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Is JAK2 V617F mutation more than a diagnostic index?
Available online at www.sciencedirect.com
Leukemia Research 33 (2009) 67–73
Is JAK2 V617F mutation more than a diagnostic index? A meta-analysis of clinical outcomes in essential thrombocythemia Issa J. Dahabreh a,b , Katerina Zoi a , Stavroula Giannouli b , Christine Zoi a,b , Dimitrios Loukopoulos a , Michael Voulgarelis b,∗ b
a Hematology Research Laboratory, Biomedical Research Foundation, Academy of Athens, Athens, Greece Department of Pathophysiology, Medical School, National University of Athens, 75 Mikras Asias Street, 11527 Athens, Greece
Received 28 March 2008; received in revised form 2 May 2008; accepted 6 June 2008 Available online 15 July 2008
Abstract A systematic review and meta-analysis was carried out to compare the frequency of clinically significant outcomes between JAK2 V617F positive and wild type patients with essential thrombocythemia (ET). JAK2 V617F positivity in patients with ET was associated with a clear increase in the odds of thrombosis [OR = 1.83 (95% CI, 1.32–2.53), p < 0.0001], and much higher odds of transformation to polycythemia vera [OR = 7.67 (95% CI, 2.04–28.87), p = 0.0009]. The mean difference of the white blood cell count between JAK2 positive and negative patients was associated with an increased odds ratio for thrombosis (p = 0.02). The JAK2 V617F mutation in patients with ET is associated with an increased risk of adverse cardiovascular outcomes via an increase in the leukocyte count. © 2008 Elsevier Ltd. All rights reserved. Keywords: Essential thrombocythemia; JAK2 V617F; Thrombosis; Systematic review; Meta-analysis
1. Introduction A point mutation in the jak2 gene, resulting in a substitution of valine for phenylalanine (JAK2 V617F), has been identified as a unifying event for typical chronic myeloproliferative disorders, rarely occurring in atypical myeloproliferative disorders or other myeloid malignancies [1–4]. Although present in the vast majority of polycythemia vera (PV) patients, it is seen in significantly fewer essential thrombocythemia (ET) patients, with an estimated prevalence of approximately 50%. ET is characterized by megakaryocytic hyperplasia, splenomegaly and its clinical course is complicated by hemorrhagic and thrombotic episodes as well as the potential for transformation to acute leukemia. It has been suggested that the JAK2 mutational status identifies two distinct subtypes of ET regarding their hematological and clinical characteristics [5]. Studies have consistently demonstrated that the JAK2 ∗
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0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2008.06.006
V617F positive ET confers a “polycythemic” laboratory profile, with higher hemoglobin values, leukocytosis, and lower erythropoietin levels [6,7]. These findings are consistent with animal models demonstrating that JAK2 V617F skews the myeloproliferative phenotype towards a polycythemic variant, thereby suggesting that this mutation, even if not pathogenetic, is a phenotype-defining mutation in myeloproliferative disorders [8–10]. On the contrary, results regarding clinically significant characteristics, such as the rate of thrombosis, splenomegaly and disease progression to PV, myelofibrosis or leukemia have been relatively inconsistent, mostly due to the small sample size of reported studies and the low number of events that have been recorded. In an effort to investigate whether JAK2 V617F positivity is associated with specific clinical characteristics, we performed a systematic review and meta-analysis to identify and pool studies comparing the two molecularly defined subtypes of ET. Furthermore, we used meta-regression analysis to evaluate the influence of thrombocytosis, leukocytosis and polycythemia on the incidence of thrombosis.
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2. Methods 2.1. Study eligibility and data abstraction We used the MEDLINE, EMBASE and KoreaMed online databases (January, 2005 to February 2008) to identify published studies investigating the JAK2 mutational status of patients with ET. To increase the yield of our search strategy we contacted experts in the field of myeloproliferative disorders, searched the reference lists of review articles and retrieved clinical studies. Studies were considered eligible if the enrollment of patients was based on standardized diagnostic criteria, such as those proposed by the Polycythemia Vera Study Group (PVSG) or the World Health Organization (WHO, 2001). Studies selecting patients for a specific clinical outcome (e.g., thrombosis) or reporting only on patients positive for the JAK2 V617F mutation were excluded from this analysis. Case reports, studies of familial myeloproliferative disorders, reviews and animal studies were also excluded. Studies were considered eligible when they reported on at least one of the following endpoints: major thrombosis (including transient ischemic attacks, acute coronary syndromes, strokes, peripheral arterial thombosis, deep venus thrombosis and pulmonary embolism and venous thromboembolism), splenomegaly, evolution to polycythemia vera, myelofibrosis, or acute myeloid leukemia. When multiple reports from the same patient cohort were available, the one with the largest number of patients was used. Two reviewers (IJD and MV) independently abstracted and tabulated the following information for each eligible study: first author, journal and year of publication, number of patients included, diagnostic criteria for ET and method of mutational analysis. Furthermore, the number of JAK2 V617F positive and negative patients was recorded as well as the number of patients from each group that during the course of disease received cytoreductive treatment (hydroxyurea or anagrelide), experienced thrombosis (either arterial or venous), or a major hemorrhagic event, developed splenomegaly, or had disease transformation into PV, myelofibrosis or myeloid leukemia. Although our study protocol called for subgroup analysis by specific cytoreductive treatments (i.e., hydroxyurea versus anagrelide), the limited number of trials providing such information prevented valid comparisons.
