Essential Thrombocytopenia/JAK Mutation Causing Thrombosis: A Case Report and Review of Literature

  

    

    
CVT [46] (n=706)
    
MPNs [46] (n=2, 143)
    
MPNs [45] (n = 5,500)
  
  

    

    
n (%)
    
n (%)
    
n (%)
  
  

    
MPNs diagnosis
    

    

    

  
  

    
total
    
27 (3.8)
    

    

  
  

    
before
    
8 (1.1)
    

    

  
  

    
concomitantly
    
4 (0.6)
    

    

  
  

    
after
    
15 (2.1)
    

    

  
  

    
CVT
    

    

    

  
  

    
total
    

    
9 (0.4)
    

  
  

    
before
    

    
3 (0,14)
    

  
  

    
concomitantly
    

    
3 (0,14)
    

  
  

    
After
    

    
3 (0,14)
    

  
  

    
CVT-MPNs subgroup
    

    

    

  
  

    
ET
    

    
3/964 (0.3)
    

  
  

    
PV
    

    
5/735 (0.7)
    

  
  

    
PMF
    

    
1/ 444 (0.2)
    

  
  

    
MPNs and no thrombosis
    

    

    
178
  
  

    
ET
    

    

    
105 (59)
  
  

    
PV
    

    

    
51 (28)
  
  

    
PMF
    

    

    
21 (12)
  
  

    
post-ET or post-PV    myelofibrosis
    

    

    
1 (1)
  
  

    
JAK2V617F,in ET
    

    

    
53 (55)
  
  

    
MPNs and venous thrombosis    (other than CVT)
    

    

    
87
  
  

    
ET
    

    

    
53 (61)
  
  

    
PV
    

    

    
24 (28)
  
  

    
PMF
    

    

    
7 (8)
  
  

    
post-ET or post-PV    myelofibrosis
    

    

    
3 (3)
  
  

    
JAK2V617F, in ET
    

    

    
32 (78)
  
  

    
MPNs and cerebral vein    thrombosis
    

    

    
48
  
  

    
ET
    

    

    
30 (63 )
  
  

    
PV
    

    

    
11 (23)
  
  

    
PMF
    

    

    
6 (12)
  
  

    
post-ET or post-PV    myelofibrosis
    

    

    
1 (2)
  
  

    
JAK2V617F, in ET
    

    

    
19 (76)
  
  

    
CVT diagnosis
    

    

    

  
  

    
before
    

    

    
8 (17)
  
  

    
concomitantly
    

    

    
22 (46)
  
  

    
after
    

    

    
18 (38)
  

Table 4:  Cerebral venous thrombosis and myeloproliferative neoplasms relationship.

Janus kinase 2 (JAK2) V617F mutation

In 2005, the somatic gain-of-function mutation (due to a homozygous G ? T transversion) in the Janus Kinase 2 (JAK2) gene, determining the replacement of valine by phenylalanine at codon 617 (V617F), and contributing to the clonal expansion of hematopoietic cells in MPNs was identified [44]. Since then, the role of JAK2 mutation, both in better understanding the pathophysiology and contributing to the diagnosis of MPNs has increased.

A direct effect of JAK2 V617F mutation on PLTs function has long been suspected, although attempts to find a clear relationship have been elusive. A recent study by Hobbs and colleagues [45] based on the application of the JAK2 V617F knock-in mouse model (in which hematopoietic cells are heterozygous for human JAK2 V617F) revealed some important information about the relationship between JAK2 V617F, megakaryocyte and PLTs biology. The presence of JAK2 V617F mutation in megakaryocytes resulted in a hypersensitive signalling through the Thrombopoietin (TPO)/ Myeloproliferative Leukemia virus oncogene (MPL) pathway leading to increased activation of signal transducer, activator of transcription and extracellular signal-regulated kinase. JAK2 V617F-positive megakaryocytes exhibited increased ploidy and mobility as compared to wild-type JAK2 megakaryocytes. Finally, JAK2 V617F-expressing megakaryocytes exhibited an increased pro-platelet formation. Authors [45] concluded that JAK2 V617F-positive megakaryocytes are characterized by increased differentiation, greater migratory ability and pro-platelet formation.

