Original Study
JAK2 V617F Mutation Status of 232 Patients Diagnosed With Chronic Myeloproliferative Neoplasms Kadriye Bahriye Payzin,1 Kaan Savasoglu,2 Inci Alacacioglu,3 Fusun Ozdemirkiran,1 Belgin Berber Mutlu,2 Sadi Bener,4 Aylin Orgen Calli,4 Betul Bolat Kucukzeybek,4 Saliha Haksun5 Abstract The aim of this study was to investigate the presence of Janus kinase 2 (JAK2) V617F mutation in patients with BCR-ABL negative chronic myeloproliferative neoplasms (CMPNs) in our center. JAK2 V617F mutation frequencies in our PV and ET patients were similar to those reported previously. JAK2 V617F mutation frequency in our PMF patients was greater than in previous reports. Introduction/Background: The aim of this study was to investigate the presence of Janus kinase 2 (JAK2) V617F mutation in patients with break point cluster region-abelson negative chronic myeloproliferative neoplasms (CMPNs) in our center. Patients and Methods: We compared patients with and without the mutation, and also patients with the homozygous and heterozygous mutation, in terms of different clinical and laboratory features. Results: The JAK2 V617F mutation was detected in 77 (95%), 88 (68%), and 17 (77%) of polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) patients, respectively. Among JAK2 V617Fpositive patients, the homozygous genotype was found in 39 (50.6%) of the 77 PV, 23 (26.1%) of the 88 ET, and 11 (64.7%) of the 17 PMF patients. Bleeding was seen in 14 (6%) of all patients. Upper gastrointestinal bleeds were the most common, seen in 11 patients. Out of 232 CMPN patients, 44 (19%) had thrombosis. The most common thrombotic event was transient ischemic attack (52%). Progression to myelofibrosis was seen in 1 (1.2%) PV and 3 (2.3%) ET patients, and progression to acute leukemia was seen in 2 (2.5%) PV and 3 (2.3%) ET patients. Three patients with PV (3.7%), 3 with ET (2.7%), and 5 with PMF (2.7%) died during follow-up. Conclusion: JAK2 V617F mutation frequencies in our PV and ET patients were similar to those reported previously. JAK2 V617F mutation frequency in our PMF patients was greater than in previous reports. All of our PV patients with thrombosis and most of our ET patients with thrombosis (76.1%) were JAK2 V617F mutation-positive. This mutation seems to be correlated with thrombosis risk. Clinical Lymphoma, Myeloma & Leukemia, Vol. -, No. -, --- ª 2014 Elsevier Inc. All rights reserved. Keywords: Essential thrombocythemia, JAK2 mutation, MPNs, Polycythemia vera, Primary myelofibrosis
1
Division of Hematology, Department of Internal Medicine, Ataturk Training Hospital, Izmir, Turkey 2 Medical Genetics Laboratory, Ataturk Training Hospital, Izmir, Turkey 3 Department of Hematology, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey 4 Department of Pathology, Ataturk Training Hospital, Izmir, Turkey 5 Department of Biochemistry, Ataturk Training Hospital, Izmir, Turkey Submitted: Jan 28, 2014; Revised: Feb 19, 2014; Accepted: Feb 24, 2014 Address for correspondence: Kadriye Bahriye Payzin, MD, Division of Hematology, Department of Internal Medicine, Ataturk Training Hospital, Izmir, Turkey E-mail contact: [email protected]
2152-2650/$ - see frontmatter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clml.2014.02.013
Introduction Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are classified in the break point cluster region-abelson (BCR-ABL1) negative myeloproliferative neoplasm (MPN) group according to World Health Organization (WHO) 2008 classification.1 Their disease-causing mutations remain unidentified despite many mutations described beginning in 2005. PV patients mostly harbor Janus kinase 2 (JAK2; 9p24) mutation (96% displaying mutation in exon 14, JAK2 V617F; 3% displaying mutation in exon 12; JAK2).2,3
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JAK2 V617F Mutation Status in CMPN Janus kinase 2 V617F also occurs in ET (55%) and PMF (65%), but JAK2 exon 12 mutations are rare in ET and PMF.4 Myeloproliferative leukemia virus oncogene (MPL) (MPL515W>L/K) mutations are found in approximately 4% of ET, 8% of PMF, and rarely in PV.5 According to the 2008 WHO classification, the existence of these mutations is the major criterion in diagnosis of chronic MPNs.1 Nonetheless, evaluation of bone marrow histopathology is still required to define the specific morphological patterns, which are essential for the diagnosis of ET and PMF. Other mutations related to the tet oncogene family member 2 (TET2), additional sex comb-like 1 (ASXL1), and isocitrate dehydrogenase (IDH1/2) genes are occasionally observed in MPNs.6 Because the molecular pathogenesis of MPNs with nonmutated JAK2 is still obscure and diagnosis remains a challenge, the mutation status of the gene encoding calreticulin (CALR) was studied recently by Nangalia et al.7 CALR mutations were found in most patients with MPNs with nonmutated JAK2.7 Leukocytosis, splenomegaly, thrombohemorrhagic complications, vasomotor disturbances, pruritus, and a small risk of disease progression into acute myeloid leukemia or myelofibrosis are other disease features.4 In previous studies, the importance of mutational status of JAK2 was evaluated. Correlations have been found between high JAK2 V617F allele burden and leukocytosis, spleen size, myelofibrosis severity, fibrotic transformation, and disease duration.8 Many researchers have reported thrombosis risk in JAK2 V617F-positive ET patients to be greater than that of JAK2 V617F-negative ET patients.9-11 The effect of the JAK2 V617F mutation on clinical parameters of PMF such as prognosis and the need for transfusion is unclear.12,13 JAK2 V617F mutated status has not been shown to have an effect on survival time, but patients with low JAK2 V617F allele burden have been reported to have shorter survival times compared with those with high JAK2 V617F allele burden and wild type JAK2.14 In this study, we aimed to investigate the relationship between JAK2 status/quantitation and clinic and laboratory findings in our MPNs patients.
Patients and Methods Demographic and clinical features of 233 patients diagnosed with MPNs at our hematology department between November 2000 and November 2012 were evaluated retrospectively. The median age was 63 years (range, 18 to 89 years) with a male to female ratio of 0.9 to 1 (110 men, 123 women). BCR-ABL molecular analysis and fluorescence in situ hybridization analysis for t(9;22) were made in all MPN patients and found as not amplified. During retrospective evaluation, 1 male patient was excluded from the study because of diagnosis of chronic myelomonocytic leukemia. Eighty-one patients were rediagnosed with PV (34.9%), 129 with ET (55.6%), and 22 with PMF (9.5%) according to polycythemia vera study group or WHO 2008 criteria. White blood cell (WBC), hemoglobin (Hb), and hematocrit (htc) levels, and platelet (plt) count, and serum lactic dehydrogenase (LDH) level were recorded. Spleen size was measured in centimeters below the midpoint of the left costal margin, and spleen size > 5 cm was accepted as gross splenomegaly. JAK2 V617F analysis was performed at the time or after the time of diagnosis depending on the availability of this test. Thrombotic and hemorrhagic events before or after diagnosis were recorded. Clinical thrombotic events included ischemic stroke, cerebral transient ischemic attack (TIA), acute myocardial infarction (AMI), peripheral arterial thrombosis (PAT), pulmonary thromboembolism (PE), and venous thromboembolism (VTE). Most patients received 1 or more of the following: acetyl salicylic acid (ASA), some form of myelosuppressive therapy as hydroxyurea (HU), anagrelide, interferon (IF), phosphorus-32 (P-32). Patients with PV were phlebotomized to maintain an htc level 45%. This study was approved by the local ethics committee. Quantification of JAK2 V617F mutation was detected using an allele-specific real-time quantitative polymerase chain reaction (RQ-PCR) assay (JAK2 MutaQuant Kit, Ipsogen). RQ-PCR was performed using a Rotor-Gene (Corbett Research). Patients with 50% mutational load and those with < 50% mutational load were considered ‘homozygotes’ and ‘heterozygotes,’ respectively, as has been previously designated.15,16
Table 1 Characteristics of 232 Patients With MPNs
M/F Mean Age (Minimum-Maximum), Years Splenomegaly, n (%) Thrombosis, n (%) Bleeding, n (%) Diabetes Mellitus, n (%) Hypertension, n (%) Mean Hemoglobin Level ± SD, g/dL Mean Hematocrit Level ± SD, % Mean Leukocyte Count ± SD, 3 109/L Mean Thrombocyte Count ± SD, 3 109/L Mean Serum LDH ± SD, U/L
MPN
PV (81 Pts)
ET (129 Pts)
PMF (22 Pts)
109/123 59.9 (18-89) 125 (59.8) 44 (19) 14 (6) 29 (12.5) 42 (18.1) 14.6 3.0 13.6 10.3 15.2 8.0 344.7 22.9 313.7 219
30/51 62 (35-86) 46 (67.6) 23 (28.3) 4 (4.9) 12 (14.8) 15 (18.5) 17.6 1.8 53.6 5.9 15.2 5.6 646.9 268.4 297 115
65/64 58 (18-89) 57 (47.5) 21 (16.3) 10 (7.6) 16 (12.4) 25 (19.4) 13.26 2.0 39.8 8.3 13.7 5.9 971.0 319.9 271.9 110.5
14/8 62.8 (32-85) 22 (100) NR NR 1 (4.5) 2 (9) 11.3 1.9 33.6 5.5 23.9 16.6 344.6 73.5 648.1 556.2
Abbreviations: ET ¼ essential thrombocythemia; F ¼ female; LDH ¼ lactic dehydrogenase; M ¼ male; MPN ¼ myeloproliferative neoplasm; PMF ¼ primary myelofibrosis; pts ¼ patients; PV ¼ polycythemia vera.
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Clinical Lymphoma, Myeloma & Leukemia Month 2014
Kadriye Bahriye Payzin et al Statistical Analysis All statistical analysis was performed using the SPSS 18.0 statistical package. Analysis of variance test for continuous variables and c2 test for categorical variables as appropriate were used. Overall survival (OS) was defined as the time between diagnosis and death (from any causes) or end of follow-up (censored observations). OS was estimated using the Kaplan-Meier product limit method. All P values were 2-sided. A value of P < .05 was considered as significant.