Fixed effects meta-regression analysis was used to explore potential sources of heterogeneity, wherever detected [19]. The aim of meta-regression analysis is to investigate whether a particular study-level characteristic is related to the extent of the association between presence of the mutation and thrombosis. This analysis of a study-level characteristic as a source of heterogeneity in meta-analysis is based on weighted regression [19]. To avoid inflating type I error (“false positive”), we only evaluated the influence of hemoglobin concentration and mean white blood cell (WBC) and platelet counts on the OR for thrombosis, thus limiting the influence that multiple comparisons could have on our results. All statistical tests were two-tailed and statistical significance was defined as p < 0.05 for all comparisons, except heterogeneity. Given the low power of Cochran’s Q-test, heterogeneity was considered statistically significant at p < 0.1. Analysis was conducted using STATA (Version SE 10, College Station, TX, USA), Comprehensive Meta-analysis (Version 2.2, Biostat, Englewood, NJ, USA), and SPSS (Version 13.0, SPSS Inc., Chicago, IL, USA).
3. Results 3.1. Study flow and eligible studies Overall, 394 studies were reviewed and 87 were considered potentially eligible and were retrieved in full text, 17 of which were considered eligible for the meta-analysis as they provided data regarding at least one of the endpoints of interest [6,7,20–36]. These studies reported on 2436 patients of whom 1375 (56.5%) were found to be positive for the JAK2 mutation. The overall incidence of thrombosis was 26.4% (18.9% arterial and 8.3% venous), hemorrhage 5.2%, splenomegaly 14.6%, transformation to PV 1.3%, progression to myelofibrosis 3% and leukemic transformation 1%. The characteristics of the studies and incidence of selected outcome events are summarized in Table 1.
2.2. Statistical analysis The number of JAK2 V617F positive and negative patients and the number of events per group were used to calculate odds ratios (OR) and their 95% confidence intervals (CI). Betweenstudy heterogeneity and inconsistency were evaluated with the Q and I2 statistics, respectively [11]. The Begg–Mazumdar and Egger tests were used to evaluate small study effects [12,13]. OR were combined using the Mantel–Haenszel fixed effects model to estimate pooled point estimates and their confidence intervals [14,15]. Where heterogeneity was detected, the DerSimonian–Laird random effects model was also used, since it assumes genuine between study heterogeneity and thus produces more conservative measures of association [16]. Where zero events were observed standard continuity correction (k = 0.5) was employed [17]. Using an arbitrary cutoff of 100 patients, we compared larger studies with smaller ones by pooling the odds ratios for thrombosis in each subgroup separately. We compared the odds ratios for thrombosis in these two subgroups using a test for interaction [18].
3.2. Data synthesis Significant heterogeneity (p = 0.03) and moderate between-study inconsistency (I2 = 44.1%) was identified only in relation to the overall incidence of thrombosis. For this outcome, fixed and random effects models provided similar pooled point estimates, OR = 1.88 (95% CI, 1.55–2.28), p < 0.0001 and OR = 1.83 (95% CI, 1.32–2.53), p = 0.0003, respectively (Fig. 1). This indicates that in patients with ET presence of JAK2 V617F is associated with a highly statistically significant increase in the odds of thrombosis of approximately 90%. Five studies [6,7,20,21,35] including the three studies that received the highest weight in the overall meta-analysis [6,7,35] allowed a distinction between thrombotic events at diagnosis compared to those occurring during follow-up. Based on these studies, the JAK2 V617F mutation was associated with increased odds for thrombosis
Table 1 Characteristics of eligible studies and incidence of selected outcome events Patient number
Male/female
Diagnostic criteria
Method of mutation detection
JAK2 positive patients (%)
Patient age in years (JAK2 V617F/wild type)
Thrombotic events (%)
Hemorrhagic events (%)
Splenomegaly (%)
Baxter et al. (2005) Campbell et al. (2005) Cheung (2006) Horn (2006) Heller et al. (2006) Stevenson et al. (2006) Ahn et al. (2007) Alvarez-Larran et al. (2007) Andrikovics et al. (2007) Hsiao et al. (2007) Kittur et al. (2007) Ohyashiki et al. (2007) Pemmaraju et al. (2007) Rudzki et al. (2007) Speletas et al. (2007) Toyama et al. (2007) Vannucchi (2007)
51 776 60 23 50 27 24 103 71 53 176 51 80 59 111 82 639
24/27 312/464 24/36 9/14 16/34 12/15 14/11 34/69 17/54 20/33 66/110 24/27 22/58 26/33 25/17 31/51 202/437
PVSG PVSG NR WHO PVSG PVSG PVSG or WHO PVSG WHO PVSG WHO WHO PVSG WHO PVSG WHO PVSG or WHO
SNP screening and AS PCR AS PCR and BsaXI RFLP AS PCR AS PCR and BsaXI RFLP AS RT PCR and BsaXI RFLP DHPLC and direct sequencing AS PCR AS PCR AS PCR ARMS PCR AS PCR and AS qPCR SSP-SMFD qRT-PCR AS PCR AS PCR Direct sequencing and AS PCR AS PCR
29 (56.9) 414 (53.4) 29 (48.3) 17 (73.9) 24 (48.0) 10 (37.0) 11 (45.8) 44 (42.7) 41 (57.7) 35 (66.0) 96 (54.5) 32 (62.7) 38 (47.5) 38 (64.4) 77 (69.4) 58 (70.7) 382 (59.8)
54/55 60/52 46/81 62a 48/30 60/52 67/59 30/29 54/48 61.8/55.4 62/48 63.6/60.6 59/38 50.7/47.8 63.6/60.7 65/61.5 52.4/46.5b
12 (23.5) 137 (17.7) 26 (43.3) NR 12 (24.0) 7 (25.9) 10 (41.7) 22 (21.4) 15 (21.1) 17 (32.1) 70 (39.8) 10 (19.6) 26 (32.5) 24 (40.7) 45 (40.5) 16 (19.5) 188 (29.4)
NR 32 (4.1) NR NR NR NR NR 10 (9.7) 2 (2.8) NR NR NR 2 (2.5) 7 (11.9) 4 (3.6) 7 (8.5) 15 (5.8)c
NR 22 (3.6) NR 14 (60.8) NR NR 8 (47.1) NR 12 (16.9) NR 33 (18.8) 10 (19.6) NR 17 (28.8) 18 (16.2) NR 122 (19.1)
ARMS, Amplification refractory mutation system; AS, allele specific; DHPLC, denaturing high performance liquid chromatography; NA, not applicable; NR, not reported; PCR, polymerase chain reaction; PVSG, polycythemia vera study group criteria; qPCR, quantitative PCR; qRT, quantitative real time; RFLP, restriction fragment length polymorphism; RT, real time; SNP, small nuclear polymorphism; SSP-SMFD, sequence-specific primer-single molecule fluorescence detection assay; WHO, World Health Organization (2001) criteria. a Study only reported aggregate patient age. b Excludes 9 homozygous JAK2 V617F patients. c Data from Antonioli et al. (2008) [36].