The biological effects of JAK2 V617F were not limited to megakaryocytes but also affected PLTs. JAK2 V617F-PLTs exhibited increased thrombus formation, increased reactivity in response to collagen-related peptide and thrombin, and increased aggregation. Key experiments, performed by Hobbs and colleagues, allowed to exclude the possibility that the altered biological activity of JAK2 V617F-PLTs was simply due to a higher concentration of PLTs. Even considering equivalent PLTs numbers, the increased biological activity of JAK2 V617F-PLTs was still evident. PLTs reactivity to agonists was enhanced, with an increase in PLTs aggregation in vitro together with a reduced duration of bleeding in vivo. The Authors concluded that the JAK2 V617F mutation leads to intrinsic changes in both megakaryocyte and PLTs biology that extend beyond a simple increase in cell number [46]. Finally, the effect of JAK2 mutation may be not only confined to hematopoietic cells. The detection of JAK2 V617F mutant liver endothelial cells in patients with Budd- Chiari syndrome further extends the possible influence of JAK2 V617F mutation on thrombosis, although the underlying pathogenic mechanism is complex and not yet completely uncovered [46,47]. JAK2 V617F characterizes the majority of MPN patients, i.e. nearly 100% of patients with PV and about 50% of ET and PMF patients present the mutation. Its prevalence and clinical significance in the general population has been also evaluated [48]: among 10,507 participants from the Copenhagen City Heart Study with up to 17.6 years of follow-up who were screened for JAK2 mutation, the prevalence of the mutation was 0.2% and its presence was associated with increased morbidity and mortality. A subsequent study [49] by the same Authors showed a prevalence of 0,1% (n=68) among 49,488 individuals from the general population. Interestingly, an association of the JAK2 V617F mutation with age was observed, which implies that the status may change over time [49].

JAK2 mutation, venous and arterial thrombosis

The prevalence of JAK2 V617F mutation varies according to the site of thrombosis. Shetty and co-workers performed molecular screening for JAK2 mutation in a cohort of 321 patients affected by venous thrombosis in different sites. Overall, 19 cases (5.9%) carried the V617F variant of JAK2. The prevalence of the mutation were 3% (1/36) in patients with deep venous thrombosis, 8.8% (12/137) in those with Budd-Chiari syndrome, 5% in those with portal venous thrombosis (4/78), and 3% in those with CVT (2/70) [50]. In other studies the prevalence of JAK2 mutation in CVT patients was reported to range between 0% and 6.2%, with the highest frequency observed in a recent study by Passamonti and colleagues [16], in which 10 out of 152 patients (6.6%) affected by CVT turned out to be mutationcarriers (excluding the 6 patients with an overt MPNs at the time of CVT, the prevalence of the JAK2 V617F mutation was 2.7%: 4 out of 146). In particular, 6 patients met the diagnostic criteria for MPNs at the time of CVT, and 3 additional patients developed the disease during the follow-up (median duration, 7.8 years; range, 6 months to 21.3 years), giving an annual incidence of 0.26% patient-years [16], while one patient had no evidence of MPNs after 3 years of follow-up despite the JAK2 mutation. Among these patients, ET was the most frequent diagnosis (6 patients), with normal o slightly elevated PLTs count in most of the cases. It is important to consider that the JAK2 mutation was found in the absence of myeloproliferative features in 2.7% of CVT patients in the Italian cohort reported by Passamonti and colleagues [16] and in 5.9% in an Indian cohort [42,51]. In patients affected by ET, the JAK2 V617F mutation was the risk factor more frequently detected in patients with CVT [41].

