JAK2 V617F mutational frequency in essential thrombocythemia associated with splanchnic or cerebral vein thrombosis
Authors: Valerio De Stefano, Elena Rossi, Tommaso Za, Angela Ciminello, Silvia Betti, Claudia Luzzi, Giuseppe Leone, Patrizia Chiusolo
Journal: American Journal of Hematology (2011)

Splanchnic vein thrombosis (SVT) and chronic myeloproliferative neoplasms (MPN) are closely related. The JAK2 V617F mutation is a hallmark of MPN but is present also in a relevant percentage of patients with SVT and without an overt chronic MPN. To investigate in patients with essential thrombocythemia (ET) the impact of this mutation on the risk of thrombosis in unusual sites (splanchnic or cerebral veins), we carried out a retrospective cohort study including 224 patients. One hundred forty-two patients (63%) had the mutation, and 68 had suffered either arterial thromboses (n = 39), venous thromboses in a common site (n = 12), or in the eye (n = 1), SVT (n = 13), or cerebral vein thrombosis (CVT; n = 3). All patients with SVT or CVT were <60 years of age: in this age range, the relative risk associated with the mutation, and adjusted for inherited thrombophilia was 1.88 (95%CI 1.32–2.69) for SVT, and 2.26 (95%CI 1.76–2.91) for CVT. No increase in the risk of a venous thrombosis in common sites was found in patients with the mutation. In conclusion, in ET the risk of venous thrombosis associated with the JAK2 V617F mutation preferentially targets splanchnic or cerebral veins in young patients. Splanchnic vein thrombosis (SVT) and cerebral vein thrombosis (CVT) occur very rarely in the general population [1] but are closely related to myeloproliferative neoplasms (MPN). The leading cause of SVT is MPN, diagnosed in half of patients with Budd-Chiari syndrome (BCS) and one-third of patients with an extrahepatic portal vein obstruction (EHPVO) [1-5]. Conversely, thromboses of major abdominal veins has been reported in 4–10% of patients with polycythemia vera (PV) and essential thrombocythemia (ET) [6-8]. CVT is another rare clinical manifestation associated with PV and ET [1]; in a large series of patients with ET, the prevalence of CVT was 1% [7]. The JAK2 V617F mutation is the primary molecular marker of the Philadelphia-negative MPN, occurring in nearly all patients with PV and in about half of those with ET [9, 10]. Moreover, the mutation is present in about one half of patients with BCS, and in one-third of patients with EHPVO who show no additional signs of MPN [1, 11-16]. Patients with SVT and the JAK2 V617F mutation at the time of thrombosis have rate of a MPN development as high as 52% during the follow-up [16]. Preliminary data show that liver endothelial cells of the patients with BCS and PV have the JAK2 V617F mutation, suggesting that the endothelial abnormality may contribute to the prothrombotic state [17]. However, for yet unexplained reasons, in the absence of an overt MPN, the mutation is rare in patients with non-splanchnic venous thromboses [16, 18]. Recent studies have focused on the presence of the JAK2 V617F mutation among patients with CVT and without overt MPN. Two pooled analysis of the available studies demonstrated a mean prevalence of the JAK2 V617F mutation of 1.7–2.5% [16, 19]; however, in some patient series with CVT, the laboratory screening has been reported to be more fruitful for detecting the mutation [12, 20, 21]. The higher prevalence of the JAK2 V617F mutation in patients with SVT and, to a lesser extent, in patients with CVT, when compared with patients with venous thromboses in common sites, suggests a site-linked risk enhanced by the presence of the mutation. It is unclear whether the JAK2 V617F mutation is specifically associated with SVT in patients with overt ET. In this study, we retrospectively investigated the risk for SVT associated with the JAK2 V617F mutation in a cohort of 224 patients with ET. In addition, we investigated the risk of CVT in the same patients. Clinical characteristics of the patients are shown in Table I. The prevalence of SVT and CVT was 5.8 and 1.3%, respectively, in agreement with reports of other patient series with MPN [6-8]. None of the patients with SVT had an increase in red cell mass, confirming the diagnosis of true ET. Inherited thrombophilia was detected in 16 patients (7.1%): one with protein S deficiency, six heterozygous for factor V Leiden, eight heterozygous for prothrombin G20210A, and one with double heterozygosity for factor V Leiden and prothrombin G20210A. Six of the patients had no history of thrombosis, five had arterial thrombosis, one had an SVT, one had a CVT, and three had venous thromboses in common sites. In all cases with SVT and CVT no acquired or circumstantial