Results The demographic, laboratory, and clinical data of all patients are summarized in Table 1. There were significantly more female patients than male patients in the PV group (P ¼ .01). In contrast, there were significantly more male patients than female patients (P ¼ .01) in the PMF group. Mean age was similar in all 3 groups. All patients with PMF had splenomegaly compared with others (P ¼ .01). Gross splenomegaly was observed in PV and ET patients with similar frequency. Out of 232 MPN patients, 182 (78.4%) were positive for the JAK2 V617F mutation. The distribution of allele burden of mutation-positive patients is given in Table 2 and Figure 1. JAK2 V617F mutations were found in 77 patients in the PV group (95%), 88 patients in the ET group (68%), and 17 patients in the PMF group (77%) (Table 2). The PV and ET groups showed no significant differences for JAK2 V617F mutation positivity among the 2 sexes (P ¼ .8 for both). The JAK2 V617F mutation was found significantly more frequently in male patients in the PMF group (P ¼ .049). The heterozygous genotype was found significantly more frequently in the ET group, whereas the homozygous genotype was more frequent in the PMF group (P ¼ .000 and 0.003, respectively). The percentage of PV patients with the homozygous genotype was significantly greater than the percentage of ET patients with the same genotype (P ¼ .004) (Fig. 1). JAK2 V617F allele burden status according to sex is shown in Figure 2. In the PV group, there was no significant difference among male and female patients in terms of the homozygous genotype (P ¼ .2). In the ET group the homozygous genotype was found significantly more in JAK2 V617F-positive male patients, than in JAK2 V617Fpositive female patients (P ¼ .04). In the PMF group, no significant difference was found among the 2 sexes (P ¼ .06) (Fig. 2). Mean plt count was statistically greater but Hb level was lower in PV patients with the JAK2 V617F mutation. When compared with ET patients, mean spleen size, Hb and LDH level, and WBC count
were found to be significantly greater in patients with the JAK2 V617F mutation (P ¼ .006, .001, .01, and .001, respectively). When a similar comparison was made among PMF patients, mean age was older for the JAK2 V617F-positive group and mean spleen size was greater for the JAK2 V617F-negative group (P ¼ .04 for both). There were no significant differences among the 2 groups in terms of LDH and Hb levels, or WBC and plt counts (Table 3). Myeloproliferative neoplasm patients homozygous and heterozygous for the JAK2 V617F mutation were also compared for their Hb and serum LDH levels, WBC and plt counts, and spleen size (Table 4). Mean spleen size, LDH level, and WBC count were significantly greater in the JAK2 V617F homozygous PV patients (P ¼ .006, .03 and .005, respectively). Among the ET patients, mean WBC count and spleen size were significantly greater in patients homozygous for the JAK2 V617F mutation (P ¼ .001 and .001, respectively). In contrast, mean Hb level of the homozygous ET patients was significantly lower compared with the heterozygous ET patients (P ¼ .01). PMF patients with homozygous and heterozygous JAK2 V617F mutations did not show significant differences in age, spleen size, Hb and serum LDH levels, and WBC and plt counts (Table 4). Among all MPN patients, 44 (19%) had thrombosis. Thrombosis was not observed in any PMF patients. In the PV group, only 2 of the 23 patients (28.3%) with thrombosis developed it after the diagnosis (1 had PE and VTE, the other had TIA). The remaining 21 patients (28.4%) either had thrombosis for years, or were diagnosed with MPNs when searching for the cause of their thromboses. TIA was the most common vascular event in the PV group (15 patients). Portal vein thrombosis was observed in 3 patients, AMI in 2 patients, deep vein thrombosis (DVT) in 2 patients (1 with a history of pulmonary thromboembolism), and hepatic vein thrombosis (Budd-Chiari syndrome) in 1 patient. Eleven PV patients with thrombosis were homozygous for the JAK2 V617F mutation; the remaining patients were heterozygous. A homozygous JAK2 V617F mutation was found in 6 patients with TIA, all AMI and DVT patients, and 1 patient with portal vein thrombosis. There were no PV patients with thrombosis who tested negative for the JAK2 V617F mutation. When PV patients with and without thrombosis were compared, no significant differences were found in terms of age, spleen size, htc, Hb, and serum LDH levels, and WBC and plt counts. Twenty-one (16.3%) of the ET patients had thrombosis. Nineteen patients had a history of thrombosis before their ET diagnoses, and 2 patients were diagnosed with ET along with clinical thrombosis. Three of these patients were homozygous, 13 were
Table 2 Distribition of Allele Burden of 182 (78.4%) MPN Patients Who Were JAK2eV617F-Positive JAK2 V617F Allele Burden
78%-100%
50%-78%
Homozygous Genotypea
31%-50%
12.5%-31%
5%-12.5%
2%-5%
Heterozygous Genotypeb
PV: 77 (95%) ET: 88 (68%) PMF: 17 (77%)
12 (15.6) 14 (16) 5 (29.4)
27 (35) 9 (10) 6 (35.2)
39 (50.6) 23 (26) 11 (64.6)
21(27.4) 25 (28.4) 6 (35.4)
17 (22) 24 (27.2) 0
0 14 (15.9) 0
0 2 (2.5) 0
38 (49.4) 65 (74) 6 (27)
Data are presented as n (%). Abbreviations: ET ¼ essential thrombocythemia; MPN ¼ myeloproliferative neoplasm; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera. a JAK2 V617F allele burden 50%. b JAK2 V617F allele burden < 50%.
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JAK2 V617F Mutation Status in CMPN Figure 1 The Percent Interval of JAK2 V617F Mutation Alleles are Shown on the Y-Axis; 1 Represents 2%-5%, 2 Represents 5%-12.5%, 3 Represents 12.5%-31%, 4 Represents 31%-50%, 5 Represents 50%-78%, and 6 Represents 78%-100%. Patients With ‡ 50% Mutational Load and Those With < 50% Mutational Load Were Homozygotes and Heterozygotes, Respectively. The Heterozygous Genotype was Found Significantly More Frequently in the ET Group, and the Homozygous Genotype was More Frequent in the PMF Group (P [ .000 and .003, Respectively). The Percentage of PV Patients With the Homozygous Genotype was Significantly Greater Than the Percentage of ET Patients With the Same Genotype (P [ .004)
Abbreviations: ET ¼ essential thrombocythemia; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera.
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heterozygous, and 5 were negative for the JAK2 V617F mutation. A variety of thrombotic events were observed in ET patients. Starting from the most common was TIA, observed in 8 patients, PAT (2 patients with aortofemoral bypass) and AMI, both observed in 5 patients, Budd-Chiari syndrome observed in 3 patients, and PE and portal vein thrombosis, both observed in 1 patient. No significant differences were found among ET patients with or without thrombosis with respect to age, spleen size, htc, Hb, and serum LDH levels, and WBC and plt counts. There was no significant difference between PV and ET patients with regard to thrombosis frequency (P ¼ .07). Bleeding was seen in 14 (6%) MPN patients. Upper gastrointestinal bleeding was the most common, seen in 11 patients. Rectal or vaginal hemorrhage and epistaxis were also observed, each seen in 1 patient. Only 3 of the 10 ET patients with bleeding were negative for the JAK2 V617F mutation. Of the remaining 7, 3 were homozygous and 4 were heterozygous for the JAK2 V617F mutation. Although 95% of our PV patients were positive for the JAK2 V617F mutation, 1 of the 4 PV patients with bleeding were JAK2 V617F-negative. Two of the remaining patients were homozygous, and the remaining 1 was heterozygous for the JAK2 V617F mutation. Diabetes mellitus (DM) was identified in 14.8%, 12.4%, and 4.5% of the PV, ET, and PMF patients, respectively. DM was observed significantly more frequently in PV than in PMF patients
Clinical Lymphoma, Myeloma & Leukemia Month 2014
(P ¼ .02). No significant differences were found among different disease groups in terms of DM frequency. Essential hypertension was found in 18.5% of the PV patients, 19.4% in the ET patients, and 9% in the PV patients, with no significant differences among the 3 groups. Considering the time from diagnosis to the determination of the JAK2 V617F mutation status, no significant differences were found among the number of patients tested within 6 months or after 6 months of diagnosis in all MPN, PV, ET, and PMF patients (Fig. 3). The cytoreductive treatment (HU, IF, anagrelide) and antiaggregant agent were given according to risk classification5 and treatments of all patients are summarized in Table 5. There was 1 PV patient who received P-32 radioisotope treatment and is not listed in Table 5. Phlebotomy was practiced on PV patients when necessary. Patients receiving warfarin or clopidogrel either had venous/arterial thrombus or coronary angioplasty. Increased reticular fibrosis was observed in 1 patient who developed myelofibrosis (MF) secondary to PV 52 months after diagnosis and in 3 patients who developed MF secondary to ET 98, 48, and 66 months after diagnosis according to bone marrow biopsy. Eleven (4.7%) of the total 232 patients; 3 with PV (3.7%), 3 with ET (2.7%), and 5 with PMF (2.7%) died for reasons summarized in Table 6. Acute leukemia was observed in 2 PV (2.5%) and 3 ET (1.6%) patients. One PV patient developed acute myeloid leukemia (AML) in the 52nd month of follow-up and was lost to follow-up during chemotherapy. Another PV patient developed myelodysplastic syndrome (MDS) (refractory anemia with excess blasts-2) 127 months after diagnosis and azacytidine therapy was started. The patient progressed into AML and was lost to follow-up with sepsis. Three patients diagnosed with ET progressed into AML 13, 23, and 84 months after diagnosis and died despite chemotherapy. The median follow-up of the 232 MPN patients was 28 months (range, 1 to 219 months). At the end of the follow-up period, 11 patients (4.7%) had died. Estimated median OS could not be determined in the MPN, PV, or ET patient groups. Estimated mean OS was 147.8 months in the MPN group (95% confidence interval [CI], 164.9-210.7), 191.3 months in the PV group (95% CI, 155.5-227.1), and 143 months in the ET group (95% CI, 132.9-153.2). In the PMF group, estimated median and mean OS was 62 months (estimation is limited because of the longest survival time being censored) and 79.6 months (95% CI, 58.4-100.7), respectively (Fig. 4).
Discussion Janus kinase 2 V617F is the most frequent mutation in MPN patients. In ET and PMF patients, the JAK2 V617F mutation has been found in approximately 50%-60%, and the JAK2 V617F mutation was detected in approximately 95% of PV patients.17 In our study, JAK2 V617F mutation frequencies in our PV and ET patients were similar to those previously reported. Because our PMF group was small for an accurate evaluation, we reasoned that the frequency of the JAK2 V617F mutation in our PMF group would match those of larger studies if we had more PMF patients. In addition, we detected and quantified the JAK2 V617F mutation in more than 25% of the PMF patients who were at the 36th month
Kadriye Bahriye Payzin et al Figure 2 In the PV Group, There was no Significant Difference Among Male and Female Patients in Terms of the Homozygous Genotype (P [ .2). Twelve (42.8%) of the 28 Male and 27 (55.1%) of the 49 Female PV Patients Positive for JAK2 Were Homozygous. In the ET Group the Homozygous Genotype was Found in 15 (33%) of 45 JAK2-Positive Male Patients, Which was Significantly Greater Than the 8 (18.6%) of 43 JAK2-Positive Female Patients With the Same Genotype (P [ .04). In the PMF Group, 4 of 5 (80%) Female and 7 of 12 (58.3%) Male Patients had the Homozygous Genotype. Although no Significant Difference was Found Among the 2 Sexes (P [ .06), There Were More Female Than Male Patients With the Homozygous Genotype
Abbreviations: ET ¼ essential thrombocythemia; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera.
or more after their diagnosis. We were able to do that in only half of the PMF patients at the time of their diagnosis. The GIMEMA group has reported that heterozygous primary cases have progressed to a homozygous V617F status in the time from diagnosis to years after it.18 This might be one of the reasons we found a high JAK2 V617F allele burden in PMF. Polycythemia vera is defined as a disease seen predominantly in men,19 although there are PV groups reported to be predominantly female.20,21 Our PV group was also predominantly female (female to male ratio, 1.7-1). Female predominance in ET and male predominance in PMF has been reported in the literature.22 In our patient groups, we had almost an equal number of male and female ET patients, whereas the PMF patients were predominantly male. In clinical studies, patients with 50% JAK2 mutant allele burden are considered homozygous for the mutation, whereas patients with < 50% are considered heterozygous. Although evidence of greater JAK2 V617F allele burden in PV than ET have been reported in a few studies, data on allele burden in PMF remain to be validated.16,22-24 In our study, homozygous JAK2 V617F mutation was also more frequently found in PV compared with ET patients (50.6% and 26.1%, respectively, P ¼ .004).