The JAK2 V617F mutation is also common in patients with SVT (between 71% and 100% of the affected subjects) [15]. Up to 25% of these patients may have latent MPNs and present with normal blood counts. In this setting the presence of the JAK2 mutation was reported to be a strong predictor for the subsequent diagnosis of MPNs during follow-up [15,52-54]. These findings differ among different populations. In a recent study on thrombotic risk factors for Budd- Chiari syndrome (BCS) in a Chinese population, the JAK2 V617F mutation was found in 4 of the 169 patients tested. Overt MPNs were diagnosed in 5 patients (PV, 3; ET, 1; PMF, 1). In a previous study by Yoo and colleagues [55] on a Korean cohort, among the 26 patients with SVT, two patients (7.7%) had overt ET at the time of thrombosis and the JAK2 V617F mutation was detected in 3 (11.5%, including the two patients with ET). JAK-2 V617F mutation is an independent risk factor for thrombosis. Systematic reviews and meta-analyses on patients affected by PV, ET, or PMF show that JAK-2 V617F carriers have a 2-3 fold increased risk of thrombosis compared non carriers [11,56-58].

Finally, a thrombotic risk score (International Prognostic Score of thrombosis in Essential Thrombocythemia - IPSET thrombosis) was developed and validated in a large group of ET patients, who were divided into 3 risk groups of low (1.03 % patient-years), intermediate (2.35 % patient-years) and high (3.56 % patient-years) thrombosis risk on the basis of age above 60 years, thrombosis history, cardiovascular risk factors, and the presence of JAK-2 V617F mutation [11,59].

MPL and calreticulin (CALR) mutations: A gain-of-function mutation in the Myeloproliferative Leukemia virus (MPL) gene, which encodes the TPO receptor, has been identified in patients with ET and MPF in multiple sites within exon 10 and most of these result in the activation of MPL, even in the absence of TPO [60].MPLW515L/K has been shown to promote tumorigenesis in vivo [61], and a higher mutant allele frequency is associated with progression to myelofibrosis [60,62].

In 2013, Klampfl and colleagues [63] and Nangalia and colleagues. [64] described heterozygous Calreticulin (CALR) mutations as the second most prevalent acquired nucleotide changes in ET and PMF.

CALR mutations were mutually exclusive of JAK2 and MPL mutations. CALR mutational frequencies are estimated to range between 15% and 24% in ET, and between 25% and 35% in PMF [65]. In terms of prognosis, CALR mutations were associated with better thrombosis-free survival in ET and overall survival in PMF [65]. Klampf and colleagues [63] were the first to notice a two-fold difference in cumulative incidence of thrombosis between JAK2 and CALR mutated ET (13% vs 6.3% at 5 years, 21% vs 11% at 10 years, and 27.1% and 12.8% at 15 years, respectively). In contrast, Nangalia and colleagues [64] did not find a difference in thrombosis rates between JAK2 and CALR mutated patients. A subsequent study by Rotunno and colleagues [66] which included 576 ET patients, reported a significantly longer thrombosis-free survival in CALR mutated and triple-negative patients, compared to JAK2 or MPL mutated cases. JAK2 mutated ET patients had a two-fold increased risk of thrombosis compared to those with CALR mutations, and the difference remained significant after adjusting for age in the analysis by Rumi and colleagues [67]. Finally, Gangat and colleagues [65] analyzed on 300 ET subjects describing mutational frequencies of 53% for JAK2, 32% CALR, 3% MPL, and 12% 'triple-negative'. Triple negative ET patients (who carried the wild type genotype of JAK2, calreticulin-CALR and MPL) showed better thrombosisfree survival compared to JAK2 mutated cases, and these findings remained significant after correction for age and previous thrombosis in multivariable analysis. These observations further underline the importance of JAK2 mutational status in the clinical assessment of these patients [65].

