Clinical significance of V617F mutation of the JAK2 gene in patients with chronic myeloproliferative disorders Ana L. Basquiera1, Ne´stor W. Soria2, Ricardo Ryser3, Miriam Salguero4, Beatriz Moiraghi5, Federico Sackmann6, Ana G. Sturich1, Adriana Borello1, Adriana Berretta1, Miriam Bonafe´2, Jose´ Moreno Barral2, Emilio D. Palazzo1 and Juan J. Garcı´a1 1
Hospital Privado Centro Me´dico de Co´rdoba, Co´rdoba, Argentina Ca´tedra de Bioquı´mica y Biologı´a Molecular, Facultad de Ciencias Me´dicas, Universidad Nacional de Co´rdoba, Co´rdoba, Argentina 3 Hospital Espan˜ol, Co´rdoba, Argentina 4 Clı´nica Ve´lez Sarsfield, Co´rdoba, Argentina 5 Hospital Ramos Mejı´a, Buenos Aires, Argentina 6 FUNDALEU, Buenos Aires, Argentina 2
Objective: To determine the prevalence of JAK2 V617F mutation and its clinical correlation in patients with chronic myeloproliferative disorders (CMD): polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). Materials and methods: Detection of JAK2 V617F mutation by allele specific-PCR. Results: One hundred and three patients with CMD were included in the study. JAK2 V617F distribution was PV 40/45 (89%), ET 30/43 (69%), and IMF 7/15 (47%). In PV and ET patients only, 18 had thrombosis at diagnosis and 12 during follow-up (these were microvascular: 11, venous: 7 and arterial: 12); of these 28/70 (40%) were JAK2pos versus 2/18 (11%) JAK2neg; P50.02. In a median of 4 years, two patients with PV JAK2pos evolved to myelofibrosis and one patient with PV presented in leukemic transformation (JAK2pos before and after transformation); six patients died: four patients with IMF and two patients with PV. Conclusions: We found an association between JAK2 V617F and thrombotic events in patients with PV and ET. Keywords: JAK2, myeloproliferative disorders, thrombosis, survival, acute leukaemia
Introduction Classic chronic myeloproliferative disorders (CMD) which are Philadelphia chromosome-negative include three different entities: polycythemia vera (PV),
Correspondence to: Ne´stor W. Soria, Ca´tedra de Bioquı´mica y Biologı´a Molecular, Facultad de Ciencias Me´dicas, Universidad Nacional de Co´rdoba, Haya de la Torre s/n. 2 Piso Pabello´n Argentina, Ciudad Universitaria, (5016) Co´rdoba, Argentina E-mail:
[email protected] ß W. S. Maney & Son Ltd 2009 Received 29 March 2009; accepted 30 May 2009 DOI 10.1179/102453309X12473408860226
essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). These conditions are clonal diseases of the stem cell characterized by the proliferation of one or more of the three hematopoietic bone marrow lines (granulocytic, erythroid or megakaryocytic).1 The mutation of the gene Janus tyrosine-kinase 2 (JAK2) was described in 2005. It leads to a gain of function, through a change of G into T in nucleoside 1849 of exon 14, resulting in the substitution of valine by phenylalanine in position 617 (JAK2 V617F) of
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the pseudokinase domain (JH2) of the protein. JAK2 V617F is found in .80% of the patients with PV and in 40–60% of those with ET and IMF.2–4 The occurrence of this mutation is rare in other myeloid diseases, including atypical CMD, myelodysplastic disorders, chronic myelomonocytic leukaemia, chronic neutrophilic leukaemia and eosinophilic syndromes.5 The mutated JAK2 gene codifies a tyrosine-kinase protein that keeps active in a constitutive manner, and confers the ability to proliferate autonomously in hematopoietic cellular lines, supporting the formation of erythroid colonies independently of erythropoietin in vitro.2 PV and ET are chronic diseases, in which the main causes of morbidity and mortality are thrombotic and hemorrhagic events, and less frequently, the progression to myelofibrosis (MF) and/or transformation to acute leukaemia (AL).6 In contrast IMF is associated to a worse prognosis and a mean survival of 3.5– 10 years due to infections, bleeding, organ failure, portal hypertension and leukemic transformation.6 It has not been defined yet whether the presence of JAK2 V617F mutation or the burden of the mutated allele modifies the phenotypic expression or the prognosis of classic CMD.7 Our objective was to determine the prevalence of V617F mutation of gene JAK2 in patients with PC, ET and IMF, and to correlate the positivity of this mutation with the clinical outcome. In addition, we searched for new mutations of gen JAK2 that could explain the myeloproliferative phenotype in patients negative for V617F.