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Study
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Fig. 1. Forest plot for the overall incidence of thrombosis. Each study is shown by the point estimate of the odds ratio (square proportional to the weight of each study) and 95% confidence interval for the odds ratio (extending lines); the pooled odds ratio and 95% confidence interval by random effects calculations is depicted as a diamond. Values higher than 1 indicate that JAK2 V617F positive patients are at a higher risk for thrombosis. Studies are arranged by year of publication.
both at ET diagnosis, OR = 2.05 (95% CI, 1.53–2.74), p < 0.0001, and during follow-up, OR = 1.47 (95% CI, 1.07–2.02), p = 0.02. Small study effects were not significant for any outcome, indicating that small and larger studies provide consistent evidence of an association between presence of JAK2 V617F and thrombosis [37]. When studies enrolling less than 100 patients were compared to larger studies, the results were similar. The random effects OR for thrombosis based on small studies was 2.34 (95% CI, 1.23–4.48), p = 0.01, while the OR based on large studies was 1.76 (95% CI, 1.35–2.29), p < 0.0001; interaction p-value = 0.42. For remaining outcomes, as no heterogeneity was detected, all comparisons between JAK2 V617F positive and negative patients are presented as fixed effects OR. Discrepancies between fixed and random models were minimal (data not shown). JAK2 V617F positivity was associated with an increased risk for both arterial [OR = 1.68 (95% CI, 1.31–2.15), p < 0.0001] and venous [OR = 2.5 (95% CI, 1.71–3.66), p < 0.0001] thromboses. On the contrary, no difference was observed in hemorrhagic events between JAK2 V617F positive and wild type patients, OR = 0.90 (95% CI, 0.58–1.41), p = 0.73. Though splenomegaly appeared to be associated with JAK2 positivity, the association was marginal, OR = 1.32 (95% CI, 1–1.75), p = 0.06. Surprisingly, the mutational status was not associated with the use
of cytoreductive therapy, OR = 1.08 (95% CI, 0.79–1.50), p = 0.67. JAK2 V617F did not appear to influence the odds for progression of the disease to acute leukemia, OR = 1.44 (95% CI, 0.54–3.82), p = 0.62, or myelofibrosis, OR = 0.64 (95% CI, 0.33–1.23), p = 0.24. On the contrary, there was a clear association between the presence of JAK2 V617F and evolution of ET into PV; OR = 7.67 (95% CI, 2.04–28.87), p = 0.0009. Notably, from a total of 1267 ET patients with available data, 16 (1.3%) experienced disease transformation to PV, all of whom were positive for the JAK2 V17F mutation. 3.3. Meta-regression analysis As previously discussed, there was significant betweenstudy heterogeneity regarding the influence of JAK2 V617F on the odds of thrombosis. To explore this heterogeneity, we employed meta-regression analysis to investigate the effect of the mean difference in hemoglobin concentration, WBC and platelet counts on the odds ratio of thrombosis between JAK2 V617F positive and wild type patients. The WBC count mean difference was significantly associated with increased odds for thrombosis in JAK2 mutant patients (p = 0.02; Fig. 2). On the contrary, the mean difference in hemoglobin concentration or platelet count did not appear to affect the risk of thrombosis (p = 0.11 and p = 0.60, respectively).
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Fig. 2. Relationship between the differences in the mean white blood cell (WBC) count between JAK2 positive and wild type patient groups and the odds ratio for thrombosis. Differences (JAK2 positive minus wild type) were calculated by subtracting the mean WBC count at diagnosis of the wild type group of each trial from that of the group of JAK2 V617F positive patients. The continuous line is the fitted regression line of the mean WBC count difference on the logarithm of the OR (log OR) for thrombosis. Each study is represented by a circle proportional to its weight in the meta-analysis. The association between log OR and the WBC mean difference is significant (p = 0.02).