When searching for JAK2 mutations in stroke patients?: Because of its pathogenic role in the thrombotic process, screening for JAK2 mutation might be a reasonable diagnostic approach in selected clinical situations, such as stroke in young adults and cryptogenic stroke, even in the presence of normal blood count. In line with this view, Richard and colleagues [68] found PLTs count < 600 x 10⁹/L in 5 out of 14 patients with ischemic stroke who were subsequently diagnosed as affected by ET, further emphasizing the difficulty of diagnosing the disease at stroke onset, and suggested that searching for the JAK2 V617F mutation might be considered in selected stroke patients irrespective of PLT count. In contrast, Xavier and co-workers [69] recently concluded that routine testing for JAK2 V617F is not recommended for patients with venous thrombosis (except for those with SVT) and for all patients with arterial thrombosis, because of the low prevalence of JAK2 V617F in these clinical situations. Nevertheless, they also observed that the value of testing for JAK2 V617F mutation in patients with CVT is yet to be established. Actually, while JAK2 V617F mutation is reported to range from 12% to 74% in SVT patients lacking clinical evidence of MPN, studies that evaluated the frequency of JAK2 V617F mutation in patients with venous thrombosis at other sites (including lower limb deep venous thrombosis, pulmonary embolism, and retinal vein occlusion), found absence or very low prevalence (0.1-3%) of this genetic variant [69]. Similarly, mutation frequency in patients affected by arterial thrombosis (ischemic stroke, acute myocardial infarction, and peripheral arterial thrombosis) is very low (1.1% according to a meta-analysis of 6 studies including 535 subjects) [70].

Although these data could be considered an argument against molecular screening, the role of JAK2 V617F in arterial thrombosis may still be considered. Zerjavic and co-workers detected JAK2 V617F mutation in 1.4% of patients with venous thrombosis and in 3.3% of patients with ischemic stroke from a cohort of 444 patients with venous peripheral thrombosis and/or pulmonary embolism and 60 patients with ischemic stroke of arterial origin. When compared to a control group of 2,430 subjects, the Authors found a significant association between JAK2 V617F mutation and both venous thrombosis (OR 5.53, CI 1.77 to 17.2, p = 0.0053) and ischemic stroke (OR 13.9, CI 2.75 to 70.5, p = 0.0145) [71]. In line with these findings, all the 11 patients who complicated with stroke out of the 102 affected by ET enrolled in a recent longitudinal study [8], showed the presence of JAK2 V617F mutation, suggesting a major role of this genetic variant in the pathogenesis of stroke of arterial origin. A systematic search for MPNs might be especially important for young patients with stroke of undetermined cause [72].

Conclusion

ET may present and complicate with any kind of stroke, and stroke may precede or follow the diagnosis of ET. Clinicians should keep in mind that stroke is a possible manifestation of MPNs, and this relation should be considered especially in those cases of ischemic stroke of arterial origin occurring at young age without plausible, alternative, causative mechanisms, as well as in cases of CVT in which no obvious risk factors are detectable, regardless of PLTs count. The link between JAK2 V617F mutation and stroke (including arterial and venous thrombosis) may extend beyond their connection with MPNs. Searching for JAK2 V617F mutation, although presently not recommended as a screening tool in all patients with stroke, may lead to earlier diagnosis of ET in a considerable number of patients, especially in those with normal or slightly elevated PLTs count and no obvious manifestation of the disease.