Materials and methods Study population
Patients with classic CMD from five different Departments of Haematology were prospectively studied between April 2006 and March 2007, for evaluation of JAK2 V617F mutation. All the patients fulfilled the diagnostic criteria of the World Health Organization (WHO) for CMD including PV, ET and IMF.8 The diagnosis of CMD could be recent or previous to the recruiting date. The following data were registered from each patient: date of diagnosis, clinical and laboratory findings, bone marrow biopsy results at diagnosis, complications along the disease, previous therapies and condition at follow-up. The follow-up was performed by the primary care physician with clinical and laboratory controls at least every 6 months or according to complications emergence. Therapy was
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selected by each physician. Healthy individuals with normal hemogram were used as controls. The centralization and analysis of the data were performed in the Hospital Privado Centro Me´dico de Co´rdoba; the study was approved by the Bioethics Committee of the same Institution and was performed according to the principles established in the Helsinki’s declaration. All the patients agreed and gave written informed consent before participating in this study. Study of JAK2 V617F mutation and DNA sequencing
Analysis of the V617F mutation of gene JAK2 in peripheral blood was performed in all patients using granulocytes and whole blood.9 In five patients, bone marrow was analyzed as well. In addition, the mutation was studied in mouth swab samples from the first 15 patients and in lymphocytes from the first 31 patients. Cellular separation of was performed with an initial purification by Ficoll-Hypaque method and a second one using the Dynabeads Kit (Invitrogen). For DNA extraction from the different samples, we used the modified CTAB method. The study of JAK2 V617F mutation was performed in the first 50 patients and in 11 healthy controls by allele specific (AS) chain polymerase reaction (PCR) and PCR with restriction enzyme BsaXI, according to Baxter et al.2 Since we noticed a higher sensitivity for AS-PCR (two positive cases for AS-PCR only), in the following patients we only performed AS-PCR. In those patients negative for V617F mutation, we sequenced genomic DNA with primers designed to amplify the codifying exons and joints 12, 13, 14, 15, 16, 17, 18 and 19 of gene JAK2. The PCR products were sequenced directly in both directions with automatic sequencer and the sequences were manually analyzed. DNA extraction and PCR studies were performed in the School of Medicine of the Universidad Nacional de Co´rdoba. Episode definition
Thrombotic events included arterial, venous and microvascular episodes. Arterial events included transient ischemia, non-hemorrhagic brain strokes, acute myocardial infarction, unstable angina and peripheral arterial occlusion. Venous events included deep venous thrombosis, pulmonary thromboembolism and thrombosis of the abdominal veins.10 Microvascular ischemia was considered as microvascular events and registered separately. Important haemorrhages were defined as intracranial or retroperitoneal bleeding, evident haemorrhages with haemoglobin decrease of >2 g/dl, or any other
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bleeding that required transfusion of at least 2 units of red blood cells. Other events were considered mild hemorrhages.10 Statistical analysis
Continuous variables were analyzed with MannWhitney U or T test. Categorical variables were compared with chi-square test of Fisher’s exact test and categorized according to the mutation state. For the analysis of thrombotic events, we only considered the first event of each patient; whether it appeared at onset or during follow-up. The value of white blood cells (109/l) at diagnosis was considered as continuous variable and as categorical variable classified in the following groups ,5, 5–7.5, 7.5–10 and >10. The differences between the numbers of vascular events in both groups were calculated with Cusick’s method. The correlation between the number of vascular events, mutation and other co-variables was evaluated with logistic regression. The term overall survival was defined as time between diagnosis of CMD until death for any reason or date of last contact; survival free of thrombosis was defined as time from diagnosis until the appearance of arterial, venous or microvascular events or date of last contact. The distribution time until the event were estimated by the Kaplan-Meier method and compared through log-rank test. We considered significant a P,0.05; all the P values were two-tailed and at a 95% confidence interval (CI). Data were analyzed with StatsDirect Statistical Software, version 2.5.6.