risk factor for thrombosis was detected besides the diagnosis of ET. The JAK2 V617F mutation was found in 142 patients (63.3%).The distribution of the JAK2 V617F mutation after stratification of the patients according to clinical manifestations is shown in Table II. All patients with an SVT or CVT were younger than 60 years at the time of the event. The median age of the patients with SVT was 34.5 years (range 21–53) and seven were men (54%). The median age of the three patients with CVT was 31 years (range 21–33), and all were men. The presence of the JAK2 V617F mutation increased the relative risk (RR) for overall thrombosis by 45%. The risk was significant for both arterial and venous thromboses. However, after having grouped the patients according to the site of venous thrombosis, the risk associated with the JAK2 V617F mutation was significantly increased for venous thromboses of unusual sites (i.e., SVT or CVT) but not for venous thromboses of common sites (Table II). The results were not substantially changed after the exclusion of patients with inherited thrombophilia (Table II). After excluding patients older than 60 years of age or with thrombophilia, the risk of thrombosis of unusual sites associated with the JAK2 V617F mutation further increased (RR for SVT 1.88, 95%CI 1.32–2.69, RR for CVT 2.26, 95%CI 1.76–2.91). The risk of venous thrombosis of common sites was not significantly increased (Table III). No significant increase in the risk of arterial or venous thrombosis was found among patients older than 60 years with the JAK2 V617F mutation, confirming our previous findings in a smaller cohort of patients with ET (data not shown) [22]. Among the patients with SVT, the JAK2 V617F allele burden was higher than 75% in two of them (15.3%), and no significant difference was found when compared to patients without thrombosis (5.1%, P = 0.17). None of the patients with CVT had a mutated allele burden >75%. In our series, the presence of the mutation was associated with an increased risk of thrombosis, with an equal magnitude for both arterial and venous thromboses, in agreement with previous reports [23-25]. However, previously published reports about the risk for thromboses associated with the JAK2 V617F mutation have not distinguished the specific sites of arterial or venous thromboses nor have they stratified patients according to age [23-25]. We previously demonstrated that the increased risk of thrombosis associated with the JAK2 V617F mutation is further enhanced by the presence of inherited thrombophilia [22]. In our patient series presently analyzed, the risk associated with the JAK2 V617F mutation for venous thrombosis was no more statistically significant after excluding patients with a thrombosis of an unusual site (SVT and CVT). In patients younger than 60 years of age, presence of the JAK2 V617F mutation was associated with an increased risk of venous thrombosis in common sites with borderline statistical significance, which was lost after adjusting for the presence of an inherited thrombophilia. Conversely, in younger patients, the JAK2 V617F mutation was found to be associated with nearly a doubled risk for both SVT and CVT when compared with the patients without the mutation, independent of the presence of an inherited thrombophilia. In particular, the median age of the patients with SVT was 34 years at the time of the event; this is consistent with a median age of 38 years reported in a series of 19 patients with SVT from a cohort of ET patients [8]. In our series, we noticed a slight prevalence of males among patients with SVT, in contrast to the preponderance of females (89%) in the aforementioned report [8]. This discrepancy in the sex distribution could suggest a misdiagnosis of PV among our patients with SVT, in whom hemodilution could occur [26]. However, to rule out a masked PV, red cell mass was measured in all patients with SVT, confirming the diagnosis of ET in every case. In a meta-analysis of the available case-control studies evaluating the association between JAK2 V617F mutation and SVT in the absence of overt MPN, the mutation was found to produce an increase in risk for SVT as high as 54-fold [16]. In this study, focused on patients with overt ET, the association of the mutation with SVT resulted significantly increased but much weaker, being nearly double in comparison with the patients without the mutation. This could be explained by the presence in overt ET of additional factors, possibly in part yet unknown, playing a major role in pathogenesis of thrombosis. The patients we investigated were consecutively diagnosed at our center. The overall cohort demonstrated typical characteristics of ET in regards