis in V617F+ group (11 vs. 1 events)†Age, HbYesRelatively young cohort (78%≤ 60 years). Majority of thrombotic events occurred prior to or at diagnosis of ET
Finazzi et al, 2007179ET58∼66YesHigher overall rate of thrombosis in V617F+ group (33% vs. 17%)*Hb, WCCND77 further patients had PV, in whom the thrombotic rate was 43% (not significantly different to V617F+ ET group)
Ohyashiki et al, 2007c 49ET63 YesHigher overall rate of thrombosis in V617F+ group (29% vs. 6%)Hb, WCCNDAlso analysed 34 PV patients: no association between homo- or heterozygosity for JAK2 V617F and thrombotic risk
Carobbio et al, 2007277ET 55 NoNo difference in postdiagnosis rate of thrombosis (hazard ratio = 1·4, 95% CI 0·7 to 3·0)Hb, WCCNo 
Kittur et al, 2007176ET 55 59YesHigher rate of VTE after diagnosis in V617F+ group (11% vs. 3%)*. No difference in prediagnosis VTE (11% vs. 6%), or arterial events at any time (38% vs. 29%).Age, Hb, WCCYesSignificance on multivariable analysis was lost if history of prior thrombosis was included.
Alvarez-Larran et al, 2007103ET < 40 years old 43120NoNo difference in postdiagnosis rate of thrombosis (9 vs. 13 events).HbNoMajor thrombotic events only included
Vannucchi et al, 2007b173PV100 24YesPatients with a V617F-allele burden >75% had a higher risk of thrombosis at diagnosis and during follow-up combined (P = 0·004), and at diagnosis alone (P = 0·003), compared with those whose allele burden was <25%.Haematocrit, WCCYes 
Vannucchi et al, 2007a962323 PV, 639 ET 73 60YesIncreased rate of arterial and venous thrombosis in V617F-homozgygous ET patients (n = 14) compared with all other ET patients, at diagnosis and during follow-up*.Age, HctYesSee text
Hsiao et al, 200753ET 66 35YesHigher overall rate of thrombosis in V617F+ group (43% vs. 11%)*Hb, WCCNDThose with thrombosis were significantly older and had significantly higher WCC than those without

Vannucchi et al (2007a) retrospectively analysed 962 patients with chronic MPD (323 PV, 639 ET), subdivided into three groups with respect to their JAK2V617F status: wild-type, heterozygous (percentage of mutant allele in granulocytic cells <50%), and homozygous (mutant allele >50%). All the patients with PV were V617F-positive (68% heterozygous), as were 60% of the ET patients (of these, 96% were heterozygous). Over a median follow-up of 5·0 years, a significant excess of thrombotic events was noted in the homozygous patients with ET compared with their heterozygous and wild-type counterparts, and indeed compared with the PV patients. The significant difference in overall thrombotic rates was observed both at the time of diagnosis, largely due to a marked excess of venous events in the homozygous group, and during follow-up, when both arterial and venous events were more common in the homozygous ET group.

Compared with wild-type patients, JAK2V617F homozygosity was independently associated with the occurrence of cardiovascular events in a multivariate analysis that incorporated established risk factors for thrombosis in ET (age, history of prior thrombosis, leucocytosis, and sex), though it should be emphasised that this group only represented 2·2% of the total patient cohort. More generally, the heterozygous and homozygous ET patients, when considered together, did have a significantly higher incidence of thrombosis at diagnosis (but not during follow up), compared with their wild-type counterparts. They were, however, also older and exhibited a higher haematocrit, than the wild-type patients, which may have confounded this comparison (Vannucchi et al, 2007a). In a retrospective study of 176 patients with ET, Kittur et al (2007) also observed an increased rate of venous thrombotic events in the 96 patients (55%) who were positive for the JAK2V617F mutation (11% vs. 3%, P = 0·02). This association was, however, confined to venous events after diagnosis. Again, a positive correlation between JAK2V617F mutation status and haemoglobin, age and white cell count was noted.