Results Prevalence of JAK2 V617F mutation
One hundred and three of the 113 patients evaluated, fulfilled the WHO criteria for CMD and were included in the study, classified as PV n545, ET n543 and IMF n515. Of the 10 excluded patients, nine had PV and one ET. All of them were negative for JAK2 V617F mutation. The results reported herein include follow-up of patients with diagnosis of CMD until 31 March 2008 (median: 4 years, range: 0.1–26.2 years). Of the total patients included, 44 (43%) had diagnosis of CMD 2 years previous to the study, while in 30 (29%) and 29 (28%) cases, the diagnosis was performed 2– 5 years and .5 years before recording these data, respectively. The prevalence of the mutation was: PV 40/45 (89%), ET 30/43 (69%) and IMF 7/15 (47%). Mutation was negative in controls (0/11), samples from mouth swabs (0/15) and lymphocytes from patients with CMD (0/31). In the five patients who
V617F mutation of the JAK2 gene in myeloproliferative disorders
underwent the mutation study in bone marrow, the results were similar to those from peripheral blood samples. The characteristics at diagnosis of the 103 patients according to the presence or absence of the mutation are shown in Table 1. DNA sequencing was performed in 28 patients V617F negative (including the 10 patients excluded from the study). Only three polymorphisms of simple nucleotide (SNP) were found in five patients already described in the SNP database of the NCBI. Association of JAK2 V617F with thrombotic and hemorrhagic events
From the 88 patients with PV and ET, 18 (20.4%) developed a thrombotic event at diagnosis, more frequently those with ET (66.7%) compared to those with PV (33.3%). During the follow-up, 12 (13.6%) patients developed at least one thrombotic event, with an inverse distribution compared to the moment of diagnosis. A detailed description of these events is shown in Table 2. Globally, the difference between JAK2 V617F positive and negative patients was significant: 28/70 (40%) JAK2pos versus 2/18 (11.1%) JAK2neg (RR 3.21; 95% CI: 1.16–13.21; P50.02). This analysis did not include recurrent events: one episode of rethrombosis in a patient with ET JAK2 V617F positive who had thrombosis of the portal vein at diagnosis, and posterior events in two JAK2 V617F patients with PV who had more than one event (venous and arterial) during the follow-up (the three patients were anticoagulated at recurrence and two of them were under cytoreductive therapy). Including these cases, the difference between patients JAK2 V617F positive and negative was higher (P50.007). Only one fatal thrombotic event occurred in a female patient with PV JAK2pos. The percentage of thrombotic events at diagnosis depending on the white blood cell count (109/l), was ,550%, 5–7.5510%, 7.5–10525% and >10541%; (P50.01). Considering other known risk factors for thrombosis in patients with PV and ET (age, history of thrombosis, smoking habit, sex),11 females were significantly associated to higher risk of thrombosis (RR 2.09; 95% CI: 1.14–3.98; p50.02) (Table 3). In the multivariate analysis including leukocytosis >10,000, sex and mutation state, the positivity for JAK2 V617F was an independent predictor of thrombosis (OR 10.43; 95% CI: 1.25–87.14; P50.03). Survival free of thrombotic events was 12.1 years for JAK2 V617F positive patients and was not reached in negative patients (HR of thrombosis 4.08; 95% CI: 1.73–9.59; P50.0217) (Fig. 1).