References

1. Harrison CN, Bareford D, Butt N, Campbell P, Conneally E, Drummond M, et al. Guideline for investigation and management of adults and children presenting with a thrombocytosis. Br J Haematol. 2010; 149: 352-375.
2. Beer PA, Erber WN, Campbell PJ, Green AR. How I treat essential thrombocythemia. Blood. 2011; 117: 1472-1482.
3. Beer PA, Green AR. Essential thrombocythemia. Chapter 87. 8th edn. Prchal JT, Kaushansky K, Lichtman MA, et al, editors. In: Williams’s hematology. New York: McGraw-Hill. 2010; 1237–1248.
4. Meier B, Burton JH. Myeloproliferative disorders. Emerg Med Clin North Am. 2014; 32: 597-612.
5. Cortelazzo S, Viero P, Finazzi G, D'Emilio A, Rodeghiero F, Barbui T. Incidence and risk factors for thrombotic complications in a historical cohort of 100 patients with essential thrombocythemia. J Clin Oncol. 1990; 8: 556-562.
6. Fenaux P, Simon M, Caulier MT, Lai JL, Goudemand J, Bauters F. Clinical course of essential thrombocythemia in 147 cases. Cancer. 1990; 66: 549-556.
7. Bellucci S, Janvier M, Tobelem G, Flandrin G, Charpak Y, Berger R, et al. Essential thrombocythemias. Clinical evolutionary and biological data. Cancer. 1986; 58: 2440-2447.
8. Posfai E, Marton I, Szoke A, Borbenyi Z, Vecsei L, Csomor A, et al. Stroke in essential thrombocythemia. J Neurol Sci. 2014; 336: 260-262.
9. Landolfi R, Di Gennaro L, Falanga A. Thrombosis in myeloproliferative disorders: pathogenetic facts and speculation. Leukemia. 2008; 22: 2020-2028.
10. Landolfi R, Di Gennaro L, Barbui T, De Stefano V, Finazzi G, Marfisi R, et al. Leukocytosis as a major thrombotic risk factor in patients with polycythemia vera. Blood. 2007; 109: 2446-2452.
11. Artoni A, Bucciarelli P, Martinelli I. Cerebral thrombosis and myeloproliferative neoplasms. Curr Neurol Neurosci Rep. 2014; 14: 496.
12. Radaelli F, Colombi M, Calori R, Zilioli VR, Bramanti S, Iurlo A, et al. Analysis of risk factors predicting thrombotic and/or haemorrhagic complications in 306 patients with essential thrombocythemia. Hematol Oncol. 2007; 25: 115-120.
13. Palandri F, Polverelli N, Catani L, Ottaviani E, Baccarani M, Vianelli N. Impact of leukocytosis on thrombotic risk and survival in 532 patients with essential thrombocythemia: a retrospective study. Ann Hematol. 2011; 90: 933-938.
14. Bazzan M, Tamponi G, Schinco P, Vaccarino A, Foli C, Gallone G, et al. Thrombosis-free survival and life expectancy in 187 consecutive patients with essential thrombocythemia. Ann Hematol. 1999; 78: 539-543.
15. Austin SK, Lambert JR. The JAK2 V617F mutation and thrombosis. Br J Haematol. 2008; 143: 307-320.
16. Passamonti SM, Biguzzi E, Cazzola M, Franchi F, Gianniello F, Bucciarelli P, et al. The JAK2 V617F mutation in patients with cerebral venous thrombosis. J Thromb Haemost. 2012; 10: 998-1003.
17. Denninger MH, Chait Y, Casadevall N, Hillaire S, Guillin MC, Bezeaud A, et al. Cause of portal or hepatic venous thrombosis in adults: the role of multiple concurrent factors. Hepatology. 2000; 31: 587-591.
18. Briere JB. Budd-Chiari syndrome and portal vein thrombosis associated with myeloproliferative disorders: diagnosis and management. Semin Thromb Hemost. 2006; 32: 208-218.
19. Wolanskyj AP, Lasho TL, Schwager SM, McClure RF, Wadleigh M, Lee SJ, et al. JAK2 mutation in essential thrombocythaemia: clinical associations and long-term prognostic relevance. Br J Haematol. 2005; 131: 208-213.