Six further studies suggested the possibility of an association between JAK2 mutation status and overall thrombotic risk – i.e. combined venous and arterial events, both before and after diagnosis (Kralovics et al, 2005a; Zhao et al, 2005; Cheung et al, 2006; Heller et al, 2006; Finazzi et al, 2007; Hsiao et al, 2007; Ohyashiki et al, 2007b; Vannucchi et al, 2007b). Kralovics et al (2005a) evaluated 244 patients with MPD (128 with PV, 93 with ET, and 23 with myelofibrosis), of whom 117 (48%) had the JAK2V617F mutation. The rate of thrombotic complications was significantly higher in this group than the wild-type group (26% vs. 15%, P = 0·03), even though a significantly greater proportion of the V617F-positive group had received cytoreductive therapy. However, they also noted that patients with the mutation had significantly longer disease duration than those without, and were significantly older at the time of sampling. Both of these factors could have contributed to the increased thrombotic rate in this patient group, and so the effect of the JAK2 mutation alone could not be isolated.

Indeed, potentially confounding factors were found in all eight of the studies in which an association between JAK2 mutation status and thrombosis was observed. In seven studies, the JAK2V617F-positive subgroup had significantly higher haemoglobin concentrations at diagnosis; in five, a significantly higher white cell count or neutrophil count was noted at diagnosis; and in four, patients with the mutation were found to be significantly older than those without. The association between JAK2V617F mutation status and leucocyte count is especially noteworthy, since a number of groups have found that a relative leucocytosis (white cell count above the population median) at diagnosis is a strong, independent predictor of thrombotic complications, especially in otherwise ‘low-risk’ patients (Wolanskyj et al, 2006; Carobbio et al, 2007).

By contrast, a number of studies have failed to demonstrate any relationship between JAK2 mutation status and vascular outcome. Wolanskyj et al (2005) followed up 150 patients with ET: 73 (49%) were V617F-positive, and these did not show a higher incidence of thrombosis (combined arterial and venous) than their V617F-negative counterparts, despite a significantly higher haemoglobin concentration, leucocyte count and median age. The former group had an excess of deaths during the median follow-up of 11·4 years (21 vs. 11 deaths, P = 0·03), with a non-significant trend to inferior survival, but this was accounted for by the significantly higher median age. The authors concede that the study was not sufficiently powered to detect relatively small differences in complication rate (e.g. between 5% and 15%). Antonioli et al (2005) similarly found that, of 130 patients with ET, the V617F-positive group (n = 74) did not appear to have a significantly higher risk of thrombosis than the V617F-negative group (17 vs. 13 events). Again, venous and arterial events were grouped together.

Retrospective analysis of the large Italian ET cohort supports these findings (Carobbio et al, 2007). 129 thrombotic events – arterial or venous – were recorded prior to or at diagnosis in 439 patients. During follow-up, a further 78 events were diagnosed in 67 patients. Multivariate analysis demonstrated that ‘standard risk factors’ (age ≥ 60 years, previous thrombosis), as well as leucocytosis, were independently associated with thrombotic events. The JAK2V617F mutation status was evaluated in 277 patients (38 vascular events) but, on multivariate analysis, V617F-positivity was not found to independently predict thrombosis during follow-up (hazard ratio = 1·4, 95% confidence interval 0·7 to 3·0).

Overall, there is conflicting evidence for the importance of the JAK2V617F mutation status in predicting the risk of thrombosis in patients with MPD. Nine studies have noted an association between mutation status and thrombotic risk (albeit confined to venous events in two of these); however, all were hindered by the presence of potentially confounding factors. In the three series where multivariate analysis was performed (Heller et al, 2006; Kittur et al, 2007; Vannucchi et al, 2007a), the association did persist after adjusting for these factors, but there were caveats in each. In Vannucchi et al (2007a), the independent association was only with rare homozygous-mutant cases, in Kittur et al (2007) the association was lost if prior thrombosis was incorporated into the model, and in Heller et al (2006), the majority of the thrombotic events occurred prior to or at diagnosis. Four studies refuted the association between JAK2V617F mutation status and thrombotic risk (Wolanskyj et al, 2005; Tefferi et al, 2006; Alvarez-Larran et al, 2007; Carobbio et al, 2007), though these tended to include fewer patients, and analysed arterial and venous events together. Consequently, they may not have detected small differences in rates of vascular events between the V617F-positive and the V617F-negative groups, particularly differences confined to either arterial or venous subtypes. There are no comprehensive studies evaluating the influence on thrombotic risk of co-existing inherited common thrombophilic states in patients with the JAK2V617F mutation.