Patients with the JAK2 V617F mutation were shown to have a tendency for higher Hb levels, higher WBC counts, and lower plt counts.9,10 In a study conducted in multiple centers, JAK2 V617F allele burden in PV patients was shown to be correlated with leukocytosis, greater htc values, larger spleen size, greater cell count, and fibrosis development.3,25 In a PV group of 105 patients, higher JAK2 V617F allele burden has been shown to be correlated with greater splenomegaly and higher WBC count, but not to age, sex, or htc levels.8 We found WBC count, LDH level, and spleen size to be greater in homozygous PV patients with high allele burden than in heterozygous PV patients with lower allele burden. In ET, several studies have shown that V617F-positive patients presented with older age at diagnosis, higher Hb level and WBC count, but lower plt count.3,9 In PMF, we found the same differences, except for the lower plt count. In our ET group, we found that patients with the JAK2 V617F mutation were younger, with larger splenomegaly, and higher Hb and WBC counts, compared with patients without the mutation. Although thrombosis seems to be more common in our PV patients than in our ET patients, this was not statistically significant (28% and 16%, respectively; P ¼ .07). None of our PMF patients had clinical thrombosis before or after diagnosis. We reported 44
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JAK2 V617F Mutation Status in CMPN Table 3 Comparison of Mean Serum LDH and Hemoglobin Level, Mean age, and Mean Leukocyte and Thrombocyte Count in Patients With (Positive) and Without (Negative) the JAK2 V617F Mutation LDH, U/L
Age, Years
Spleen Size, cm
Hemoglobin, g/dL
Leukocyte Count, 3 109/L
Thrombocyte Count, 3 109/L
301 115
62 11.8
3.1 3.6
17.5 1.7
15.3 5.5
660 264
194 42
62.2 18.4
0.8 1.4
19.6 2.2
11 5.1
312 128
.2
.9
.2
.02a
JAK2 V617Fþ
288 111
52.2 16.6
1.9 2.3
13.6 2
15 6.3
961 342
JAK2 V617F
236 101
60.8 15.9
0.7 1.2
12.4 1.6
10.9 3.2
991 268
.2
.006a
.006a
.001a
.001a
.6
Patient Population PV JAK2 V617Fþ
JAK2 V617F P
.02a
.1
ET
P PMF JAK2 V617Fþ
561 352
65.6 11
17.3 4.2
640 369
JAK2 V617F
874 924
53.2 13.5
12.3 7.4
10.7 0.9
8.5 3.8
647 276
.2
.04a
.04a
.4
.1
.9
P
6.6 4.1
11.5 2
Abbreviations: ET ¼ essential thrombocythemia; LDH ¼ lactic dehydrogenase; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera. a P < .05 is statistically significant.
ASA and HU treatment and had slightly higher than normal plt count. This patient also had additional conditions such as essential hypertension and DM, which could lead to vascular complications. In ET treatment, age older than 60 years is considered as high-risk, but in younger patients we also observed other conditions that could lead to vascular events. We therefore suggest more frequent monitoring of these patients for antiaggregant and cytoreductive therapies, and for the management of their other diseases. Some researchers have found thrombosis risk to be greater in JAK2 V617F-positive ET patients compared with JAK2 V617F-negative ET patients.9-11,16,26 Our results agree with these reports. All of our PV patients with thrombosis and most of our ET patients with thrombosis (76.1%) were JAK2 V617F mutation-positive. Recent studies in PV and ET have indicated that the frequency of thrombosis progressively increased with the amount of allele burden
(19%) cases of thrombosis among our 232 MPN patients. TIA was the most common thrombotic event in our PV and ET patients. It was observed in a little more than half (52%) of the 44 patients with thrombosis complications. Peripheral arterial thrombosis was not observed in PV, whereas it was observed in 5 (23.8%) of our 21 ET patients. Among our 23 PV patients with thrombosis, 21 had a history of thrombosis years before their PV diagnoses. Despite phlebotomy, ASA, and cytoreductive treatment, 1 patient developed PE and another developed TIA. Thrombosis was observed at the time of diagnosis in 3 of our 21 ET patients. In these 3 patients, JAK2 V617F allele burden was quantified as 78% to 100% in the 69-year-old male patient with PAT, and 12.5% to 31% in the 41-year-old male patient with PAT and the 18-year-old female patient with Budd-Chiari syndrome. A 56-year-old ET patient developed a TIA while she was receiving
Table 4 Comparison of Mean Serum LDH and Hemoglobin Level, Mean Age, and Mean Leukocyte and Thrombocyte Count in Patients With the JAK2 V617F Homozygous Genotype and Patients With the JAK2 V617F Heterozygous Genotype JAK2 V617F Mutation Genotype
Age, Years
LDH, U/L
Spleen Size, cm
Leukocyte Hemoglobin, g/dL Count, 3 109/L
Thrombocyte Count, 3 109/L
PV Homozygous
329 143
64 10.7
4.2 4.4
17.5 2
17.1 4.8
644 281
Heterozygous
271 64
60 10.6
1.8 1.9
17.4 1.4
13.5 5.7
675 249
.03a
.1
.006a
.8
.005a
.6
Homozygous
313 122
63 17.1
3.5 2.5
12.8 2.4
20.8 7
Heterozygous
280 107
60 15.4
1.4 1.9
13.9 1.8
12.8 4.5
938 308
.2
.9
.001a
.01a
.001a
.2
P ET
P
1026 422
PMF Homozygous
542 351
64 12
7.1 4
11.1 1.7
31.7 18
590 398
Heterozygous
583 385
68 70.5
5.6 4.5
12.1 2.6
18.3 10
732 344
.8
.5
.5
.3
.1
.4
P
6
-
Abbreviations: ET ¼ essential thrombocythemia; LDH ¼ lactic dehydrogenase; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera. a P < .05 is statistically significant.
Clinical Lymphoma, Myeloma & Leukemia Month 2014
Kadriye Bahriye Payzin et al Figure 3 Patients Identified as JAK2 Mutation Homozygous (1) and Heterozygous (2) in the First 6 Months After Diagnosis, and Patients Identified as Homozygous (3) and Heterozygous (4) After 6 Months or Later. The Frequency of JAK2 Mutation Positivity and the Frequency of Various Allele Burden Levels in our CMPNs Series did not Significantly Change in the Beginning or Later
Abbreviations: CMPNs ¼ chronic myeloproliferative neoplasms; et ¼ essential thrombocythemia; mf ¼ myelofibrosis; pv ¼ polycythemia vera.
in ET and PV.3,27-29 It has been reported that the highest rate of vascular complications was seen in groups with allele burden > 50%, found in most of PV patients and in a small proportion of ET patients.3 We had similar results in our patients. Homozygous genotype for the JAK2 V617F mutation was found in almost half (48.7%) of our PV patients with thrombosis. We identified the heterozygous JAK2 V617F mutation in 13 of 21 (61.9%), which is more than half of our ET patients with thrombosis. The homozygous genotype was found in 3 of our ET patients with thrombosis, whereas 5 of the ET patients with thrombosis were negative for the JAK2 V617F mutation. In their large study of 806 patients, Campbell et al have found that the JAK2 V617F mutation has a relatively small effect on
vascular complications that are observed in patients before their ET diagnoses.9 This group also observed that ET patients with the JAK2 V617F mutation do not have an increased risk of arterial or venous thrombosis after entry into the trial. Similarly, in a retrospective study of 867 ET patients, the GIMEMA group found that the JAK2 V617F mutation at diagnosis was not a predictor for occurrence of thrombosis during follow-up.30 We found that almost one-quarter (23.8%) of our ET patients with thrombosis were negative for the JAK2 V617F mutation. Additionally, we quantified the allele burden as < 31% (12.5%-31% in 6 patients, 2%-5% in 1 patient, and < 2% in 1 patient) in more than half (57%) of our JAK2 V617F-positive patients with thrombosis. These low values made us question the effect of the degree of JAK2 V617F allele burden on vascular events in ET patients. Although it is accepted that the occurrence of thrombosis in PV or ET is not directly linked to increased htc or plt counts, leukocytosis has recently been associated with increased risk of major vascular events and eventually could represent a major target of cytoreductive therapy.31 It is reported that a leukocyte count > 8.4 109/L in ET is also strictly associated with an increased risk of thrombosis as recently shown by Barbui et al.32,33 Several studies have confirmed that standard risk factors (age > 60 years and/or previous thrombosis) and leukocytosis (> 11.3 109/L in the GIMEMA study) were significantly associated with an increased risk of events in follow-up.9,34-36 Although we observed an increased frequency of thrombosis in patients with > 11 109/L WBC count consistent with the previous reports, we could not show the correlation of age > 60 years with increased frequency of thrombosis in PV and ET. Few data have been published on the influence of JAK2 V617F mutation on bleeding risk in MPNs. In PV patients, no correlation has been found between homozygous and heterozygous genotype and the occurrence of bleeding complications.15 In a study of 106 ET patients, minor bleeds from various mucocutaneous sites were observed in 7 patients, 6 of which were JAK2 V617F-negative. Analysis from this study indicated that JAK2 V617F does not have a protective role on hemorrhagic risk.31 In our study, gastrointestinal system (GIS) bleeding was observed in all PV patients with bleeding. GIS hemorrhage or epistaxis was observed in ET patients with bleeding. We have thus concluded, different from previous reports, that JAK2 V617F negativity is not correlated with bleeding complications in MPNs. We think that in most patients, taking antiaggregants or anticoagulants are more important in bleeding complications than is the JAK2 V617F mutation. In the
Table 5 Therapy and Follow-Up Data of 232 MPN Patients
Characteristic a
PV (81) ET (129)a PMF (22) Total: 232
HU 74 91 19 184
(91%) (71%) (86%) (79%)
ASA/Warfarin/ Clopidogrel 69 111 13 193
(85%); (86%); (59%); (83%);
5/1 5/4 0/0 10/5
Anagrelide 3 23 4 30
(3.7%) (18%) (18.2%) (13%)
Interferon 4 6 5 15
(4.9%) (4.6%) (22.7%) (6.5%)
Acute Leukemia 2 (2.5%) 3 (1.6%) e e
Died 3 3 5 11
(3.7%) (2.3%) (2.7%) (4.7%)
Median Time From Diagnosis, Months (Range) 38 24.5 36 28
(1-219) (1-152) (1-112) (1-219)
Abbreviations: ASA ¼ acetyl salicylic acid; ET ¼ essential thrombocythemia; HU ¼ hydroxyurea; MF ¼ myelofibrosis; MPN ¼ myeloproliferative neoplasm; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera. a MF secondary to PV, 1 patient (1.2%), and MF secondary to ET, 3 patients (2.3%).