20. Arellano-Rodrigo E, Alvarez-Larran A, Reverter JC, Colomer D, Villamor N, Bellosillo B, et al. Platelet turnover, coagulation factors, and soluble markers of platelet and endothelial activation in essential thrombocythemia: relationship with thrombosis occurrence and JAK2 V617F allele burden. Am J Hematol. 2009; 84: 102-108.
21. Conlan MG, Haire WD. Low protein S in essential thrombocythemia with thrombosis. Am J Hematol. 1989; 32: 88-93.
22. Wieczorek I, MacGregor IR, Ludlam CA. Low proteins C and S and activation of fibrinolysis in treated essential thrombocythemia. Am J Hematol. 1995; 49: 277-281.
23. Bucalossi A, Marotta G, Bigazzi C, Galieni P, Dispensa E. Reduction of antithrombin III, protein C, and protein S levels and activated protein C resistance in polycythemia vera and essential thrombocythemia patients with thrombosis. Am J Hematol. 1996; 52: 14-20.
24. Marchetti M, Castoldi E, Spronk HM, van Oerle R, Balducci D, Barbui T, et al. Thrombin generation and activated protein C resistance in patients with essential thrombocythemia and polycythemia vera. Blood. 2008; 112: 4061-4068.
25. Dienava-Verdoold I, Marchetti MR, te Boome LC, Russo L, Falanga A, Koene HR, et al. Platelet-mediated proteolytic down regulation of the anticoagulant activity of protein S in individuals with haematological malignancies. Thromb Haemost. 2012; 107: 468-476.
26. Trifa AP, Cucuianu A, Popp RA, Coada CA, Costache RM, Militaru MS, et al. The relationship between factor V Leiden, prothrombin G20210A, and MTHFR mutations and the first major thrombotic episode in polycythemia vera and essential thrombocythemia. Ann Hematol. 2014; 93: 203–209.
27. Sun T, Zhang L. Thrombosis in myeloproliferative neoplasms with JAK2V617F mutation. Clin Appl Thromb Hemost. 2013; 19: 374-381.
28. D'Ambrosio D, Della-Morte D, Gargiulo G, Rossetti M, Rundek T, Rengo F, et al. Intrapetrous internal carotid artery dissection and essential thrombocythemia: what relationship? A case report. Cases J. 2008; 1: 354.
29. Verdure P, Lefaucheur R, Guegan-Massardier E, Triquenot-Bagan A, Gerardin E, Maltete D. Bilateral vertebral artery dissection and essential thrombocythemia with JAK2 mutation. Rev Neurol (Paris). 2012; 168: 543-544.
30. Freilinger T, Saam T, Duering M, Dichgans M, Peters N. Internal carotid artery dissection and ischemic cerebral infarction in the setting of essential thrombocythemia. Clin Appl Thromb Hemost. 2011; 17: 138-140.
31. Miller TD, Farquharson MH. Essential thrombocythaemia and its neurological complications. Pract Neurol. 2010; 10: 195-201.
32. Adam R, Priglinger M, Harrington T, Gottlieb D, Krause M. An unusual cause of cerebella hemorrhage in a young patient: essential thrombocythemia. J Stroke Cerebrovasc Dis. 2014; 23: 373-374.
33. Kalala F, Mamara A, Ioannou M, Speletas M. Transient ischemic attacks as the first presentation of JAK2-V617F positive chronic myeloproliferative neoplasm. Hematol Rep. 2012; 4: e12.
34. Falanga A, Marchetti M. Thrombotic disease in the myeloproliferative neoplasms. Hematology Am Soc Hematol Educ Program. 2012; 2012: 571-581.
35. Stam J. Thrombosis of the cerebral veins and sinuses. N Engl J Med. 2005; 352: 1791-1798.
36. Ferro JM, Canhao P, Stam J, Bousser MG, Barinagarrementeria F; ISCVT Investigators. Prognosis of cerebral vein and dural sinus thrombosis: results of the International Study on Cerebral Vein and Dural Sinus Thrombosis (ISCVT). Stroke. 2004; 35: 664-670.