4. Discussion The discovery of the JAK2 V617F mutation, in providing an insight into the pathogenesis of chronic myeloproliferative disorders, has revolutionized the diagnostic approach to these myeloid malignancies [38,39]. Despite the significance of JAK2 V617F in disease classification, reports regarding its clinical relevance have been controversial, mostly due to the small size of cohorts and low incidence of outcome events. We attempted to address such restrictions by conducting a meta-analysis of published studies. Evidence based management of ET is guided by an accurate assessment of cardiovascular risk, primarily defined by patient age and previous history of thrombosis [40]. Our data suggest that JAK2 V617F is a marker associated with thrombosis and highlight the need for prospective studies to evaluate the potential role of mutational status in designing a rational treatment strategy. Furthermore, our analysis demonstrated that leukocytosis influences the risk of thrombosis as the mean difference of WBC count between JAK2 positive and JAK2 negative patients was associated with an increased odds ratio for thrombosis. The fact that JAK2 mutational status did not appear to be associated with bleeding events provides an indication that coagulation defects in ET are multifactorial and cannot be explained by JAK2 V617F alone. Disease progression into myelofibrosis or leukemia did not appear to be influenced by the presence of JAK2 mutation, although the number of studies reporting on these outcomes and the available follow-up periods may be inadequate to allow definitive conclusions. On the other hand, the association of JAK2 V617F positivity with an eight-fold increase in the risk of transformation of ET to PV is in agreement with the notion that JAK2 V617F in ET confers a rather “polycythemic” myeloproliferative phenotype [7,42–44]. Interestingly, large retrospective studies have identified leukocytosis as an independent risk factor for thrombosis among patients with ET [45,46]. To this effect, using
meta-regression analysis we demonstrated that the higher the difference of mean WBC count at diagnosis between JAK2 V617F positive and wild type patients, the higher the odds for thrombosis for patients carrying the mutation. Notably, we found no such association with hemoglobin concentration or platelet count. Taken together, these findings indicate that JAK2 V617F mutation may partly exert its effect on the risk of thrombosis through an increase in the leukocyte count. Several studies have demonstrated that among patients with ET, those positive for the JAK2 V617F mutation show evidence of elevated CD11b and tissue factor expression by neutrophils, platelet P-selectin expression and persistent formation of platelet–leukocyte aggregates, all contributing to higher risk for thrombosis [47–49]. The increased number and activation of WBCs conferred by JAK2 V617F may enhance their interaction with the endothelium, particularly at sites of inflammation or injury, thus increasing the risk of thrombosis [48,50]. Recently, interest has focused on the potential association between JAK2 V617F allele burden and thrombosis [6,31,36]. Studies have thus far been somewhat inconsistent and further research is warranted. The triple association between leukocytosis, JAK2 V617F mutation and increased thrombotic risk may also explain why anagrelide, a platelet-specific drug, was found to be inferior compared to hydroxyurea [51]. The later, being a non-selective cytoreductive agent, is quite effective in reducing the risk of thrombosis in patients with ET, targeting both platelets and leukocytes [52]. This notion is further supported by studies demonstrating that hydroxyurea inhibits the P-selectin-mediated tissue factor expression by neutrophils and reduces the formation of platelet–neutrophil aggregates [53]. The surprising finding that cytoreductive treatment was administered at the same rate to JAK2 positive and wild-type patients possibly reflects the fact that management decisions are often not based solely on the assessment of thrombotic history and patient age but also take into account other factors,
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such as co-existing cardiovascular risk factors or thrombocytosis. Although based on a comprehensive literature search, the present analysis is limited by the unavailability of individual patient data that would allow time-to-event analysis and a direct investigation of any interaction between JAK2 mutational status and factors such as leukocytosis, disease duration or established cardiovascular risk factors. Our results should therefore be primarily viewed in the context of a hypothesis pending prospective validation. In conclusion, based on the totality of available published evidence, our analysis indicates that the presence of JAK2 mutation in patients with ET is associated with an increased risk of adverse cardiovascular outcomes, and suggests that its effect on thrombotic risk may be primarily mediated by an increase in the leukocyte count.
Conflict of interest The authors have no conflict of interest to report.
Acknowledgements The authors received no financial or other support for the research reported in this manuscript. Contributors. I.J.D. conceived of the study and performed the data analysis. I.J.D. and M.V. designed the study protocol and performed the data extraction. S.G., K.Z., C.Z. and D.L. assessed study eligibility for the meta-analysis and provided valuable clinical and laboratory perspective. All authors interpreted the results of the statistical analysis. I.J.D. drafted the manuscript and all authors revised it critically. M.V. is the guarantor of the study.