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JAK2 V617F Mutation Status in CMPN Table 6 Data of 11 CMPN Patients Who Died During the Follow-Up Period Patient Number Diagnosis
Age, Years
Sex
JAK2 V617F Genotype
Therapy
1 2 3
PV PV PV
66 59 70
M F F
Homozygous Homozygous Heterozygous
HU, IF, ASA HU, ASA, Phlebotomy Phlebotomy
4 5 6 7
ET ET ET PMF
71 57 73 67
F M M M
Heterozygous Heterozygous Homozygous Heterozygous
8 9 10
PMF PMF PMF
61 62 78
M M M
Homozygous Homozygous Heterozygous
HU, ASA, anagrelide HU, ASA HU, ASA HU, splenoctomy, lenalidomide HU, splenectomy HU, splenectomy HU
11
PMF
60
F
Heterozygous
HU
Events Before Death AML MDS (RAEB-2) Portal hypertension, renal insuffiency, pneumonia AML AML AML Sepsis Heart failure Heart failure Coronary artery disease, renal failure, pneumonia Renal failure, diabetes mellitus, essential hypertension
Follow-Up, Months 53 132 1 93 54 35 56 72 62 1 61
Abbreviations: AML ¼ acute myeloid leukemia; ASA ¼ acetyl salicylic acid; CMPN ¼ chronic myeloproliferative neoplasm; ET ¼ essential thrombocythemia; F ¼ female; HU ¼ hydroxyurea; IF ¼ interferon; M ¼ male; MDS ¼ myelodysplastic syndrome; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera; RAEB ¼ refractory anemia with excess blasts.
PV and ET group, frequencies of clinical bleeding in patients homozygous and heterozygous for the JAK2 mutation were similar, consistent with results of previous studies. Hematologic transformations to MF, MDS, or AML are recognized long-term complications of PV and ET.37 One (1.2%) of our 81 PV patients progressed into MF secondary to PV, and 3 (2.3%) of our 129 ET patients progressed into MF secondary to ET. Our results were consistent with a study of 605 ET patients in which 17 (2.8%) had progressed to MF. In the same study, acute leukemia was seen in 14 (2.3%) patients, whereas in our study, slightly fewer
Figure 4 Kaplan-Meier Overall Survival Curves of PV, ET, and PMF Patients. PMF Patients Were Found to Have Shorter Survival Times Than PV and ET Patients
patients, 3 (1.6%) in the ET group and 1 (1.2%) in the PV group, progressed into acute leukemia. Leukemic clones emerging from V617F-positive chronic MPN can be either V617F-positive or V617F-negative.38 V617F-positive AML more frequently occurs after transition to the MF phase, whereas V617F-negative AML seems to arise directly from chronicphase JAK2-positive MPN.39 In our ET patient group, 1 progressed into AML after MF development. The remaining 2 patients directly developed AML. Three ET and 1 PV patient who had acute leukemia also had the JAK2 V617F mutation. The question of a possible effect of the JAK2 V617F mutation on OS of patients with MPN has not been clearly answered by previously published studies, which have provided conflicting results.27 So far, we only have the median survival in our PMF patients (62 months; 95% CI, 57.9-66). In the PV and ET groups, along with all MPN patients, we have not yet reached median survival statistically because of less patient loss in these goups. A subset of patients with PV, ET, and PMF harbor mutations in night lighteinducible and clock-regulated genes and the Casitas B-cell lymphoma gene, TET2, ASXL1, and IDH,40 but pathogenetic contributions of these and other mutations are unclear at this time.41 Nowadays, calreticulin mutations have been described. These mutations have been observed in approximately 70% of patients with ET or PMF who do not carry mutations in either the JAK2 or MPL gene, and have not been observed in patients with PV. Because all of these mutations are still being researched, we could not study these in our patients.
Conclusion
8
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Abbreviations: Cum ¼ cumulative; ET ¼ essential thrombocythemia; ex ¼ exitus; PMF ¼ primary myelofibrosis; pts ¼ patients; PV ¼ polycythemia vera.
Clinical Lymphoma, Myeloma & Leukemia Month 2014
Thrombosis was found in 19% of all of our MPN patients. TIA was the most common thrombotic event, seen in 52% of the patients with thrombosis. When our center began assessing patients for the JAK2 V617F mutation, we quantified the JAK2 V617F mutation in patients diagnosed at that time and previously. Among those 2 groups with a similar number of patients, there were no
Kadriye Bahriye Payzin et al significant differences in JAK2 V617F positivity or quantification values. However, our results were based on different patients assessed at different times for the JAK2 V617F mutation. Our results are limited by this, and it is apparent that JAK2 V617F assessments need to be done at certain time points after diagnosis to determine the effects of follow-up times and cytoreductive treatments on JAK2 V617F positivity and allele burden quantification. With longer follow-up and larger patient populations, this study aimed to investigate our patients’ progression into myelofibrosis or acute leukemia, the effect of clinical and laboratory parameters on patient survival, and finally to compare our results with current literature findings.
Clinical Practice Points In this study, we aimed to investigate the presence of JAK2
V617F in CMPNs patients in our center. JAK2 V617F mutation frequencies of our PV and ET patients were similar to those reported previously. JAK2 V617F mutation frequency of our PMF patients was higher than in previous reports. We attribute this difference to our low number of PMF patients, and think that it will match other reports if we have a larger data set. Transient ischemic attack was the most common thrombotic event in our PV and ET patients. In ET treatment age over 60 is considered a standard risk, but in younger patients we also observed other conditions such as essential hypertension, diabetes mellitus, hereditary thrombophilia that could lead to vascular events. We therefore suggest more frequent monitoring of these patients for antiaggregant and cytoreductive therapies, and for the management of their other diseases.
Disclosure The authors have stated that they have no conflicts of interest.
References 1. Tefferi A, Vardiman JW. Classification and diagnosis of myeloproliferative neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia 2008; 22:14-22. 2. Pardanani A, Lasho TL, Finke C, et al. Prevalence and clinicopathologic correlates of JAK2 exon 12 mutations in JAK2 V617F-negative polycythemia vera. Leukemia 2007; 21:1960-3. 3. Vannucchi AM, Antonioli E, Guglielmelli P, et al. Clinical correlates of JAK2 V617F presence or allele burden in myeloproliferative neoplasms: a critical reappraisal. Leukemia 2008; 22:1299-307. 4. Tefferi A. Polycythemia vera and essential thrombocythemia: 2012 update on diagnosis, risk stratification, and management. Am J Hematol 2012; 87:285-93. 5. Pardanani A, Lasho TL, Finke CM, et al. Infrequent occurrence of MPL exon 10 mutations in polycythemia vera and post-polycythemia vera myelofibrosis. Am J Hematol 2011; 86:701-2. 6. Martínez-Avilés L, Besses C, Álvarez-Larrán A, Torres E, Serrano S, Bellosillo B. TET2, ASXL1, IDH1, IDH2, and c-CBL genes in JAK2- and MPL-negative myeloproliferative neoplasms. Ann Hematol 2012; 91:533-41. 7. Nangalia J, Massie CE, Baxter EJ, et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med 2013; 369:2391-405. 8. Silver RT, Vandris K, Wang YL, et al. JAK2(V617F) allele burden in polycythemia vera correlates with grade of myelofibrosis, but is not substantially affected by therapy. Leuk Res 2011; 35:177-82. 9. Campbell PJ, Scott LM, Buck G, et al. Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study. Lancet 2005; 366:1945-53. 10. Finazzi G, Rambaldi A, Guerini V, et al. Risk of thrombosis in patients with essential thrombocythemia and polycythemia vera according to JAK2 V617F mutation status. Haematologica 2007; 92:135-6. 11. Wolansky AP, Lasho TL, Schwager SM, et al. JAK2 V617F mutation in essential thrombocythaemia: clinical associations and long-term prognostic relevance. Br J Haematol 2005; 131:208-13.