JAK2V617F in the absence of known MPD

  1. Top of page
  2. Summary
  3. JAK2V617F and myeloproliferative disorders
  4. Predicting thrombotic events in MPD
  5. JAK2V617F and thrombosis in MPD
  6. JAK2V617F in the absence of known MPD
  7. Conclusion
  8. References

Splanchnic vein thrombosis

Splanchnic vein thrombosis (SVT) is frequently an early or presenting feature of an undiagnosed MPD; being identified as the underlying disorder in 28–49% of cases of hepatic vein thrombosis and 14–35% of patients with portal vein thrombosis (Chait et al, 2005; Briere, 2006). The precise prevalence of an unequivocal MPD in this setting is unclear as often the MPD may not become apparent until several years later, or despite suggestive features of an MPD, fulfilment of conventional diagnostic criteria may be lacking. In fact, 25–65% of SVT cases are thought to have what is frequently termed ‘latent’ or ‘masked’ MPD in which patients present with SVT with normal blood counts, rarely meet the diagnostic criteria for MPD, but have bone marrow histology or erythroid cell colony characteristics suggestive of an underlying MPD (Valla et al, 1985; Landolfi et al, 2007; Regina et al, 2007).

Applying well-defined diagnostic criteria in this setting is further complicated by the SVT itself; splenomegaly may be secondary to portal hypertension, hypersplenism may mask a thrombocytosis, haemodilution and variceal bleeding may mask an erythrocytosis, and a reactive erythrocytosis secondary to tissue ischaemia may exist. Consequently, identification of abnormal megakaryopoiesis and endogenous erythroid colony formation has been suggested as the most reliable means of MPD diagnosis in SVT (Chait et al, 2005). However, given that JAK2V617F is strongly related to endogenous erythroid colony formation (Baxter et al, 2005; James et al, 2005), it raises the question of whether JAK2 identification may be a useful, simpler diagnostic tool in identifying SVT patients with ‘latent’ MPD.

Several retrospective studies have investigated the relationship between JAK2 and SVT (Table III). The JAK2V617F prevalence ranged from 17–58% in a variety of intra-abdominal thromboses, demonstrating that the JAK2V617F mutation is frequently found in SVT. JAK2V617F was particularly common in patients with overt MPD at the time of the SVT, ranging from 71–100%. This is higher than the JAK2V617F mutation frequency of 50–79% reported in MPD patients with common thromboses (Campbell et al, 2005; Wolanskyj et al, 2005; Cheung et al, 2006; Tefferi et al, 2006), and suggests that the association for JAK2 and thrombotic risk may be more pronounced for thrombosis at unusual sites (eg. intra-abdominal thrombosis). This is further supported by Colaizzo et al (2007a) who evaluated the JAK2V617F mutation in a variety of types of venous thromboembolism (deep vein, retinal vein, SVT and cerebral vein thrombosis) and observed that the JAK2V617F mutation was restricted to 27/93 (29%) of SVT patients and not present in thrombotic disease of other locations. The prevalence of JAK2V617F seems to be highest in patients with thrombosis of the portal vein, and interestingly, in isolated mesenteric vein thrombosis JAK2V617F has not been reported (Primignani et al, 2006; Landolfi et al, 2007). If there is a link between thrombosis and JAK2V617F then, in cases of ‘latent’ MPD, it could be that the JAK2 mediated platelet and leucocyte activation play a more important role in the hypercoagulable state that results in SVT, as opposed to thrombosis at other sites.

Table III.   Studies addressing the association between JAK2V617F and splanchnic vein thrombosis.
StudySVT typePts (n)JAK2 V617F n (%)Overt MPD at thrombosis n (%)No overt MPD at thrombosis n (%)Comments
JAK2 V617FJAK2WTJAK2 V617FJAK2 WT
  1. *6 cases with a diagnosis of MPD did not have JAK2 analysis performed.

  2. Pts, patients; MPD, myeloproliferative disorder; WT, wild-type; SVT, splanchnic vein thrombosis; HVT, hepatic vein thrombosis; BCS, Budd-Chiari syndrome; PVT, portal vein thrombosis; MVT, mesenteric vein thrombosis; EHPVO, extrahepatic portal vein obstruction; SMAT, superior mesenteric artery thrombosis; BM, bone marrow.