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0.03 0.18 0.65
21.3 (3.4) 65.9 (6.5) 359.5 (266.8) 98.5 (49.3) 84 (9.5) 54.5 (2.1) 2 (40) 1 (20) 0 1 5.8 (2.3–9.4)
18.9 (2)
58.6 (6.8)
604 (387)
132 (122)
84.2 (8.5)
207.9 (80) 24 (60)
12 (30)
2
4
3.4 (0.2–25.4)
5 1
.0.99 .0.99 2.3 (0.15–16.9)
0
.0.99
0.2
146.2 11 (36.7)
83.9 (10.3)
95.2 (195.3)
101.8 (367.8)
44.4 (11.1)
13.5 (1.6)
13.6 (6)
57.3 (17) 14/16
n530
JAK2pos
0.009 0.44
0.96
0.03
0.01
7.2 (2.9)
12.3 (5.7)
0.02 0.14
P
48.4 (14.4) 5/0
n55
60.1 (9.6) 23/17
n540
JAK2neg
2.2 (0.7–24.8)
2
2
0
67 1 (7.8)
90.9 (5)
43.7 (21.4)
126.8 (746.4)
39.7 (3.8)
12.7(1.4)
9.9 (4.4)
45.1 (25.2) 3/10
n513
JAK2neg
Essential thrombocythemia
0.36
0.23
0.95
–
0.01 0.05
0.06
0.15
0.58
0.03
0.03
0.05
0.07 0.16
P
7.1 (2.1–9.4)
1
7
1
223{ 19.41
92 (8.2)
69.5 (34.1)
368.4 (190.1)
31.7 (7.3)
10.1 (2.9)
7.9 (2.1)
60.1 (13.8) 3/4
n57
JAK2pos
4.7 (0.07 –19)
1
8
0
54 (24) 20.4
89.4 (9.8)
59.2 (49.7)
335.3 (265.6)
30.3 (9.9)
9.9 (3.5)
9.3 (5.7)
59.2 (14.1) 6/2
n58
JAK2neg
Idiopathic myelofibrosis
0.53
0.93
–
0.46
– 0.8
0.76
0.68
0.78
0.75
0.91
0.61
0.90 0.26
P
*JAK2pos and JAK2neg mean positivity and negativity for JAK2 V617F mutation, respectively; SD5standard deviation; MCV5median corpuscular volume; ALP5alkaline phosphatase. {Represents time from diagnosis until last control. {Data from four patients (one from JAK2 V617F positive group and three from the negative group) 1Media of the spleen size (cm) by ultrasound. One splenectomized patient in JAK2neg group.
Age, years (SD) Sex, males/females, n White blood cells (109/l), media (SD) Hemoglobin (g/dl), media (SD) Hematocrit (%), media (SD) Platelets (109/l), media (SD) Reticulocytes (109/l), media (SD) MCV (fl), media (SD) ALP media (SD) Splenomegaly, n (%) Aquagenic pruritus, n (%) Bone marrow fibrosis, n Abnormal karyotype, n Length of disease, years, median (range){
Variable
JAK2pos
Polycythemia vera
Table 1 Characteristics of the 103 patients with CMD at diagnosis*
Basquiera et al. V617F mutation of the JAK2 gene in myeloproliferative disorders
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V617F mutation of the JAK2 gene in myeloproliferative disorders
Table 2 Vascular events at diagnosis and during follow-up according to positivity for JAK2 V617F*
Variable Patients with vascular events at diagnosis Total Arterial thrombosis Venous thrombosis Microvascular Patients with vascular events during follow-up Total Arterial thrombosis Venous thrombosis Microvascular Time until first event, months, media Total of patients who presented events{
Polycythemia vera
Essential thrombocythemia
JAK2pos
JAK2neg
JAK2pos
JAK2neg
n540
n55
n530
n513
Total
P{
11 3 3 5
1 1 0 0
18 5 4 9
NS
3 2 1 0 67.5 14
0 0 0 0 – 1
12 7 3 2
NS
30
0.02
6 1 1 4
0 0 0 0
8 4 2 2 75.4 14
1 1 0 0 19.6 1
*JAK2pos and JAK2neg mean positivity and negativity for JAK2 V617F mutation, respectively; NS5not significant. {Univariated analysis of patients with Polycythemia vera and Essential thrombocytemia, JAK2V167F positive versus negative patients. {Numbers correspond to the first event of the patients (diagnosis or follow-up).
Within the 103 patients, 17 had hemorrhagic events, six of which were major episodes (6/103; 5.8%). We did not find significant differences between the result of the mutation and the presence of total or major hemorrhagic events. Therapy
Seventy patients underwent cytoreductive therapy (alone or in combination) during the course of disease (33/45 with PV, 33/43 with ET and 4/15 with IMF). The need of therapy was higher in JAK2 V617F positive patients compared to negative ones (61/77 versus 13/26; RR 1.49; 95% CI: 1.12–2.22; P50.006).