37. Wasay M, Bakshi R, Bobustuc G, Kojan S, Sheikh Z, Dai A, et al. Cerebral venous thrombosis: analysis of a multicenter cohort from the United States. J Stroke Cerebrovasc Dis. 2008; 17: 49-54.
38. Dentali F, Ageno W. Cerebral vein thrombosis. Intern Emerg Med. 2010; 5: 27-32.
39. Coutinho JM, Zuurbier SM, Aramideh M, Stam J. The incidence of cerebral venous thrombosis: a cross-sectional study. Stroke. 2012; 43: 3375-3377.
40. Martinelli I, Passamonti SM, Rossi E, De Stefano V. Cerebral sinus-venous thrombosis. Intern Emerg Med. 2012; 7 Suppl 3: S221-225.
41. Martinelli I, De Stefano V, Carobbio A, Randi ML, Santarossa C, Rambaldi A, et al. Cerebral vein thrombosis in patients with Philadelphia-negative myeloproliferative neoplasms. An European Leukemia Net study. Am J Hematol. 2014; 89: E200-205.
42. Dentali F, Ageno W, Rumi E, Casetti I, Poli D, Scoditti U, et al. Cerebral venous thrombosis and myeloproliferative neoplasms: results from two large databases. Thromb Res. 2014; 134: 41-43.
43. Bousser MG, Ferro JM. Cerebral venous thrombosis: an update. Lancet Neurol. 2007; 6: 162-170.
44. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005; 352: 1779-1790.
45. Hobbs CM, Manning H, Bennett C, Vasquez L, Severin S, Brain L, et al. JAK2V617F leads to intrinsic changes in platelet formation and reactivity in a knock-in mouse model of essential thrombocythemia. Blood. 2013; 122: 3787-3797.
46. Fleischman AG, Tyner JW. Causal role for JAK2 V617F in thrombosis. Blood. 2013; 122: 3705-3706.
47. Sozer S, Fiel MI, Schiano T, Xu M, Mascarenhas J, Hoffman R. The presence of JAK2V617F mutation in the liver endothelial cells of patients with Budd-Chiari syndrome. Blood. 2009; 113: 5246-5249.
48. Nielsen C, Birgens HS, Nordestgaard BG, Kjaer L, Bojesen SE. The JAK2 V617F somatic mutation, mortality and cancer risk in the general population. Haematologica. 2011; 96: 450-453.
49. Nielsen C, Birgens HS, Nordestgaard BG, Bojesen SE. Diagnostic value of JAK2 V617F somatic mutation for myeloproliferative cancer in 49 488 individuals from the general population. Br J Haematol. 2013; 160: 70-79.
50. Shetty S, Kulkarni B, Pai N, Mukundan P, Kasatkar P, Ghosh K. JAK2 mutations across a spectrum of venous thrombosis cases. Am J Clin Pathol. 2010; 134: 82-85.
51. De T, Prabhakar P, Nagaraja D, Christopher R. Janus kinase (JAK) 2 V617F mutation in Asian Indians with cerebral venous thrombosis and without overt myeloproliferative disorders. J Neurol Sci. 2012; 323: 178-182.
52. Colaizzo D, Amitrano L, Tiscia GL, Scenna G, Grandone E, Guardascione MA, et al. The JAK2 V617F mutation frequently occurs in patients with portal and mesenteric venous thrombosis. J Thromb Haemost. 2007; 5: 55-61.
53. De Stefano V, Fiorini A, Rossi E, Za T, Farina G, Chiusolo P, et al. Incidence of the JAK2 V617F mutation among patients with splanchnic or cerebral venous thrombosis and without overt chronic myeloproliferative disorders. J Thromb Haemost. 2007; 5: 708-714.
54. Dentali F, Squizzato A, Brivio L, Appio L, Campiotti L, Crowther M, et al. JAK2V617F mutation for the early diagnosis of Ph- myeloproliferative neoplasms in patients with venous thromboembolism: a meta-analysis. Blood. 2009; 113: 5617-5623.
55. Yoo EH, Jang JH, Park KJ, Gwak GY, Kim HJ, Kim SH, et al. Prevalence of overt myeloproliferative neoplasms and JAK2 V617F mutation in Korean patients with splanchnic vein thrombosis. Int J Lab Hematol. 2011; 33: 471-476.
56. Ziakas PD. Effect of JAK2 V617F on thrombotic risk in patients with essential thrombocythemia: measuring the uncertain. Haematologica. 2008; 93: 1412-1414.