Kiladjian et al, 2008BCS PVT241  94 (39)62/72 (86)10/72 (14)30/164 (18)134/164 (82)MPD diagnosis refers to bone marrow MPD features only. 101 patients had additional EEC studies combined with bone marrow biopsies JAK2V617F was detected in 96·5% of patients with both positive, 58% of those with only 1 positive feature and in 7% with neither. Higher trend for JAK2V617F prevalence in BCS compared to PVT.
Patel et al, 2006HVT41  24 (58)10/11 (91) 1/11 (9)14/30 (47)16/30 (53)Of those without overt MPD, 5/17 JAK2 wt and 4/14 JAK2V617F had features suggestive of MPD. Bone marrow hyperplasia seen in 16 cases (12 with JAK2V617F). JAK2 mutation patients had higher haemoglobin concentration.
Smalberg et al, 2006BCS PVT407/17 (41) 7/13 (54)*–*  ≥10 (≥37)2 patients diagnosed with MPD did not meet WHO criteria. JAK2 analysis in 17 patients only. No new MPD observed on follow up. No difference in survival between MPD and non-MPD patients.
Colaizzo et al, 2007bPVT MVT99  17 (17)  7/9 (78) 2/9 (22)10/90 (11)80/90 (89)At 41 months median follow up 5 pts developed MPD (3 with JAK2V617F).
Landolfi et al, 2007HVT PVT SVT94  32 (34)15/15 (100)0/15 (0)17/79 (22)62/79 (78)No Follow up reported.
Primignani et al, 2006EHPVO BCS93  34 (37)   29 (71)  12 (29) 2/33 (6)31/33 (94)MPD diagnosis based on BM histology. The sensitivity bias of JAK2 corrected by clonality analysis of haemopoiesis. Estimated prevalence of MPD in SVT as 53%.
Regina et al, 2007PVT HVT44   8 (18)    0 (0)   0 (0) 8/44 (18)36/44 (82)All JAK2V617F positive cases had BM Hyperplasia and EEC. Follow up of 8 JAK2V617F patients –3 developed MPD, 1 death, 4 no evidence of MPD.
McMahon et al, 2007HVT PVT MVT SMAT42   7 (17)  3/3 (100) 0/3 (0) 4/39 (10)35/39 (90)All cases underwent small intestinal or multivisceral transplants. Shorter survival noted in JAK2V617F patients.
Boissinot et al, 2006SVT45  14 (31)14/17 (82)3/17 (18) 0/28 (0)28/28 (100)MPD refers to “latent” (JAK2V617F positive and/or EEC/EMC) and with BM histology consistent with MPD in 11/12 cases.

The studies in Table III highlight that the JAK2V617F mutation was also observed in a portion of patients not suffering from an overt MPD at the time of the thrombotic event (0–47%). Many authors have speculated that patients with the JAK2 mutation, but without overt MPD may have ‘latent’ or ‘early’ MPD and be at much higher risk of developing overt disease at a later date. Kiladjian et al (2008) and Primignani et al (2006) both evaluated the sensitivity of JAK2 mutation screening in SVT and revealed that there was good correlation with histological diagnosis via bone marrow. Long follow-up studies are limited, but suggest that the presence of JAK2 may portend, but is not synonymous with, the subsequent development of a MPD. In one study, with a median follow up of 49 months, only four of 14 cases with the JAK2 mutation had developed MPD (Patel et al, 2006) whilst in another study with 41 months follow up, 7/10 patients with JAK2V617F still had no evidence of MPD (Colaizzo et al, 2007b). This suggests either that very long latent periods for MPD, or that a portion of JAK2V617F patients with SVT do not suffer from an overt MPD.

Ultimately, prospective studies with long follow-up are needed to clarify the role of JAK2 testing in this setting. It is currently unclear if detection of the JAK2 mutation might improve diagnosis, and assist in therapeutic decisions at the time of the thrombotic event (eg.cytoreduction). However, given the evidence of aspirin efficacy in MPD, some authors are advocating the use of aspirin in combination with anticoagulant therapy for patients with SVT, carrying the JAK2 mutation (Michiels et al, 2007). Based on recent data, Kiladjian et al (2008) has developed an algorithm in which JAK2 screening can be the initial test in establishing a diagnosis of MPD, sparing many SVT patients, from a bone marrow biopsy. In addition, the sensitivity, standardisation and convenience of JAK2 analysis (compared to endogenous erythroid colony formation assays) makes it a rational investigation in patients with idiopathic SVT.