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[42] Tefferi A, Elliott M. Thrombosis in myeloproliferative disorders: prevalence, prognostic factors, and the role of leukocytes and JAK2V617F. Semin Thromb Hemost 2007;33:313–20. [43] Dupont S, Masse A, James C, Teyssandier I, Lecluse Y, Larbret F, et al. The JAK2 617V > F mutation triggers erythropoietin hypersensitivity and terminal erythroid amplification in primary cells from patients with polycythemia vera. Blood 2007;110:1013–21. [44] Jamieson CH, Gotlib J, Durocher JA, Chao MP, Mariappan MR, Lay M, et al. The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation. Proc Natl Acad Sci USA 2006;103:6224–9. [45] Carobbio A, Finazzi G, Guerini V, Spinelli O, Delaini F, Marchioli R, et al. Leukocytosis is a risk factor for thrombosis in essential thrombocythemia: interaction with treatment, standard risk factors, and Jak2 mutation status. Blood 2007;109:2310–3. [46] Tefferi A, Gangat N, Wolanskyj A. The interaction between leukocytosis and other risk factors for thrombosis in essential thrombocythemia. Blood 2007;109:4105. [47] Falanga A, Marchetti M, Vignoli A, Balducci D, Russo L, Guerini V, et al. V617F JAK-2 mutation in patients with essential thrombocythemia: relation to platelet, granulocyte, and plasma hemostatic and inflammatory molecules. Exp Hematol 2007;35:702–11. [48] Falanga A, Marchetti M, Evangelista V, Vignoli A, Licini M, Balicco M, et al. Polymorphonuclear leukocyte activation and hemostasis in patients with essential thrombocythemia and polycythemia vera. Blood 2000;96:4261–6. [49] Arellano-Rodrigo E, Alvarez-Larran A, Reverter JC, Villamor N, Colomer D, Cervantes F. Increased platelet and leukocyte activation as contributing mechanisms for thrombosis in essential thrombocythemia and correlation with the JAK2 mutational status. Haematologica 2006;91:169–75. [50] McIntyre TM, Prescott SM, Weyrich AS, Zimmerman GA. Cell-cell interactions: leukocyte–endothelial interactions. Curr Opin Hematol 2003;10:150–8. [51] Harrison CN, Campbell PJ, Buck G, Wheatley K, East CL, Bareford D, et al. Hydroxyurea compared with anagrelide in high-risk essential thrombocythemia. N Engl J Med 2005;353:33–45. [52] Barbui T, Finazzi G. When and how to treat essential thrombocythemia. N Engl J Med 2005;353:85–6. [53] Maugeri N, Giordano G, Petrilli MP, Fraticelli V, de Gaetano G, Cerletti C, et al. Inhibition of tissue factor expression by hydroxyurea in polymorphonuclear leukocytes from patients with myeloproliferative disorders: a new effect for an old drug? J Thromb Haemost 2006;4:2593–8.
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Is JAK2 V617F mutation more than a diagnostic index? A meta-analysis of clinical outcomes in essential thrombocythemia Issa J. Dahabreh a,b , Katerina Zoi a , Stavroula Giannouli b , Christine Zoi a,b , Dimitrios Loukopoulos a , Michael Voulgarelis b,∗ b
a Hematology Research Laboratory, Biomedical Research Foundation, Academy of Athens, Athens, Greece Department of Pathophysiology, Medical School, National University of Athens, 75 Mikras Asias Street, 11527 Athens, Greece
Received 28 March 2008; received in revised form 2 May 2008; accepted 6 June 2008 Available online 15 July 2008
Abstract A systematic review and meta-analysis was carried out to compare the frequency of clinically significant outcomes between JAK2 V617F positive and wild type patients with essential thrombocythemia (ET). JAK2 V617F positivity in patients with ET was associated with a clear increase in the odds of thrombosis [OR = 1.83 (95% CI, 1.32–2.53), p < 0.0001], and much higher odds of transformation to polycythemia vera [OR = 7.67 (95% CI, 2.04–28.87), p = 0.0009]. The mean difference of the white blood cell count between JAK2 positive and negative patients was associated with an increased odds ratio for thrombosis (p = 0.02). The JAK2 V617F mutation in patients with ET is associated with an increased risk of adverse cardiovascular outcomes via an increase in the leukocyte count. © 2008 Elsevier Ltd. All rights reserved. Keywords: Essential thrombocythemia; JAK2 V617F; Thrombosis; Systematic review; Meta-analysis
1. Introduction A point mutation in the jak2 gene, resulting in a substitution of valine for phenylalanine (JAK2 V617F), has been identified as a unifying event for typical chronic myeloproliferative disorders, rarely occurring in atypical myeloproliferative disorders or other myeloid malignancies [1–4]. Although present in the vast majority of polycythemia vera (PV) patients, it is seen in significantly fewer essential thrombocythemia (ET) patients, with an estimated prevalence of approximately 50%. ET is characterized by megakaryocytic hyperplasia, splenomegaly and its clinical course is complicated by hemorrhagic and thrombotic episodes as well as the potential for transformation to acute leukemia. It has been suggested that the JAK2 mutational status identifies two distinct subtypes of ET regarding their hematological and clinical characteristics [5]. Studies have consistently demonstrated that the JAK2 ∗
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0145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2008.06.006
V617F positive ET confers a “polycythemic” laboratory profile, with higher hemoglobin values, leukocytosis, and lower erythropoietin levels [6,7]. These findings are consistent with animal models demonstrating that JAK2 V617F skews the myeloproliferative phenotype towards a polycythemic variant, thereby suggesting that this mutation, even if not pathogenetic, is a phenotype-defining mutation in myeloproliferative disorders [8–10]. On the contrary, results regarding clinically significant characteristics, such as the rate of thrombosis, splenomegaly and disease progression to PV, myelofibrosis or leukemia have been relatively inconsistent, mostly due to the small sample size of reported studies and the low number of events that have been recorded. In an effort to investigate whether JAK2 V617F positivity is associated with specific clinical characteristics, we performed a systematic review and meta-analysis to identify and pool studies comparing the two molecularly defined subtypes of ET. Furthermore, we used meta-regression analysis to evaluate the influence of thrombocytosis, leukocytosis and polycythemia on the incidence of thrombosis.