12. Campbell PJ, Griesshammer M, Dohner K, et al. V617F mutation in JAK2 is associated with poorer survival in idiopathic myelofibrosis. Blood 2006; 107:2098-100. 13. Tefferi A, Lasho TL, Schwager SM, et al. The JAK2(V617F) tyrosine kinase mutation in myelofibrosis with myeloid metaplasia: lineage specificity and clinical correlates. Br J Haematol 2005; 131:320-8. 14. Guglielmelli P, Barosi G, Specchia G, et al. Identification of patients with poorer survival in primary myelofibrosis based on the burden of JAK2 V617F mutated allele. Blood 2009; 114:1477-83. 15. Tefferi A, Lasho TL, Schwager SM, et al. The clinical phenotype of wild-type, heterozygous, and homozygous JAK2V617F in polycythemia vera. Cancer 2006; 106:631-5. 16. Vannucchi AM, Antonioli E, Guglielmelli P, et al. Clinical profile of homozygous JAK2 617V>F mutation in patients with polycythemia vera or essential thrombocythemia. Blood 2007; 110:840-6. 17. Cross NC. Genetic and epigenetic complexity in myeloproliferative neoplasms. Hematology Am Soc Hematol Educ Program 2011; 2011:208-14. 18. Barosi G, Bergamaschi G, Marchetti M, et al. JAK2 V617F mutational status predicts progression to large splenomegaly and leukemic transformation in primary myelofibrosis. Blood 2007; 110:4030-6. 19. McNally RJ, Rowland D, Roman E, Cartwright RA. Age and sex distributions of hematological malignancies in the U.K. Hematol Oncol 1997; 15:173-89. 20. Stein BL, Williams DM, Wang NY, et al. Sex differences in the JAK2 V617F allele burden in chronic myeloproliferative disorders. Haematologica 2010; 95:1090-7. 21. Johansson P, Kutti J, Andreasson B, et al. Trends in the incidence of chronic Philadelphia chromosome negative (Ph-) myeloproliferative disorders in the city of Goteborg, Sweden, during 1983-99. J Intern Med 2004; 256:161-5. 22. Passamonti F, Rumi E, Pietra D, et al. Relation between JAK2 (V617F) mutation status, granulocyte activation and constitutive mobilization of CD34positive cells into peripheral blood in myeloproliferative disorders. Blood 2006; 107:3676-82. 23. Tefferi A, Strand JJ, Lasho TL, et al. Bone marrow JAK2 V617F allele burden and clinical correlates in polycythemia vera. Leukemia 2007; 21:2074-5. 24. Kittur J, Knudson RA, Lasho TL, et al. Clinical correlates of JAK2V617F allele burden in essential thrombocythemia. Cancer 2007; 109:2279-84. 25. Vannucchi AM, Antonioli E, Guglielmelli P, et al. Prospective identification of high-risk polycythemia vera patients based on JAK2(V617F) allele burden. Leukemia 2007; 21:1952-9. 26. Cheung B, Radia D, Pantelidis P, Yadegarfar G, Harrison C. The presence of the JAK2 V617F mutation is associated with a higher haemoglobin and increased risk of thrombosis in essential thrombocythaemia. Br J Haematol 2006; 132:244-5. 27. Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005; 7:387-97. 28. Vainchenker W, Constantinescu SN. A unique activating mutation in JAK2 (V617F) is at the origin of polycythemia vera and allows a new classification of myeloproliferative diseases. Hematology Am Soc Hematol Educ Program 2005: 195-200. 29. Prchal JF, Axelrad AA. Letter: bone-marrow responses in polycythemia vera. N Engl J Med 1974; 290:1382. 30. Carobbio A, Finazzi G, Antonioli E, et al. JAK2 V617F allele burden and thrombosis: a direct comparison in essential thrombocythemia and polycythemia vera. Exp Hematol 2009; 37:1016-21. 31. Barbui T, Carobbio A, Rambaldi A, Finazzi G. Perspectives on thrombosis in essential thrombocythemia and polycythemia vera: is leukocytosis a causative factor? Blood 2009; 114:759-63. 32. Patriarca A, Pompetti F, Malizia R, et al. Is the absence of JAK2 mutation a risk factor for bleeding in essential thrombocythemia? An analysis of 106 patients. Blood Transfus 2010; 8:21-7. 33. Barbui T, Barosi G, Grossi A, et al. Practice guidelines for the therapy of essential thrombocythemia. A statement from the Italian Society of Hematology, the Italian Society of Experimental Hematology and the Italian Group for Bone Marrow Transplantation. Haematologica 2004; 89:215-32. 34. Wilkins BS, Erber WN, Bareford D, et al. Bone marrow pathology in essential thrombocythemia: interobserver reliability and utility for identifying disease subtypes. Blood 2008; 111:60-70. 35. Levine RL, Loriaux M, Huntly BJ, et al. The JAK2 V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia. Blood 2005; 106:3377-9. 36. Moliterno AR, Williams DM, Rogers O, Isaacs MA, Spivak JL. Phenotypic variability within the JAK2 V617F-positive MPD: roles of progenitor cell and neutrophil allele burdens. Exp Hematol 2008; 36:1480-6. 37. Campbell PJ, Green AR. The myeloproliferative disorders. N Engl J Med 2006; 355:2452-66. 38. Theocharides A, Boissinot M, Girodon F, et al. Leukemic blasts in transformed JAK2 V617F-positive myeloproliferative disorders are frequently negative for the JAK2 V617F mutation. Blood 2007; 110:375-9. 39. Beer PA, Delhommeau F, LeCouédic JP, et al. Two routes to leukemic transformation after a JAK2 mutation-positive myeloproliferative neoplasm. Blood 2010; 115:2891-900. 40. Tefferi A, Vainchenker W. Myeloproliferative neoplasms: molecular pathophysiology, essential clinical understanding, and treatment strategies. J Clin Oncol 2011; 29:573-82. 41. Vainchenker W, Delhommeau F, Constantinescu SN, Bernard OA. New mutations and pathogenesis of myeloproliferative neoplasms. Blood 2011; 118:1723-35.
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JAK2 V617F Mutation Status of 232 Patients Diagnosed With Chronic Myeloproliferative Neoplasms Kadriye Bahriye Payzin,1 Kaan Savasoglu,2 Inci Alacacioglu,3 Fusun Ozdemirkiran,1 Belgin Berber Mutlu,2 Sadi Bener,4 Aylin Orgen Calli,4 Betul Bolat Kucukzeybek,4 Saliha Haksun5 Abstract The aim of this study was to investigate the presence of Janus kinase 2 (JAK2) V617F mutation in patients with BCR-ABL negative chronic myeloproliferative neoplasms (CMPNs) in our center. JAK2 V617F mutation frequencies in our PV and ET patients were similar to those reported previously. JAK2 V617F mutation frequency in our PMF patients was greater than in previous reports. Introduction/Background: The aim of this study was to investigate the presence of Janus kinase 2 (JAK2) V617F mutation in patients with break point cluster region-abelson negative chronic myeloproliferative neoplasms (CMPNs) in our center. Patients and Methods: We compared patients with and without the mutation, and also patients with the homozygous and heterozygous mutation, in terms of different clinical and laboratory features. Results: The JAK2 V617F mutation was detected in 77 (95%), 88 (68%), and 17 (77%) of polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) patients, respectively. Among JAK2 V617Fpositive patients, the homozygous genotype was found in 39 (50.6%) of the 77 PV, 23 (26.1%) of the 88 ET, and 11 (64.7%) of the 17 PMF patients. Bleeding was seen in 14 (6%) of all patients. Upper gastrointestinal bleeds were the most common, seen in 11 patients. Out of 232 CMPN patients, 44 (19%) had thrombosis. The most common thrombotic event was transient ischemic attack (52%). Progression to myelofibrosis was seen in 1 (1.2%) PV and 3 (2.3%) ET patients, and progression to acute leukemia was seen in 2 (2.5%) PV and 3 (2.3%) ET patients. Three patients with PV (3.7%), 3 with ET (2.7%), and 5 with PMF (2.7%) died during follow-up. Conclusion: JAK2 V617F mutation frequencies in our PV and ET patients were similar to those reported previously. JAK2 V617F mutation frequency in our PMF patients was greater than in previous reports. All of our PV patients with thrombosis and most of our ET patients with thrombosis (76.1%) were JAK2 V617F mutation-positive. This mutation seems to be correlated with thrombosis risk. Clinical Lymphoma, Myeloma & Leukemia, Vol. -, No. -, --- ª 2014 Elsevier Inc. All rights reserved. Keywords: Essential thrombocythemia, JAK2 mutation, MPNs, Polycythemia vera, Primary myelofibrosis
1
Division of Hematology, Department of Internal Medicine, Ataturk Training Hospital, Izmir, Turkey 2 Medical Genetics Laboratory, Ataturk Training Hospital, Izmir, Turkey 3 Department of Hematology, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey 4 Department of Pathology, Ataturk Training Hospital, Izmir, Turkey 5 Department of Biochemistry, Ataturk Training Hospital, Izmir, Turkey Submitted: Jan 28, 2014; Revised: Feb 19, 2014; Accepted: Feb 24, 2014 Address for correspondence: Kadriye Bahriye Payzin, MD, Division of Hematology, Department of Internal Medicine, Ataturk Training Hospital, Izmir, Turkey E-mail contact: [email protected]
2152-2650/$ - see frontmatter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clml.2014.02.013
Introduction Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are classified in the break point cluster region-abelson (BCR-ABL1) negative myeloproliferative neoplasm (MPN) group according to World Health Organization (WHO) 2008 classification.1 Their disease-causing mutations remain unidentified despite many mutations described beginning in 2005. PV patients mostly harbor Janus kinase 2 (JAK2; 9p24) mutation (96% displaying mutation in exon 14, JAK2 V617F; 3% displaying mutation in exon 12; JAK2).2,3
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JAK2 V617F Mutation Status in CMPN Janus kinase 2 V617F also occurs in ET (55%) and PMF (65%), but JAK2 exon 12 mutations are rare in ET and PMF.4 Myeloproliferative leukemia virus oncogene (MPL) (MPL515W>L/K) mutations are found in approximately 4% of ET, 8% of PMF, and rarely in PV.5 According to the 2008 WHO classification, the existence of these mutations is the major criterion in diagnosis of chronic MPNs.1 Nonetheless, evaluation of bone marrow histopathology is still required to define the specific morphological patterns, which are essential for the diagnosis of ET and PMF. Other mutations related to the tet oncogene family member 2 (TET2), additional sex comb-like 1 (ASXL1), and isocitrate dehydrogenase (IDH1/2) genes are occasionally observed in MPNs.6 Because the molecular pathogenesis of MPNs with nonmutated JAK2 is still obscure and diagnosis remains a challenge, the mutation status of the gene encoding calreticulin (CALR) was studied recently by Nangalia et al.7 CALR mutations were found in most patients with MPNs with nonmutated JAK2.7 Leukocytosis, splenomegaly, thrombohemorrhagic complications, vasomotor disturbances, pruritus, and a small risk of disease progression into acute myeloid leukemia or myelofibrosis are other disease features.4 In previous studies, the importance of mutational status of JAK2 was evaluated. Correlations have been found between high JAK2 V617F allele burden and leukocytosis, spleen size, myelofibrosis severity, fibrotic transformation, and disease duration.8 Many researchers have reported thrombosis risk in JAK2 V617F-positive ET patients to be greater than that of JAK2 V617F-negative ET patients.9-11 The effect of the JAK2 V617F mutation on clinical parameters of PMF such as prognosis and the need for transfusion is unclear.12,13 JAK2 V617F mutated status has not been shown to have an effect on survival time, but patients with low JAK2 V617F allele burden have been reported to have shorter survival times compared with those with high JAK2 V617F allele burden and wild type JAK2.14 In this study, we aimed to investigate the relationship between JAK2 status/quantitation and clinic and laboratory findings in our MPNs patients.