Haematological complications and survival
At follow-up, two JAK2 V617F positive patients with PV evolved to myelofibrosis (14.1 and 15.6 years from diagnosis, respectively). One patient with PV developed transformation to acute myeloid leukaemia after 20.2 years from diagnosis. In this patient the mutation study was positive before and after transformation. Four patients with diagnosis of IMF and two patients with PV died: three patients positive for mutation and three negatives (HR 0.38; 95% CI: 0.06–2.30; P50.27). Causes of death were: infection (two cases), disease progression (two cases), leukemic
Table 3 Risk factors for thrombotic events in patients with polycytemia vera and essential thrombocytemia*
Variable White blood cells ,10,000 >10,000 Age ,60 years >60 years Sex Males Females Smoking habit Absent Present Thrombosis history Absent Present JAK2 V617F mutation Negative Positive
Thrombosis
Absence of thrombosis
Univariated analysis
Multivariated analysis
Nu
Nu
Relative risk (95% CI)
P{
Relative risk (95% CI)
P{
8 21
31 24
1 2.27 (1.18 to 4.59)
0.01
1 2.32 (0.81 to 6.58)
NS
11 19
33 25
1 1.72 (0.95 to 3.21)
NS
–
–
10 20
35 23
1 2.09 (1.14 to 3.98)
0.02
1 2.26 (0.83 to 6.18)
NS
21 9
47 11
1 1.46 (0.76 to 2.54)
NS
–
–
25 5
56 3
1 2.02 (0.93 to 3.37)
–
–
NS
2 28
16 42
1 3.6 (1.16 to 13.21)
0.02
1 10.43 (1.25 to 87.14)
0.03
*Thrombotic events including arterial, venous and microvascular episodes, at diagnosis and during follow-up; NS5not significant. {Chi-square test. {Logistic regression.
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Figure 1 Survival time free of thrombotic events of patients with polycytemia vera and essential thrombocythemia according to V617F JAK2 mutation. Circles and squares represent events
transformation (one case) and fatal thrombotic event (one case). Considering only the patients with IMF (n515), we did not find significant differences in overall survival between JAK2 V617F positive and negative patients (HR 0.34; 95% CI: 0.05–2.44; P50.39). The subtype of CMD (IMF) was the only factor that affected survival (P50.0016).
Discussion The prevalence of the mutation in our study was similar to data described previously for PV and IMF and slightly higher for ET.3 We could not exclude the possibility that some incipient cases of PV with thrombocytosis had been previously diagnosed as ET, since we did not perform a central revision of the samples of bone marrow biopsy or analyzed the formation of erythroid colonies in vitro.12 As previously described, patients with ET, positive for the mutation were older at diagnosis and had higher haemoglobin and white blood cell levels compared to negative patients.13,14 A greater age at diagnosis and a higher level of white blood cells were also characteristics of JAK2 V617F positive patients with PV in this study. Wolanskyj et al. have suggested that the relationship between age and incidence of JAK2 mutation could be associated to the influence of age over genetic instability.13 In addition, in PV and ET as well, we demonstrated, with a higher number of patients, a raise of leukocyte alkaline phosphatase in JAK2 V617F positive patients.15 This and other markers of leukocyte activation are typical of patients that carry the mutation and there is a clear correlation between the charge of the mutated allele and the markers level.16
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In patients with IMF, we did not find significant differences in clinical or laboratory characteristics comparing JAK2 V617F positive and negative patients; even though the number of patients of this group was limited. Two large cohorts of patients with IMF have demonstrated a greater age at diagnosis, higher haemoglobin level and elevated frequency of aquagenic pruritus in patients positive for the mutation.17 In our study, JAK2 V617F positive patients with PV and ET had more thrombotic events compared to negative ones. We considered that the coupled study of the two subtypes of CMD was valid, since they have similar clinical and biological features and a comparable rate of thrombotic events in JAK2 V617F positive patients.13,16 Since the mutation was described for the first time, several authors have attempted to demonstrate a correlation between JAK2 V617F and thrombotic events. Even though some of them have reported a lack of correlation13,14 others have demonstrated a significant association.16,18,19 This lack of agreement could be due to several factors. In this and other studies,16,18,19 patients with thrombosis at diagnosis and during follow-up were included in the analysis. However, other authors have analyzed the events, but not the patients themselves (the treatment effects are involved),14 and others have considered only the events at the moment of diagnosis.13 The administration of cytoreductive therapy and/or antiaggregants reduces the incidence of thrombotic