57. Dahabreh IJ, Zoi K, Giannouli S, Zoi C, Loukopoulos D, Voulgarelis M. Is JAK2 V617F mutation more than a diagnostic index? A meta-analysis of clinical outcomes in essential thrombocythemia. Leuk Res. 2009; 33: 67-73.
58. Lussana F, Caberlon S, Pagani C, Kamphuisen PW, Buller HR, Cattaneo M. Association of V617F Jak2 mutation with the risk of thrombosis among patients with essential thrombocythaemia or idiopathic myelofibrosis: a systematic review. Thromb Res. 2009; 124: 409-417.
59. Barbui T, Finazzi G, Carobbio A, Thiele J, Passamonti F, Rumi E, et al. Development and validation of an International Prognostic Score of thrombosis in World Health Organization-essential thrombocythemia (IPSET-thrombosis). Blood. 2012; 120: 5128-5133.
60. Takei H, Morishita S, Araki M, Edahiro Y, Sunami Y, Hironaka Y, et al. Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay. PLoS One. 2014; 9: e104958.
61. Pikman Y, Lee BH, Mercher T, McDowell E, Ebert BL, Gozo M, Cuker A. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med. 2006; 3: e270.
62. Rumi E, Pietra D, Guglielmelli P, Bordoni R, Casetti I, Milanesi C, et al. Acquired copy-neutral loss of heterozygosity of chromosome 1p as a molecular event associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms. Blood. 2013; 121: 4388-4395.
63. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013; 369: 2379-2390.
64. Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC, et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med. 2013; 369: 2391-2405.
65. Gangat N, Wassie EA, Lasho TL, Finke C, Ketterling RP, Hanson CA, et al. Mutations and thrombosis in essential thrombocythemia: prognostic interaction with age and thrombosis history. Eur J Haematol. 2014.
66. Rotunno G, Mannarelli C, Guglielmelli P, Pacilli A, Pancrazzi A, Pieri L, et al. Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia. Blood. 2014; 123: 1552-1555.
67. Rumi E, Pietra D, Ferretti V, Klampfl T, Harutyunyan AS, Milosevic JD, et al. JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes. Blood. 2014; 123: 1544-1551.
68. Richard S, Perrin J, Baillot PA, Lacour JC, Ducrocq X. Ischaemic stroke and essential thrombocythemia: a series of 14 cases. Eur J Neurol. 2011; 18: 995-998.
69. Xavier SG, Gadelha T, Rezende SM, Zalcberg IR, Spector N. JAK2V617F mutation in patients with thrombosis: to screen or not to screen? Int J Lab Hematol. 2011; 33: 117-124.
70. Dentali F, Squizzato A, Appio L, Brivio L, Ageno W. JAK2V617F mutation in patients with arterial thrombosis in the absence of overt myeloproliferative disease. J Thromb Haemost. 2009; 7: 722-725.
71. Zerjavic K, Zagradisnik B, Stangler Herodez S, Lokar L, Glaser Krasevac M, Kokalj Vokac N. Is the JAK2 V617F mutation a hallmark for different forms of thrombosis? Acta Haematol. 2010; 124: 49-56.
72. Alvarez-Larran A, Cervantes F, Bellosillo B, Giralt M, Julia A, Hernandez-Boluda JC, et al. Essential thrombocythemia in young individuals: frequency and risk factors for vascular events and evolution to myelofibrosis in 126 patients. Leukemia. 2007; 21: 1218-1223.
73. Saposnik G, Barinagarrementeria F, Brown RD, Bushnell CD, Cucchiara B, Cushman M, et al. Diagnosis and management of cerebral venous thrombosis: a statement for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2011; 42: 1158-1192.
74. Bousser MG, Ferro JM. Cerebral venous thrombosis: an update. Lancet Neurol. 2007; 6: 162-170.