Patients and Methods Demographic and clinical features of 233 patients diagnosed with MPNs at our hematology department between November 2000 and November 2012 were evaluated retrospectively. The median age was 63 years (range, 18 to 89 years) with a male to female ratio of 0.9 to 1 (110 men, 123 women). BCR-ABL molecular analysis and fluorescence in situ hybridization analysis for t(9;22) were made in all MPN patients and found as not amplified. During retrospective evaluation, 1 male patient was excluded from the study because of diagnosis of chronic myelomonocytic leukemia. Eighty-one patients were rediagnosed with PV (34.9%), 129 with ET (55.6%), and 22 with PMF (9.5%) according to polycythemia vera study group or WHO 2008 criteria. White blood cell (WBC), hemoglobin (Hb), and hematocrit (htc) levels, and platelet (plt) count, and serum lactic dehydrogenase (LDH) level were recorded. Spleen size was measured in centimeters below the midpoint of the left costal margin, and spleen size > 5 cm was accepted as gross splenomegaly. JAK2 V617F analysis was performed at the time or after the time of diagnosis depending on the availability of this test. Thrombotic and hemorrhagic events before or after diagnosis were recorded. Clinical thrombotic events included ischemic stroke, cerebral transient ischemic attack (TIA), acute myocardial infarction (AMI), peripheral arterial thrombosis (PAT), pulmonary thromboembolism (PE), and venous thromboembolism (VTE). Most patients received 1 or more of the following: acetyl salicylic acid (ASA), some form of myelosuppressive therapy as hydroxyurea (HU), anagrelide, interferon (IF), phosphorus-32 (P-32). Patients with PV were phlebotomized to maintain an htc level 45%. This study was approved by the local ethics committee. Quantification of JAK2 V617F mutation was detected using an allele-specific real-time quantitative polymerase chain reaction (RQ-PCR) assay (JAK2 MutaQuant Kit, Ipsogen). RQ-PCR was performed using a Rotor-Gene (Corbett Research). Patients with 50% mutational load and those with < 50% mutational load were considered ‘homozygotes’ and ‘heterozygotes,’ respectively, as has been previously designated.15,16
Table 1 Characteristics of 232 Patients With MPNs
M/F Mean Age (Minimum-Maximum), Years Splenomegaly, n (%) Thrombosis, n (%) Bleeding, n (%) Diabetes Mellitus, n (%) Hypertension, n (%) Mean Hemoglobin Level ± SD, g/dL Mean Hematocrit Level ± SD, % Mean Leukocyte Count ± SD, 3 109/L Mean Thrombocyte Count ± SD, 3 109/L Mean Serum LDH ± SD, U/L
MPN
PV (81 Pts)
ET (129 Pts)
PMF (22 Pts)
109/123 59.9 (18-89) 125 (59.8) 44 (19) 14 (6) 29 (12.5) 42 (18.1) 14.6 3.0 13.6 10.3 15.2 8.0 344.7 22.9 313.7 219
30/51 62 (35-86) 46 (67.6) 23 (28.3) 4 (4.9) 12 (14.8) 15 (18.5) 17.6 1.8 53.6 5.9 15.2 5.6 646.9 268.4 297 115
65/64 58 (18-89) 57 (47.5) 21 (16.3) 10 (7.6) 16 (12.4) 25 (19.4) 13.26 2.0 39.8 8.3 13.7 5.9 971.0 319.9 271.9 110.5
14/8 62.8 (32-85) 22 (100) NR NR 1 (4.5) 2 (9) 11.3 1.9 33.6 5.5 23.9 16.6 344.6 73.5 648.1 556.2
Abbreviations: ET ¼ essential thrombocythemia; F ¼ female; LDH ¼ lactic dehydrogenase; M ¼ male; MPN ¼ myeloproliferative neoplasm; PMF ¼ primary myelofibrosis; pts ¼ patients; PV ¼ polycythemia vera.
2
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Clinical Lymphoma, Myeloma & Leukemia Month 2014
Kadriye Bahriye Payzin et al Statistical Analysis All statistical analysis was performed using the SPSS 18.0 statistical package. Analysis of variance test for continuous variables and c2 test for categorical variables as appropriate were used. Overall survival (OS) was defined as the time between diagnosis and death (from any causes) or end of follow-up (censored observations). OS was estimated using the Kaplan-Meier product limit method. All P values were 2-sided. A value of P < .05 was considered as significant.
Results The demographic, laboratory, and clinical data of all patients are summarized in Table 1. There were significantly more female patients than male patients in the PV group (P ¼ .01). In contrast, there were significantly more male patients than female patients (P ¼ .01) in the PMF group. Mean age was similar in all 3 groups. All patients with PMF had splenomegaly compared with others (P ¼ .01). Gross splenomegaly was observed in PV and ET patients with similar frequency. Out of 232 MPN patients, 182 (78.4%) were positive for the JAK2 V617F mutation. The distribution of allele burden of mutation-positive patients is given in Table 2 and Figure 1. JAK2 V617F mutations were found in 77 patients in the PV group (95%), 88 patients in the ET group (68%), and 17 patients in the PMF group (77%) (Table 2). The PV and ET groups showed no significant differences for JAK2 V617F mutation positivity among the 2 sexes (P ¼ .8 for both). The JAK2 V617F mutation was found significantly more frequently in male patients in the PMF group (P ¼ .049). The heterozygous genotype was found significantly more frequently in the ET group, whereas the homozygous genotype was more frequent in the PMF group (P ¼ .000 and 0.003, respectively). The percentage of PV patients with the homozygous genotype was significantly greater than the percentage of ET patients with the same genotype (P ¼ .004) (Fig. 1). JAK2 V617F allele burden status according to sex is shown in Figure 2. In the PV group, there was no significant difference among male and female patients in terms of the homozygous genotype (P ¼ .2). In the ET group the homozygous genotype was found significantly more in JAK2 V617F-positive male patients, than in JAK2 V617Fpositive female patients (P ¼ .04). In the PMF group, no significant difference was found among the 2 sexes (P ¼ .06) (Fig. 2). Mean plt count was statistically greater but Hb level was lower in PV patients with the JAK2 V617F mutation. When compared with ET patients, mean spleen size, Hb and LDH level, and WBC count
were found to be significantly greater in patients with the JAK2 V617F mutation (P ¼ .006, .001, .01, and .001, respectively). When a similar comparison was made among PMF patients, mean age was older for the JAK2 V617F-positive group and mean spleen size was greater for the JAK2 V617F-negative group (P ¼ .04 for both). There were no significant differences among the 2 groups in terms of LDH and Hb levels, or WBC and plt counts (Table 3). Myeloproliferative neoplasm patients homozygous and heterozygous for the JAK2 V617F mutation were also compared for their Hb and serum LDH levels, WBC and plt counts, and spleen size (Table 4). Mean spleen size, LDH level, and WBC count were significantly greater in the JAK2 V617F homozygous PV patients (P ¼ .006, .03 and .005, respectively). Among the ET patients, mean WBC count and spleen size were significantly greater in patients homozygous for the JAK2 V617F mutation (P ¼ .001 and .001, respectively). In contrast, mean Hb level of the homozygous ET patients was significantly lower compared with the heterozygous ET patients (P ¼ .01). PMF patients with homozygous and heterozygous JAK2 V617F mutations did not show significant differences in age, spleen size, Hb and serum LDH levels, and WBC and plt counts (Table 4). Among all MPN patients, 44 (19%) had thrombosis. Thrombosis was not observed in any PMF patients. In the PV group, only 2 of the 23 patients (28.3%) with thrombosis developed it after the diagnosis (1 had PE and VTE, the other had TIA). The remaining 21 patients (28.4%) either had thrombosis for years, or were diagnosed with MPNs when searching for the cause of their thromboses. TIA was the most common vascular event in the PV group (15 patients). Portal vein thrombosis was observed in 3 patients, AMI in 2 patients, deep vein thrombosis (DVT) in 2 patients (1 with a history of pulmonary thromboembolism), and hepatic vein thrombosis (Budd-Chiari syndrome) in 1 patient. Eleven PV patients with thrombosis were homozygous for the JAK2 V617F mutation; the remaining patients were heterozygous. A homozygous JAK2 V617F mutation was found in 6 patients with TIA, all AMI and DVT patients, and 1 patient with portal vein thrombosis. There were no PV patients with thrombosis who tested negative for the JAK2 V617F mutation. When PV patients with and without thrombosis were compared, no significant differences were found in terms of age, spleen size, htc, Hb, and serum LDH levels, and WBC and plt counts. Twenty-one (16.3%) of the ET patients had thrombosis. Nineteen patients had a history of thrombosis before their ET diagnoses, and 2 patients were diagnosed with ET along with clinical thrombosis. Three of these patients were homozygous, 13 were
Table 2 Distribition of Allele Burden of 182 (78.4%) MPN Patients Who Were JAK2eV617F-Positive JAK2 V617F Allele Burden
78%-100%
50%-78%
Homozygous Genotypea
31%-50%
12.5%-31%
5%-12.5%
2%-5%
Heterozygous Genotypeb
PV: 77 (95%) ET: 88 (68%) PMF: 17 (77%)
12 (15.6) 14 (16) 5 (29.4)
27 (35) 9 (10) 6 (35.2)
39 (50.6) 23 (26) 11 (64.6)
21(27.4) 25 (28.4) 6 (35.4)
17 (22) 24 (27.2) 0
0 14 (15.9) 0
0 2 (2.5) 0
38 (49.4) 65 (74) 6 (27)
Data are presented as n (%). Abbreviations: ET ¼ essential thrombocythemia; MPN ¼ myeloproliferative neoplasm; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera. a JAK2 V617F allele burden 50%. b JAK2 V617F allele burden < 50%.
Clinical Lymphoma, Myeloma & Leukemia Month 2014
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JAK2 V617F Mutation Status in CMPN Figure 1 The Percent Interval of JAK2 V617F Mutation Alleles are Shown on the Y-Axis; 1 Represents 2%-5%, 2 Represents 5%-12.5%, 3 Represents 12.5%-31%, 4 Represents 31%-50%, 5 Represents 50%-78%, and 6 Represents 78%-100%. Patients With ‡ 50% Mutational Load and Those With < 50% Mutational Load Were Homozygotes and Heterozygotes, Respectively. The Heterozygous Genotype was Found Significantly More Frequently in the ET Group, and the Homozygous Genotype was More Frequent in the PMF Group (P [ .000 and .003, Respectively). The Percentage of PV Patients With the Homozygous Genotype was Significantly Greater Than the Percentage of ET Patients With the Same Genotype (P [ .004)
Abbreviations: ET ¼ essential thrombocythemia; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera.