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2. Methods 2.1. Study eligibility and data abstraction We used the MEDLINE, EMBASE and KoreaMed online databases (January, 2005 to February 2008) to identify published studies investigating the JAK2 mutational status of patients with ET. To increase the yield of our search strategy we contacted experts in the field of myeloproliferative disorders, searched the reference lists of review articles and retrieved clinical studies. Studies were considered eligible if the enrollment of patients was based on standardized diagnostic criteria, such as those proposed by the Polycythemia Vera Study Group (PVSG) or the World Health Organization (WHO, 2001). Studies selecting patients for a specific clinical outcome (e.g., thrombosis) or reporting only on patients positive for the JAK2 V617F mutation were excluded from this analysis. Case reports, studies of familial myeloproliferative disorders, reviews and animal studies were also excluded. Studies were considered eligible when they reported on at least one of the following endpoints: major thrombosis (including transient ischemic attacks, acute coronary syndromes, strokes, peripheral arterial thombosis, deep venus thrombosis and pulmonary embolism and venous thromboembolism), splenomegaly, evolution to polycythemia vera, myelofibrosis, or acute myeloid leukemia. When multiple reports from the same patient cohort were available, the one with the largest number of patients was used. Two reviewers (IJD and MV) independently abstracted and tabulated the following information for each eligible study: first author, journal and year of publication, number of patients included, diagnostic criteria for ET and method of mutational analysis. Furthermore, the number of JAK2 V617F positive and negative patients was recorded as well as the number of patients from each group that during the course of disease received cytoreductive treatment (hydroxyurea or anagrelide), experienced thrombosis (either arterial or venous), or a major hemorrhagic event, developed splenomegaly, or had disease transformation into PV, myelofibrosis or myeloid leukemia. Although our study protocol called for subgroup analysis by specific cytoreductive treatments (i.e., hydroxyurea versus anagrelide), the limited number of trials providing such information prevented valid comparisons.
Fixed effects meta-regression analysis was used to explore potential sources of heterogeneity, wherever detected [19]. The aim of meta-regression analysis is to investigate whether a particular study-level characteristic is related to the extent of the association between presence of the mutation and thrombosis. This analysis of a study-level characteristic as a source of heterogeneity in meta-analysis is based on weighted regression [19]. To avoid inflating type I error (“false positive”), we only evaluated the influence of hemoglobin concentration and mean white blood cell (WBC) and platelet counts on the OR for thrombosis, thus limiting the influence that multiple comparisons could have on our results. All statistical tests were two-tailed and statistical significance was defined as p < 0.05 for all comparisons, except heterogeneity. Given the low power of Cochran’s Q-test, heterogeneity was considered statistically significant at p < 0.1. Analysis was conducted using STATA (Version SE 10, College Station, TX, USA), Comprehensive Meta-analysis (Version 2.2, Biostat, Englewood, NJ, USA), and SPSS (Version 13.0, SPSS Inc., Chicago, IL, USA).
3. Results 3.1. Study flow and eligible studies Overall, 394 studies were reviewed and 87 were considered potentially eligible and were retrieved in full text, 17 of which were considered eligible for the meta-analysis as they provided data regarding at least one of the endpoints of interest [6,7,20–36]. These studies reported on 2436 patients of whom 1375 (56.5%) were found to be positive for the JAK2 mutation. The overall incidence of thrombosis was 26.4% (18.9% arterial and 8.3% venous), hemorrhage 5.2%, splenomegaly 14.6%, transformation to PV 1.3%, progression to myelofibrosis 3% and leukemic transformation 1%. The characteristics of the studies and incidence of selected outcome events are summarized in Table 1.
2.2. Statistical analysis The number of JAK2 V617F positive and negative patients and the number of events per group were used to calculate odds ratios (OR) and their 95% confidence intervals (CI). Betweenstudy heterogeneity and inconsistency were evaluated with the Q and I2 statistics, respectively [11]. The Begg–Mazumdar and Egger tests were used to evaluate small study effects [12,13]. OR were combined using the Mantel–Haenszel fixed effects model to estimate pooled point estimates and their confidence intervals [14,15]. Where heterogeneity was detected, the DerSimonian–Laird random effects model was also used, since it assumes genuine between study heterogeneity and thus produces more conservative measures of association [16]. Where zero events were observed standard continuity correction (k = 0.5) was employed [17]. Using an arbitrary cutoff of 100 patients, we compared larger studies with smaller ones by pooling the odds ratios for thrombosis in each subgroup separately. We compared the odds ratios for thrombosis in these two subgroups using a test for interaction [18].
3.2. Data synthesis Significant heterogeneity (p = 0.03) and moderate between-study inconsistency (I2 = 44.1%) was identified only in relation to the overall incidence of thrombosis. For this outcome, fixed and random effects models provided similar pooled point estimates, OR = 1.88 (95% CI, 1.55–2.28), p < 0.0001 and OR = 1.83 (95% CI, 1.32–2.53), p = 0.0003, respectively (Fig. 1). This indicates that in patients with ET presence of JAK2 V617F is associated with a highly statistically significant increase in the odds of thrombosis of approximately 90%. Five studies [6,7,20,21,35] including the three studies that received the highest weight in the overall meta-analysis [6,7,35] allowed a distinction between thrombotic events at diagnosis compared to those occurring during follow-up. Based on these studies, the JAK2 V617F mutation was associated with increased odds for thrombosis