events in PV and ET.20 When the events of the follow-up of not randomized studies with therapeutic variations are included in the study, this approach could modify the results. Another issue to consider is the difference between the numbers of JAK2 V617F positive versus negative patients, especially in PV, which increases the number of patients required to reach a significant difference between both groups. Finally, the different definitions of thrombotic events used in clinical assays could be another reason for the lack of definition of the JAK2 association with the incidence of thrombosis in patients with CMD. In our series, the percentage of thrombotic events increased along with the rise of white blood cells, as previously described.11 Carobbio et al. have suggested that the leukocytosis and not the mutation, is the factor that increases the risk of thrombosis; however, in this study, the analysis of the mutation was performed in 60% of the studied population. The higher frequency of leukocytosis in JAK2 V617F
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positive patients with PV and ET could reflect that leukocytosis is an indirect effect of the mutational state.16 Other finding of our study was the presence of microvascular events only in JAK2 V617F positive patients. Thrombosis in the microcirculation is sensitive to aspirin and has been attributed to a platelet hyperactivation in patients with PV and ET. In addition, there is an in vivo interaction between platelets and activated polymorphonuclear leucocytes that leads to the formation of mixed aggregates of leukocytes/platelets in the circulation.21 These aggregates, platelet hypersensitivity and other variables of haemostatic activation are significantly more frequent in patients who carry the mutation and add an extra evidence of JAK2 involvement and the resulting myeloproloferative phenotype in the pathogenesis of thrombotic events.22 Thus, the incidence of thrombosis in patients with PV and ET could depend more on the activation of white blood cells and platelets mediated by JAK2 V617F than on its number.21 Even though all the cases that progressed to MF or AL were JAK2 V617F positives, the number of patients was not enough to validate a statistical analysis. In addition, the median follow-up in our study was 4 years from diagnosis of MPD, and it has been suggested that these patients should be followed during 5–10 years in order to evaluate the risk of progression to MF and/or AL.23 Progression to MF in JAK2 V617F positive patients with PV has been reproduced in animal models and clinical assays.17,24,25 On the other hand, the positivity of JAK2 and leukemic transformation is a controversial issue. Only 25% of the cases of acute myeloid leukaemia secondary to CMD Philadelphia chromosomenegative have the mutation and there is not an increase of the charge of the mutated allele along with the transformation.23 The function of JAK2 could not be essential in this context of PV and ET patients, but it is associated to an increased risk of leukaemia in IMF.14 Four out of six patients who died during the follow-up had diagnosis of IMF, and there were no differences in survival rates between JAK2-positive patients versus negative ones. Even though the follow-up of our series was not long enough to estimate survival in CMD, Wolanskyj et al. could neither demonstrate significant survival differences related to the mutation, with a median follow-up of 11.4 years.13
V617F mutation of the JAK2 gene in myeloproliferative disorders
After the description of the JAK2 V617 mutation, other mutations have been described in the JAK2 gene in patients with CMD.26 Approximately 3% of the patients with PV have a mutation in exon 12.27 In our study, we did not find this mutation or any other relevant mutations and only found polymorphisms of gene JAK2, also described by other groups.28 The lack of detection of mutations in exon 12 could be due to the size of the sample or the type of methodology used, since the threshold of PCR sequencing is lower than AS-PCR.29 Some authors have suggested that the DNA study of erythropoietin-independent erythroid colonies could be the most sensitive method.30 In conclusion, our study contributes to demonstrate that the V617F mutation of the JAK2 gene is associated to an increased risk of thrombosis independently from other known factors in patients with PV and ET. Even though we were unable to demonstrate an association between JAK2 mutation and survival patients with IMF had the worst prognosis. JAK2 V617F mutation should be incorporated in the design of future randomized studies to determine its value in the assessment of vascular risk in patients with CMD.
Acknowledgement This study was supported by a grant from the Florencio Fiorini Foundation and the Argentine Medical Association.
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