4
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heterozygous, and 5 were negative for the JAK2 V617F mutation. A variety of thrombotic events were observed in ET patients. Starting from the most common was TIA, observed in 8 patients, PAT (2 patients with aortofemoral bypass) and AMI, both observed in 5 patients, Budd-Chiari syndrome observed in 3 patients, and PE and portal vein thrombosis, both observed in 1 patient. No significant differences were found among ET patients with or without thrombosis with respect to age, spleen size, htc, Hb, and serum LDH levels, and WBC and plt counts. There was no significant difference between PV and ET patients with regard to thrombosis frequency (P ¼ .07). Bleeding was seen in 14 (6%) MPN patients. Upper gastrointestinal bleeding was the most common, seen in 11 patients. Rectal or vaginal hemorrhage and epistaxis were also observed, each seen in 1 patient. Only 3 of the 10 ET patients with bleeding were negative for the JAK2 V617F mutation. Of the remaining 7, 3 were homozygous and 4 were heterozygous for the JAK2 V617F mutation. Although 95% of our PV patients were positive for the JAK2 V617F mutation, 1 of the 4 PV patients with bleeding were JAK2 V617F-negative. Two of the remaining patients were homozygous, and the remaining 1 was heterozygous for the JAK2 V617F mutation. Diabetes mellitus (DM) was identified in 14.8%, 12.4%, and 4.5% of the PV, ET, and PMF patients, respectively. DM was observed significantly more frequently in PV than in PMF patients
Clinical Lymphoma, Myeloma & Leukemia Month 2014
(P ¼ .02). No significant differences were found among different disease groups in terms of DM frequency. Essential hypertension was found in 18.5% of the PV patients, 19.4% in the ET patients, and 9% in the PV patients, with no significant differences among the 3 groups. Considering the time from diagnosis to the determination of the JAK2 V617F mutation status, no significant differences were found among the number of patients tested within 6 months or after 6 months of diagnosis in all MPN, PV, ET, and PMF patients (Fig. 3). The cytoreductive treatment (HU, IF, anagrelide) and antiaggregant agent were given according to risk classification5 and treatments of all patients are summarized in Table 5. There was 1 PV patient who received P-32 radioisotope treatment and is not listed in Table 5. Phlebotomy was practiced on PV patients when necessary. Patients receiving warfarin or clopidogrel either had venous/arterial thrombus or coronary angioplasty. Increased reticular fibrosis was observed in 1 patient who developed myelofibrosis (MF) secondary to PV 52 months after diagnosis and in 3 patients who developed MF secondary to ET 98, 48, and 66 months after diagnosis according to bone marrow biopsy. Eleven (4.7%) of the total 232 patients; 3 with PV (3.7%), 3 with ET (2.7%), and 5 with PMF (2.7%) died for reasons summarized in Table 6. Acute leukemia was observed in 2 PV (2.5%) and 3 ET (1.6%) patients. One PV patient developed acute myeloid leukemia (AML) in the 52nd month of follow-up and was lost to follow-up during chemotherapy. Another PV patient developed myelodysplastic syndrome (MDS) (refractory anemia with excess blasts-2) 127 months after diagnosis and azacytidine therapy was started. The patient progressed into AML and was lost to follow-up with sepsis. Three patients diagnosed with ET progressed into AML 13, 23, and 84 months after diagnosis and died despite chemotherapy. The median follow-up of the 232 MPN patients was 28 months (range, 1 to 219 months). At the end of the follow-up period, 11 patients (4.7%) had died. Estimated median OS could not be determined in the MPN, PV, or ET patient groups. Estimated mean OS was 147.8 months in the MPN group (95% confidence interval [CI], 164.9-210.7), 191.3 months in the PV group (95% CI, 155.5-227.1), and 143 months in the ET group (95% CI, 132.9-153.2). In the PMF group, estimated median and mean OS was 62 months (estimation is limited because of the longest survival time being censored) and 79.6 months (95% CI, 58.4-100.7), respectively (Fig. 4).
Discussion Janus kinase 2 V617F is the most frequent mutation in MPN patients. In ET and PMF patients, the JAK2 V617F mutation has been found in approximately 50%-60%, and the JAK2 V617F mutation was detected in approximately 95% of PV patients.17 In our study, JAK2 V617F mutation frequencies in our PV and ET patients were similar to those previously reported. Because our PMF group was small for an accurate evaluation, we reasoned that the frequency of the JAK2 V617F mutation in our PMF group would match those of larger studies if we had more PMF patients. In addition, we detected and quantified the JAK2 V617F mutation in more than 25% of the PMF patients who were at the 36th month
Kadriye Bahriye Payzin et al Figure 2 In the PV Group, There was no Significant Difference Among Male and Female Patients in Terms of the Homozygous Genotype (P [ .2). Twelve (42.8%) of the 28 Male and 27 (55.1%) of the 49 Female PV Patients Positive for JAK2 Were Homozygous. In the ET Group the Homozygous Genotype was Found in 15 (33%) of 45 JAK2-Positive Male Patients, Which was Significantly Greater Than the 8 (18.6%) of 43 JAK2-Positive Female Patients With the Same Genotype (P [ .04). In the PMF Group, 4 of 5 (80%) Female and 7 of 12 (58.3%) Male Patients had the Homozygous Genotype. Although no Significant Difference was Found Among the 2 Sexes (P [ .06), There Were More Female Than Male Patients With the Homozygous Genotype
Abbreviations: ET ¼ essential thrombocythemia; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera.
or more after their diagnosis. We were able to do that in only half of the PMF patients at the time of their diagnosis. The GIMEMA group has reported that heterozygous primary cases have progressed to a homozygous V617F status in the time from diagnosis to years after it.18 This might be one of the reasons we found a high JAK2 V617F allele burden in PMF. Polycythemia vera is defined as a disease seen predominantly in men,19 although there are PV groups reported to be predominantly female.20,21 Our PV group was also predominantly female (female to male ratio, 1.7-1). Female predominance in ET and male predominance in PMF has been reported in the literature.22 In our patient groups, we had almost an equal number of male and female ET patients, whereas the PMF patients were predominantly male. In clinical studies, patients with 50% JAK2 mutant allele burden are considered homozygous for the mutation, whereas patients with < 50% are considered heterozygous. Although evidence of greater JAK2 V617F allele burden in PV than ET have been reported in a few studies, data on allele burden in PMF remain to be validated.16,22-24 In our study, homozygous JAK2 V617F mutation was also more frequently found in PV compared with ET patients (50.6% and 26.1%, respectively, P ¼ .004).
Patients with the JAK2 V617F mutation were shown to have a tendency for higher Hb levels, higher WBC counts, and lower plt counts.9,10 In a study conducted in multiple centers, JAK2 V617F allele burden in PV patients was shown to be correlated with leukocytosis, greater htc values, larger spleen size, greater cell count, and fibrosis development.3,25 In a PV group of 105 patients, higher JAK2 V617F allele burden has been shown to be correlated with greater splenomegaly and higher WBC count, but not to age, sex, or htc levels.8 We found WBC count, LDH level, and spleen size to be greater in homozygous PV patients with high allele burden than in heterozygous PV patients with lower allele burden. In ET, several studies have shown that V617F-positive patients presented with older age at diagnosis, higher Hb level and WBC count, but lower plt count.3,9 In PMF, we found the same differences, except for the lower plt count. In our ET group, we found that patients with the JAK2 V617F mutation were younger, with larger splenomegaly, and higher Hb and WBC counts, compared with patients without the mutation. Although thrombosis seems to be more common in our PV patients than in our ET patients, this was not statistically significant (28% and 16%, respectively; P ¼ .07). None of our PMF patients had clinical thrombosis before or after diagnosis. We reported 44
Clinical Lymphoma, Myeloma & Leukemia Month 2014
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JAK2 V617F Mutation Status in CMPN Table 3 Comparison of Mean Serum LDH and Hemoglobin Level, Mean age, and Mean Leukocyte and Thrombocyte Count in Patients With (Positive) and Without (Negative) the JAK2 V617F Mutation LDH, U/L
Age, Years
Spleen Size, cm
Hemoglobin, g/dL
Leukocyte Count, 3 109/L
Thrombocyte Count, 3 109/L
301 115
62 11.8
3.1 3.6
17.5 1.7
15.3 5.5
660 264
194 42
62.2 18.4
0.8 1.4
19.6 2.2
11 5.1
312 128
.2
.9
.2
.02a
JAK2 V617Fþ
288 111
52.2 16.6
1.9 2.3
13.6 2
15 6.3
961 342
JAK2 V617F
236 101
60.8 15.9
0.7 1.2
12.4 1.6
10.9 3.2
991 268
.2
.006a
.006a
.001a
.001a
.6
Patient Population PV JAK2 V617Fþ
JAK2 V617F P
.02a
.1
ET
P PMF JAK2 V617Fþ
561 352
65.6 11
17.3 4.2
640 369
JAK2 V617F
874 924
53.2 13.5
12.3 7.4
10.7 0.9
8.5 3.8
647 276
.2
.04a
.04a
.4
.1
.9
P
6.6 4.1
11.5 2
Abbreviations: ET ¼ essential thrombocythemia; LDH ¼ lactic dehydrogenase; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera. a P < .05 is statistically significant.
ASA and HU treatment and had slightly higher than normal plt count. This patient also had additional conditions such as essential hypertension and DM, which could lead to vascular complications. In ET treatment, age older than 60 years is considered as high-risk, but in younger patients we also observed other conditions that could lead to vascular events. We therefore suggest more frequent monitoring of these patients for antiaggregant and cytoreductive therapies, and for the management of their other diseases. Some researchers have found thrombosis risk to be greater in JAK2 V617F-positive ET patients compared with JAK2 V617F-negative ET patients.9-11,16,26 Our results agree with these reports. All of our PV patients with thrombosis and most of our ET patients with thrombosis (76.1%) were JAK2 V617F mutation-positive. Recent studies in PV and ET have indicated that the frequency of thrombosis progressively increased with the amount of allele burden
(19%) cases of thrombosis among our 232 MPN patients. TIA was the most common thrombotic event in our PV and ET patients. It was observed in a little more than half (52%) of the 44 patients with thrombosis complications. Peripheral arterial thrombosis was not observed in PV, whereas it was observed in 5 (23.8%) of our 21 ET patients. Among our 23 PV patients with thrombosis, 21 had a history of thrombosis years before their PV diagnoses. Despite phlebotomy, ASA, and cytoreductive treatment, 1 patient developed PE and another developed TIA. Thrombosis was observed at the time of diagnosis in 3 of our 21 ET patients. In these 3 patients, JAK2 V617F allele burden was quantified as 78% to 100% in the 69-year-old male patient with PAT, and 12.5% to 31% in the 41-year-old male patient with PAT and the 18-year-old female patient with Budd-Chiari syndrome. A 56-year-old ET patient developed a TIA while she was receiving
Table 4 Comparison of Mean Serum LDH and Hemoglobin Level, Mean Age, and Mean Leukocyte and Thrombocyte Count in Patients With the JAK2 V617F Homozygous Genotype and Patients With the JAK2 V617F Heterozygous Genotype JAK2 V617F Mutation Genotype
Age, Years
LDH, U/L
Spleen Size, cm
Leukocyte Hemoglobin, g/dL Count, 3 109/L
Thrombocyte Count, 3 109/L
PV Homozygous
329 143
64 10.7
4.2 4.4
17.5 2
17.1 4.8
644 281
Heterozygous
271 64
60 10.6
1.8 1.9
17.4 1.4
13.5 5.7
675 249
.03a
.1
.006a
.8
.005a
.6
Homozygous
313 122
63 17.1
3.5 2.5
12.8 2.4
20.8 7
Heterozygous
280 107
60 15.4
1.4 1.9
13.9 1.8
12.8 4.5
938 308
.2
.9
.001a
.01a
.001a
.2
P ET
P
1026 422
PMF Homozygous
542 351
64 12
7.1 4
11.1 1.7
31.7 18
590 398
Heterozygous
583 385
68 70.5
5.6 4.5
12.1 2.6
18.3 10
732 344
.8
.5
.5
.3
.1
.4
P
6
-
Abbreviations: ET ¼ essential thrombocythemia; LDH ¼ lactic dehydrogenase; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera. a P < .05 is statistically significant.