Table 1 Characteristics of eligible studies and incidence of selected outcome events Patient number
Male/female
Diagnostic criteria
Method of mutation detection
JAK2 positive patients (%)
Patient age in years (JAK2 V617F/wild type)
Thrombotic events (%)
Hemorrhagic events (%)
Splenomegaly (%)
Baxter et al. (2005) Campbell et al. (2005) Cheung (2006) Horn (2006) Heller et al. (2006) Stevenson et al. (2006) Ahn et al. (2007) Alvarez-Larran et al. (2007) Andrikovics et al. (2007) Hsiao et al. (2007) Kittur et al. (2007) Ohyashiki et al. (2007) Pemmaraju et al. (2007) Rudzki et al. (2007) Speletas et al. (2007) Toyama et al. (2007) Vannucchi (2007)
51 776 60 23 50 27 24 103 71 53 176 51 80 59 111 82 639
24/27 312/464 24/36 9/14 16/34 12/15 14/11 34/69 17/54 20/33 66/110 24/27 22/58 26/33 25/17 31/51 202/437
PVSG PVSG NR WHO PVSG PVSG PVSG or WHO PVSG WHO PVSG WHO WHO PVSG WHO PVSG WHO PVSG or WHO
SNP screening and AS PCR AS PCR and BsaXI RFLP AS PCR AS PCR and BsaXI RFLP AS RT PCR and BsaXI RFLP DHPLC and direct sequencing AS PCR AS PCR AS PCR ARMS PCR AS PCR and AS qPCR SSP-SMFD qRT-PCR AS PCR AS PCR Direct sequencing and AS PCR AS PCR
29 (56.9) 414 (53.4) 29 (48.3) 17 (73.9) 24 (48.0) 10 (37.0) 11 (45.8) 44 (42.7) 41 (57.7) 35 (66.0) 96 (54.5) 32 (62.7) 38 (47.5) 38 (64.4) 77 (69.4) 58 (70.7) 382 (59.8)
54/55 60/52 46/81 62a 48/30 60/52 67/59 30/29 54/48 61.8/55.4 62/48 63.6/60.6 59/38 50.7/47.8 63.6/60.7 65/61.5 52.4/46.5b
12 (23.5) 137 (17.7) 26 (43.3) NR 12 (24.0) 7 (25.9) 10 (41.7) 22 (21.4) 15 (21.1) 17 (32.1) 70 (39.8) 10 (19.6) 26 (32.5) 24 (40.7) 45 (40.5) 16 (19.5) 188 (29.4)
NR 32 (4.1) NR NR NR NR NR 10 (9.7) 2 (2.8) NR NR NR 2 (2.5) 7 (11.9) 4 (3.6) 7 (8.5) 15 (5.8)c
NR 22 (3.6) NR 14 (60.8) NR NR 8 (47.1) NR 12 (16.9) NR 33 (18.8) 10 (19.6) NR 17 (28.8) 18 (16.2) NR 122 (19.1)
ARMS, Amplification refractory mutation system; AS, allele specific; DHPLC, denaturing high performance liquid chromatography; NA, not applicable; NR, not reported; PCR, polymerase chain reaction; PVSG, polycythemia vera study group criteria; qPCR, quantitative PCR; qRT, quantitative real time; RFLP, restriction fragment length polymorphism; RT, real time; SNP, small nuclear polymorphism; SSP-SMFD, sequence-specific primer-single molecule fluorescence detection assay; WHO, World Health Organization (2001) criteria. a Study only reported aggregate patient age. b Excludes 9 homozygous JAK2 V617F patients. c Data from Antonioli et al. (2008) [36].
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Study
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Fig. 1. Forest plot for the overall incidence of thrombosis. Each study is shown by the point estimate of the odds ratio (square proportional to the weight of each study) and 95% confidence interval for the odds ratio (extending lines); the pooled odds ratio and 95% confidence interval by random effects calculations is depicted as a diamond. Values higher than 1 indicate that JAK2 V617F positive patients are at a higher risk for thrombosis. Studies are arranged by year of publication.
both at ET diagnosis, OR = 2.05 (95% CI, 1.53–2.74), p < 0.0001, and during follow-up, OR = 1.47 (95% CI, 1.07–2.02), p = 0.02. Small study effects were not significant for any outcome, indicating that small and larger studies provide consistent evidence of an association between presence of JAK2 V617F and thrombosis [37]. When studies enrolling less than 100 patients were compared to larger studies, the results were similar. The random effects OR for thrombosis based on small studies was 2.34 (95% CI, 1.23–4.48), p = 0.01, while the OR based on large studies was 1.76 (95% CI, 1.35–2.29), p < 0.0001; interaction p-value = 0.42. For remaining outcomes, as no heterogeneity was detected, all comparisons between JAK2 V617F positive and negative patients are presented as fixed effects OR. Discrepancies between fixed and random models were minimal (data not shown). JAK2 V617F positivity was associated with an increased risk for both arterial [OR = 1.68 (95% CI, 1.31–2.15), p < 0.0001] and venous [OR = 2.5 (95% CI, 1.71–3.66), p < 0.0001] thromboses. On the contrary, no difference was observed in hemorrhagic events between JAK2 V617F positive and wild type patients, OR = 0.90 (95% CI, 0.58–1.41), p = 0.73. Though splenomegaly appeared to be associated with JAK2 positivity, the association was marginal, OR = 1.32 (95% CI, 1–1.75), p = 0.06. Surprisingly, the mutational status was not associated with the use
of cytoreductive therapy, OR = 1.08 (95% CI, 0.79–1.50), p = 0.67. JAK2 V617F did not appear to influence the odds for progression of the disease to acute leukemia, OR = 1.44 (95% CI, 0.54–3.82), p = 0.62, or myelofibrosis, OR = 0.64 (95% CI, 0.33–1.23), p = 0.24. On the contrary, there was a clear association between the presence of JAK2 V617F and evolution of ET into PV; OR = 7.67 (95% CI, 2.04–28.87), p = 0.0009. Notably, from a total of 1267 ET patients with available data, 16 (1.3%) experienced disease transformation to PV, all of whom were positive for the JAK2 V17F mutation. 3.3. Meta-regression analysis As previously discussed, there was significant betweenstudy heterogeneity regarding the influence of JAK2 V617F on the odds of thrombosis. To explore this heterogeneity, we employed meta-regression analysis to investigate the effect of the mean difference in hemoglobin concentration, WBC and platelet counts on the odds ratio of thrombosis between JAK2 V617F positive and wild type patients. The WBC count mean difference was significantly associated with increased odds for thrombosis in JAK2 mutant patients (p = 0.02; Fig. 2). On the contrary, the mean difference in hemoglobin concentration or platelet count did not appear to affect the risk of thrombosis (p = 0.11 and p = 0.60, respectively).