to the sex distribution (67% females), the median age at diagnosis (55 years), the prevalence of the JAK2 V617F mutation (63%), and the rate of thrombosis (30%) [27]. The prevalences of SVT (5.8%) and CVT (1.3%) were consistent with those reported in prior investigations [7, 8]. Therefore, our results are unlikely to be biased towards a preferential recruitment of patients with thromboses of unusual sites and can be generalized for all patients with ET. In conclusion, presence of the JAK2 V617F mutation is confirmed as a risk factor for arterial and venous thromboses in patients with ET. Indeed, the associated risk appears to be relevant only in younger patients. Being older than 60 years of age is a well-recognized risk factor for thrombosis in ET patients [27] and the null contribution of the JAK2 V617F mutation to the thrombotic risk in this setting suggests that older age is a strong enough risk factor that it obscures the role of other predictors. Here, we demonstrated that in patients with ET the risk for venous thrombosis associated with the JAK2 V617F mutation preferentially targets the splanchnic or cerebral veins, reinforcing the close association between JAK2 V617F and SVT, and possibly CVT, observed in patients without overt MPN. We conducted a retrospective cohort study among patients with a diagnosis of ET consecutively admitted to our center since 2002. The diagnosis was established according to either previous [28] or updated WHO criteria [9, 10]. In all patients diagnosed before 2007, the diagnosis of ET was re-evaluated and confirmed according to the updated WHO criteria [9, 10]. After providing informed consent, all patients underwent laboratory screening for thrombophilia. Additional blood samples were taken from all patients; genomic DNA was extracted from peripheral blood granulocytes by standard procedures and archived. Additionally, all patients provided informed consent for future investigations on the archived DNA. According to our criteria, 224 out of 233 patients referred to our center between 2002 and 2009 with a stored or fresh DNA sample available for JAK2 mutational analysis were recruited for the study. This study was approved by the institutional review board. The thrombotic events of interest included those that occurred after a referral to our center and a diagnosis of ET or occurred within the two years preceding the diagnosis. These criteria were based on the knowledge that 75% of thrombotic events heralding PV occur within this interval of time [29]. Therefore, patients with a presentation during this period were considered to have the thrombosis as an inaugural manifestation of the disease. The thrombotic events of interest included ischemic stroke, transient ischemic attack (TIA), acute myocardial infarction, unstable angina pectoris, peripheral arterial thrombosis, retinal artery or vein occlusion, deep venous thrombosis (including thrombosis of cerebral and splanchnic veins), and pulmonary embolism. The definition of acute coronary syndrome encompasses acute myocardial infarction and unstable angina pectoris. The definition of cerebrovascular disease encompasses ischemic stroke and TIA. Splanchnic venous thrombosis includes occlusion of hepatic, portal, mesenteric, and splenic veins. Moreover, superficial vein thromboses diagnosed by ultrasound methods were also included as events of interest. Microcirculatory events (including vascular headaches, dizziness, visual disturbances, burning pain sensations in the palms of the hands and soles of the feet, distal paresthesia, and acrocyanosis) were not included as events of interest. As previously described, a diagnosis of thrombosis was accepted only if it was objectively proven according to previously published criteria or if the diagnosis was made in the presence of unequivocal medical documentation recording the occurrence of angina or TIA [22]. Laboratory screening for inherited and acquired thrombophilia, the presence of the JAK2 V617F mutation, and an estimate of the JAK2 mutated allele burden were performed as previously reported [22]. Screening for thrombophilia included measuring antithrombin and protein C functional activities, free protein S antigen, and fasting homocysteine; searching for the FV Leiden, for the PT G20210A, and for antiphospholipid antibodies (lupus anticoagulant, anticardiolipin antibodies, and anti-b2glycoprotein I) [22]. All patients with SVT, in whom hemodilution can occur [26], had no increase in their red cell mass evaluated by 51Cr-labeled erythrocytes, a test performed to rule out masked PV [30]. The RR with a 95% confidence interval (95% CI) was estimated by a 2 × 2 contingency table to estimate the probability of thrombosis.