Clinical Lymphoma, Myeloma & Leukemia Month 2014
Kadriye Bahriye Payzin et al Figure 3 Patients Identified as JAK2 Mutation Homozygous (1) and Heterozygous (2) in the First 6 Months After Diagnosis, and Patients Identified as Homozygous (3) and Heterozygous (4) After 6 Months or Later. The Frequency of JAK2 Mutation Positivity and the Frequency of Various Allele Burden Levels in our CMPNs Series did not Significantly Change in the Beginning or Later
Abbreviations: CMPNs ¼ chronic myeloproliferative neoplasms; et ¼ essential thrombocythemia; mf ¼ myelofibrosis; pv ¼ polycythemia vera.
in ET and PV.3,27-29 It has been reported that the highest rate of vascular complications was seen in groups with allele burden > 50%, found in most of PV patients and in a small proportion of ET patients.3 We had similar results in our patients. Homozygous genotype for the JAK2 V617F mutation was found in almost half (48.7%) of our PV patients with thrombosis. We identified the heterozygous JAK2 V617F mutation in 13 of 21 (61.9%), which is more than half of our ET patients with thrombosis. The homozygous genotype was found in 3 of our ET patients with thrombosis, whereas 5 of the ET patients with thrombosis were negative for the JAK2 V617F mutation. In their large study of 806 patients, Campbell et al have found that the JAK2 V617F mutation has a relatively small effect on
vascular complications that are observed in patients before their ET diagnoses.9 This group also observed that ET patients with the JAK2 V617F mutation do not have an increased risk of arterial or venous thrombosis after entry into the trial. Similarly, in a retrospective study of 867 ET patients, the GIMEMA group found that the JAK2 V617F mutation at diagnosis was not a predictor for occurrence of thrombosis during follow-up.30 We found that almost one-quarter (23.8%) of our ET patients with thrombosis were negative for the JAK2 V617F mutation. Additionally, we quantified the allele burden as < 31% (12.5%-31% in 6 patients, 2%-5% in 1 patient, and < 2% in 1 patient) in more than half (57%) of our JAK2 V617F-positive patients with thrombosis. These low values made us question the effect of the degree of JAK2 V617F allele burden on vascular events in ET patients. Although it is accepted that the occurrence of thrombosis in PV or ET is not directly linked to increased htc or plt counts, leukocytosis has recently been associated with increased risk of major vascular events and eventually could represent a major target of cytoreductive therapy.31 It is reported that a leukocyte count > 8.4 109/L in ET is also strictly associated with an increased risk of thrombosis as recently shown by Barbui et al.32,33 Several studies have confirmed that standard risk factors (age > 60 years and/or previous thrombosis) and leukocytosis (> 11.3 109/L in the GIMEMA study) were significantly associated with an increased risk of events in follow-up.9,34-36 Although we observed an increased frequency of thrombosis in patients with > 11 109/L WBC count consistent with the previous reports, we could not show the correlation of age > 60 years with increased frequency of thrombosis in PV and ET. Few data have been published on the influence of JAK2 V617F mutation on bleeding risk in MPNs. In PV patients, no correlation has been found between homozygous and heterozygous genotype and the occurrence of bleeding complications.15 In a study of 106 ET patients, minor bleeds from various mucocutaneous sites were observed in 7 patients, 6 of which were JAK2 V617F-negative. Analysis from this study indicated that JAK2 V617F does not have a protective role on hemorrhagic risk.31 In our study, gastrointestinal system (GIS) bleeding was observed in all PV patients with bleeding. GIS hemorrhage or epistaxis was observed in ET patients with bleeding. We have thus concluded, different from previous reports, that JAK2 V617F negativity is not correlated with bleeding complications in MPNs. We think that in most patients, taking antiaggregants or anticoagulants are more important in bleeding complications than is the JAK2 V617F mutation. In the
Table 5 Therapy and Follow-Up Data of 232 MPN Patients
Characteristic a
PV (81) ET (129)a PMF (22) Total: 232
HU 74 91 19 184
(91%) (71%) (86%) (79%)
ASA/Warfarin/ Clopidogrel 69 111 13 193
(85%); (86%); (59%); (83%);
5/1 5/4 0/0 10/5
Anagrelide 3 23 4 30
(3.7%) (18%) (18.2%) (13%)
Interferon 4 6 5 15
(4.9%) (4.6%) (22.7%) (6.5%)
Acute Leukemia 2 (2.5%) 3 (1.6%) e e
Died 3 3 5 11
(3.7%) (2.3%) (2.7%) (4.7%)
Median Time From Diagnosis, Months (Range) 38 24.5 36 28
(1-219) (1-152) (1-112) (1-219)
Abbreviations: ASA ¼ acetyl salicylic acid; ET ¼ essential thrombocythemia; HU ¼ hydroxyurea; MF ¼ myelofibrosis; MPN ¼ myeloproliferative neoplasm; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera. a MF secondary to PV, 1 patient (1.2%), and MF secondary to ET, 3 patients (2.3%).
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JAK2 V617F Mutation Status in CMPN Table 6 Data of 11 CMPN Patients Who Died During the Follow-Up Period Patient Number Diagnosis
Age, Years
Sex
JAK2 V617F Genotype
Therapy
1 2 3
PV PV PV
66 59 70
M F F
Homozygous Homozygous Heterozygous
HU, IF, ASA HU, ASA, Phlebotomy Phlebotomy
4 5 6 7
ET ET ET PMF
71 57 73 67
F M M M
Heterozygous Heterozygous Homozygous Heterozygous
8 9 10
PMF PMF PMF
61 62 78
M M M
Homozygous Homozygous Heterozygous
HU, ASA, anagrelide HU, ASA HU, ASA HU, splenoctomy, lenalidomide HU, splenectomy HU, splenectomy HU
11
PMF
60
F
Heterozygous
HU
Events Before Death AML MDS (RAEB-2) Portal hypertension, renal insuffiency, pneumonia AML AML AML Sepsis Heart failure Heart failure Coronary artery disease, renal failure, pneumonia Renal failure, diabetes mellitus, essential hypertension
Follow-Up, Months 53 132 1 93 54 35 56 72 62 1 61
Abbreviations: AML ¼ acute myeloid leukemia; ASA ¼ acetyl salicylic acid; CMPN ¼ chronic myeloproliferative neoplasm; ET ¼ essential thrombocythemia; F ¼ female; HU ¼ hydroxyurea; IF ¼ interferon; M ¼ male; MDS ¼ myelodysplastic syndrome; PMF ¼ primary myelofibrosis; PV ¼ polycythemia vera; RAEB ¼ refractory anemia with excess blasts.
PV and ET group, frequencies of clinical bleeding in patients homozygous and heterozygous for the JAK2 mutation were similar, consistent with results of previous studies. Hematologic transformations to MF, MDS, or AML are recognized long-term complications of PV and ET.37 One (1.2%) of our 81 PV patients progressed into MF secondary to PV, and 3 (2.3%) of our 129 ET patients progressed into MF secondary to ET. Our results were consistent with a study of 605 ET patients in which 17 (2.8%) had progressed to MF. In the same study, acute leukemia was seen in 14 (2.3%) patients, whereas in our study, slightly fewer
Figure 4 Kaplan-Meier Overall Survival Curves of PV, ET, and PMF Patients. PMF Patients Were Found to Have Shorter Survival Times Than PV and ET Patients
patients, 3 (1.6%) in the ET group and 1 (1.2%) in the PV group, progressed into acute leukemia. Leukemic clones emerging from V617F-positive chronic MPN can be either V617F-positive or V617F-negative.38 V617F-positive AML more frequently occurs after transition to the MF phase, whereas V617F-negative AML seems to arise directly from chronicphase JAK2-positive MPN.39 In our ET patient group, 1 progressed into AML after MF development. The remaining 2 patients directly developed AML. Three ET and 1 PV patient who had acute leukemia also had the JAK2 V617F mutation. The question of a possible effect of the JAK2 V617F mutation on OS of patients with MPN has not been clearly answered by previously published studies, which have provided conflicting results.27 So far, we only have the median survival in our PMF patients (62 months; 95% CI, 57.9-66). In the PV and ET groups, along with all MPN patients, we have not yet reached median survival statistically because of less patient loss in these goups. A subset of patients with PV, ET, and PMF harbor mutations in night lighteinducible and clock-regulated genes and the Casitas B-cell lymphoma gene, TET2, ASXL1, and IDH,40 but pathogenetic contributions of these and other mutations are unclear at this time.41 Nowadays, calreticulin mutations have been described. These mutations have been observed in approximately 70% of patients with ET or PMF who do not carry mutations in either the JAK2 or MPL gene, and have not been observed in patients with PV. Because all of these mutations are still being researched, we could not study these in our patients.
Conclusion
8
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Abbreviations: Cum ¼ cumulative; ET ¼ essential thrombocythemia; ex ¼ exitus; PMF ¼ primary myelofibrosis; pts ¼ patients; PV ¼ polycythemia vera.
Clinical Lymphoma, Myeloma & Leukemia Month 2014
Thrombosis was found in 19% of all of our MPN patients. TIA was the most common thrombotic event, seen in 52% of the patients with thrombosis. When our center began assessing patients for the JAK2 V617F mutation, we quantified the JAK2 V617F mutation in patients diagnosed at that time and previously. Among those 2 groups with a similar number of patients, there were no
Kadriye Bahriye Payzin et al significant differences in JAK2 V617F positivity or quantification values. However, our results were based on different patients assessed at different times for the JAK2 V617F mutation. Our results are limited by this, and it is apparent that JAK2 V617F assessments need to be done at certain time points after diagnosis to determine the effects of follow-up times and cytoreductive treatments on JAK2 V617F positivity and allele burden quantification. With longer follow-up and larger patient populations, this study aimed to investigate our patients’ progression into myelofibrosis or acute leukemia, the effect of clinical and laboratory parameters on patient survival, and finally to compare our results with current literature findings.
Clinical Practice Points In this study, we aimed to investigate the presence of JAK2
V617F in CMPNs patients in our center. JAK2 V617F mutation frequencies of our PV and ET patients were similar to those reported previously. JAK2 V617F mutation frequency of our PMF patients was higher than in previous reports. We attribute this difference to our low number of PMF patients, and think that it will match other reports if we have a larger data set. Transient ischemic attack was the most common thrombotic event in our PV and ET patients. In ET treatment age over 60 is considered a standard risk, but in younger patients we also observed other conditions such as essential hypertension, diabetes mellitus, hereditary thrombophilia that could lead to vascular events. We therefore suggest more frequent monitoring of these patients for antiaggregant and cytoreductive therapies, and for the management of their other diseases.
Disclosure The authors have stated that they have no conflicts of interest.
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Clinical Lymphoma, Myeloma & Leukemia Month 2014
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