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Fig. 2. Relationship between the differences in the mean white blood cell (WBC) count between JAK2 positive and wild type patient groups and the odds ratio for thrombosis. Differences (JAK2 positive minus wild type) were calculated by subtracting the mean WBC count at diagnosis of the wild type group of each trial from that of the group of JAK2 V617F positive patients. The continuous line is the fitted regression line of the mean WBC count difference on the logarithm of the OR (log OR) for thrombosis. Each study is represented by a circle proportional to its weight in the meta-analysis. The association between log OR and the WBC mean difference is significant (p = 0.02).
4. Discussion The discovery of the JAK2 V617F mutation, in providing an insight into the pathogenesis of chronic myeloproliferative disorders, has revolutionized the diagnostic approach to these myeloid malignancies [38,39]. Despite the significance of JAK2 V617F in disease classification, reports regarding its clinical relevance have been controversial, mostly due to the small size of cohorts and low incidence of outcome events. We attempted to address such restrictions by conducting a meta-analysis of published studies. Evidence based management of ET is guided by an accurate assessment of cardiovascular risk, primarily defined by patient age and previous history of thrombosis [40]. Our data suggest that JAK2 V617F is a marker associated with thrombosis and highlight the need for prospective studies to evaluate the potential role of mutational status in designing a rational treatment strategy. Furthermore, our analysis demonstrated that leukocytosis influences the risk of thrombosis as the mean difference of WBC count between JAK2 positive and JAK2 negative patients was associated with an increased odds ratio for thrombosis. The fact that JAK2 mutational status did not appear to be associated with bleeding events provides an indication that coagulation defects in ET are multifactorial and cannot be explained by JAK2 V617F alone. Disease progression into myelofibrosis or leukemia did not appear to be influenced by the presence of JAK2 mutation, although the number of studies reporting on these outcomes and the available follow-up periods may be inadequate to allow definitive conclusions. On the other hand, the association of JAK2 V617F positivity with an eight-fold increase in the risk of transformation of ET to PV is in agreement with the notion that JAK2 V617F in ET confers a rather “polycythemic” myeloproliferative phenotype [7,42–44]. Interestingly, large retrospective studies have identified leukocytosis as an independent risk factor for thrombosis among patients with ET [45,46]. To this effect, using
meta-regression analysis we demonstrated that the higher the difference of mean WBC count at diagnosis between JAK2 V617F positive and wild type patients, the higher the odds for thrombosis for patients carrying the mutation. Notably, we found no such association with hemoglobin concentration or platelet count. Taken together, these findings indicate that JAK2 V617F mutation may partly exert its effect on the risk of thrombosis through an increase in the leukocyte count. Several studies have demonstrated that among patients with ET, those positive for the JAK2 V617F mutation show evidence of elevated CD11b and tissue factor expression by neutrophils, platelet P-selectin expression and persistent formation of platelet–leukocyte aggregates, all contributing to higher risk for thrombosis [47–49]. The increased number and activation of WBCs conferred by JAK2 V617F may enhance their interaction with the endothelium, particularly at sites of inflammation or injury, thus increasing the risk of thrombosis [48,50]. Recently, interest has focused on the potential association between JAK2 V617F allele burden and thrombosis [6,31,36]. Studies have thus far been somewhat inconsistent and further research is warranted. The triple association between leukocytosis, JAK2 V617F mutation and increased thrombotic risk may also explain why anagrelide, a platelet-specific drug, was found to be inferior compared to hydroxyurea [51]. The later, being a non-selective cytoreductive agent, is quite effective in reducing the risk of thrombosis in patients with ET, targeting both platelets and leukocytes [52]. This notion is further supported by studies demonstrating that hydroxyurea inhibits the P-selectin-mediated tissue factor expression by neutrophils and reduces the formation of platelet–neutrophil aggregates [53]. The surprising finding that cytoreductive treatment was administered at the same rate to JAK2 positive and wild-type patients possibly reflects the fact that management decisions are often not based solely on the assessment of thrombotic history and patient age but also take into account other factors,
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such as co-existing cardiovascular risk factors or thrombocytosis. Although based on a comprehensive literature search, the present analysis is limited by the unavailability of individual patient data that would allow time-to-event analysis and a direct investigation of any interaction between JAK2 mutational status and factors such as leukocytosis, disease duration or established cardiovascular risk factors. Our results should therefore be primarily viewed in the context of a hypothesis pending prospective validation. In conclusion, based on the totality of available published evidence, our analysis indicates that the presence of JAK2 mutation in patients with ET is associated with an increased risk of adverse cardiovascular outcomes, and suggests that its effect on thrombotic risk may be primarily mediated by an increase in the leukocyte count.
Conflict of interest The authors have no conflict of interest to report.
Acknowledgements The authors received no financial or other support for the research reported in this manuscript. Contributors. I.J.D. conceived of the study and performed the data analysis. I.J.D. and M.V. designed the study protocol and performed the data extraction. S.G., K.Z., C.Z. and D.L. assessed study eligibility for the meta-analysis and provided valuable clinical and laboratory perspective. All authors interpreted the results of the statistical analysis. I.J.D. drafted the manuscript and all authors revised it critically. M.V. is the guarantor of the study.
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