Cerebral vein thrombosis

Like SVT, a proportion of patients with cerebral vein thrombosis (CVT) may have an unequivocal diagnosis of an underlying MPD, or have features that ultimately develop into MPD at a later date. There is scant data assessing any association between cerebral vein thrombosis and the JAK2V617F mutation. One study evaluated 45 CVT patients which had four cases of overt MPD at diagnosis. There was a high mutation prevalence of 75% (3/4) amongst these overt MPD patients with CVT, but conversely a low prevalence of 5% (2/41) in those without a diagnosis of MPD (Landolfi et al, 2007). Similarly, in a report evaluating the JAK2 mutation status in patients without overt MPD, only one of eight CVT cases, was identified as carrying the mutation (Pardanani et al, 2007). Conversely, another study observed that there were no cases positive for the JAK2V617F mutation amongst 45 patients with CVT (Colaizzo et al, 2007a). Currently any possible association between JAK2V617F and cerebral vein thrombosis is unclear, and until the strength of this possible association is clarified further, at best, JAK2 analysis should be reserved for very selected idiopathic cases of CVT.

Thrombosis at other sites

With the association of JAK2V617F and SVT, investigators have questioned whether JAK2V617F is specific for MPD, suggesting that the mutation may be independently associated with thrombosis. Over the past few years, JAK2V617F has been studied in many healthy control groups, via a variety of techniques, and no positive cases have been found (Passamonti et al, 2007). However, the largest prevalence study to date observed the JAK2V617F in almost 1% of the 3935 blood samples from Chinese donors (Xu et al, 2007). This is much higher than the prevalence of myeloproliferative disease, suggesting that JAK2 may not be pathognomonic for myeloproliferative disorders per se. Although only one case had clinical features suggestive of MPD, the JAK2V617F subgroup had significantly higher platelet and leucocyte counts. Some of these cases may be early preclinical MPD, or cases that lack other cooperating mutations or polymorphisms needed to expose an MPD phenotype, but such suggestions currently remain speculative. Confirmation of the genotype frequency is required in large prospective studies, with adequate follow up of subjects who carry the mutation. Currently the clinical significance of JAK2V617F outside of MPD is unknown.

Expert opinion is currently divided, but some suggest that patients with an idiopathic thrombophilic state be tested for JAK2V617F. Remacha et al (2007) screened 295 venous thrombosis patients, and found only one positive for the JAK2V617F mutation, with subsequent follow up suggesting that this sole patient had preclinical polycythaemia. A similar study (Pardanani et al, 2007) evaluated JAK2V617F in 210 consecutive patients referred for thrombophilia testing (all non-splanchnic vein thrombosis), who did not meet the criteria for MPD. Four cases (2%) were identified as carriers of the JAK2V617F mutation, all with a history of recurrent unprovoked thrombosis. In another report the mutation was not found in any of 110 patients with deep vein thrombosis (Colaizzo et al, 2007a). A recent study looked at 394 unprovoked VTE cases and identified six patients (1·5%) with the JAK2V617F mutation, with three cases of MPD, one undiagnosed fatality and two cases with sustained normal blood counts at 3 years follow up (Ugo et al, 2007). Taken together these studies suggest that the prevalence of JAK2V617F in idiopathic venous thrombosis may reach 2% at best, and without more convincing evidence to the contrary should not be considered as a ‘blanket’ screening tool.

Conclusion

  1. Top of page
  2. Summary
  3. JAK2V617F and myeloproliferative disorders
  4. Predicting thrombotic events in MPD
  5. JAK2V617F and thrombosis in MPD
  6. JAK2V617F in the absence of known MPD
  7. Conclusion
  8. References

The suggestion that JAK2V617F-positive patients with MPD might be at higher risk of thrombotic complications than their wild-type counterparts remains plausible, but unproven. Once large well designed prospective studies addressing venous and arterial events separately are performed, JAK2V617F mutation status may become predictive of thrombotic risk in MPD. In patients with SVT (without MPD), mutation screening may identify an undiagnosed MPD or recognise a ‘latent’ MPD. However, the role of an isolated JAK2V617F mutation in other forms of thrombosis is doubtful. Given the low frequency and undetermined clinical significance of a positive result, there is currently little evidence base to support JAK2 screening in thrombophilia investigations.

References

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  3. JAK2V617F and myeloproliferative disorders
  4. Predicting thrombotic events in MPD
  5. JAK2V617F and thrombosis in MPD
  6. JAK2V617F in the absence of known MPD
  7. Conclusion
  8. References
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