JAK2 V617F Mutation Prevalence in Myeloproliferative Neoplasms in Pernambuco,Brazil

Abstract

Background: The JAK2 V617F mutation is associated with three myeloproliferative neoplasms (MPNs): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). It generates an unregulated clonal hematopoietic progenitor and leads to abnormal increased proliferation of one or more myeloid lineages. Subjects bearing this mutation may present more frequently with complications such as thrombosis and bleeding, and no specific treatment has yet been developed for BCR-ABL-negative JAK2 V617F-negative MPNs. Aims: To determine the prevalence of JAK2 V617F in MPNs in Pernambuco, Brazil, and to compare it with previous studies. Material and Methods: 144 blood samples were collected at the Hospital of Hematology of the HEMOPE Foundation and were genotyped by polymerase chain reaction-restriction fragment length polymorphism with BsaXI enzymatic digestion. Results and Discussion: 88% (46/52) of the patients with PV, 47% (39/81) with ET, and 77% (8/11) with PMF were positive for JAK2 V617F, while more than 35% of the individuals were JAK2 V617F-negative, confirming a high prevalence of this abnormality in MPNs, more frequently with a low mutated allele burden, similar to what has been reported in other Western countries, despite differences among methods used to detect this mutation. Screening for JAK2 V617F may allow specific management of these diseases with JAK2 inhibitors in the future and highlights the need for further studies on the pathogenesis of BCR-ABL-negative JAK2 V617F-negative MPNs.

Introduction

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic diseases in which one or more myeloid lineages present with abnormally increased levels of cell proliferation. They are characterized by a multipotent progenitor cell that proliferates independently from physiological levels of growth factors such as erythropoietin (EPO) and thrombopoetin, leading to accelerated hematopoiesis and bone marrow hypercellularity (Baxter et al., 2005). Differently from chronic myeloid leukemia, BCR-ABL-negative MPNs have variable association with bleeding, thrombotic events, marrow fibrosis, and less frequently blastic transformation (Lussana et al., 2009).

JAK2 is a member of the janus kinases (JAKs) family, a group of cytoplasmic tyrosine kinases that transduce cytokine-mediated signals via the JAK-signal transducers and activators of transcription (STAT) pathway. Four JAKs have been described: JAK1, JAK2, JAK3, and TYK2 (Ward et al., 2000). When activation of a receptor by a cytokine occurs, JAK2 becomes phosphorylated and causes further phosphorylation and dimerization of the STAT5 protein. STAT5 dimers enter the nucleus and act as transcription factors for gene regulation. The JAK2 gene is localized in chromosomal region 9p24 and was first cloned in 1989 in a cDNA library of a murine cell lineage (Wilks, 1989).

The JAK2 V617F point mutation generates the substitution of a guanidine for a thymidine in the encoding sequence (1848G>T), resulting in the exchange of a valine residue for a phenylalanine at position 617 of the mutated JAK2 protein. This appears to relieve the inhibitory effect of the pseudokinase domain (JH2) on the kinase activity domain (JH1), leading to an unregulated proliferation in myeloid/erythroid cells (Baxter et al., 2005; James et al., 2005; Vainchenker et al., 2011). JAK2 constitutive activation causes hypersensitivity to several cytokines known to stimulate hematopoietic precursors, such as EPO, thrombopoietin, interleukin 3, granulocyte-macrophage colony-stimulating factor, and stem cell factor (Levine and Wernig, 2006; Monte-Mór et al., 2007). Other changes in mutated cells involve STAT5-mediated signaling, extracellular signal regulated kinases, and phosphoinositol kinase/Akt pathways (Parganas et al., 1998; Campbell and Green, 2006; Lippert et al., 2006).

This mutation is now recognized as an important oncogenic event responsible for the development of BCR-ABL-negative MPNs, such as polycythemia vera (PV) rubra, essential thrombocythemia (ET), and primary myelofibrosis (PMF). The discovery of the JAK2 V617F mutation was most important to the diagnosis of PV, as it is present in more than 90% of individuals with this MPN. This allowed better characterization of PV in contrast to other MPNs and validated the clinical diagnostic criteria for this disease. (Tefferi et al., 2007).

JAK2 V617F has been found in more than 90% of PV cases, but also in up to 60% of ET and PMF patients (Monte-Mór et al., 2007; Haferlach et al., 2008; Scott et al., 2009). Since the mutation is not universally present, other genetic events are believed to be involved in the etiopathogenesis of both ET and PMF, as well as JAK2 V617F-negative PV.

Several research groups have reported the somatic mutation V617F in the gene encoding JAK2 in patients with BCR-ABL-negative MPNs, and molecular studies in different regions of the world, such as the United States, Europe, Middle East, India, Korea, Japan, Argentina, and Southeastern Brazil, have investigated its prevalence (Baxter et al., 2005; James et al., 2005; Kravolics et al., 2005; Levine and Wernig, 2006; Monte-Mór et al., 2007; Bang et al., 2009; Basquiera et al., 2009; Sazawal et al., 2010; Ayad and Nafea, 2011; Mahfouz et al., 2011).

The aims of this study were to determine the prevalence of JAK2 V617F in patients with BCR-ABL-negative MPNs at the Hospital of Hematology at the HEMOPE Foundation, and to compare our results with previous studies.

Materials and Methods

This study was performed after approval by the local ethics committee of the HEMOPE Foundation. All samples were collected on informed consent.

Patients were recruited at the Hospital of Hematology in Recife, Pernambuco, Brazil, based on the clinical diagnosis of the main three BCR-ABL-negative MPNs (PV, ET and PMF). Between May 2009 and August 2010, 144 peripheral blood samples were collected in vacuum collecting tubes containing ethylenediaminetetraacetic acid as anticoagulant.

Peripheral blood granulocytes were separated by gradient centrifugation with Ficoll-Paque (Sigma) to enhance test sensitivity (Hermouet et al., 2007). DNA was extracted using standard phenol-chlorophorm protocol (Davis et al., 1986). We performed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with DNA amplification by specific primers (forward primer: 5′ - GGG TTT CCT CAG AAC GAA CGT TGA - 3′; reverse primer: 5′ - TCA TTG CTT TCC TTT TTC ACA A - 3′), yielding a 460 base pair (bp)-long fragment. This reaction was followed by digestion with BsaXI enzyme (New England BioLabs Inc.), and samples were analyzed by electrophoresis in 3% agarose gel. Genotypes were confirmed in 24 samples with suboptimal PCR amplification by direct DNA sequencing on a MegaBACE 1000 DNA Analysis System (GE Healthcare).

In normal subjects, the restriction enzyme BsaXI acts on specific binding sites by cutting the PCR product into three fragments: 241, 189, and 30 base pairs long. Fragments bearing the V617F mutation have their restriction site abolished. Thus, samples with mixed cellularity due to a relatively lower mutation burden present with four fragments (460, 241, 189, and 30 bp), while samples with a high mutated allele burden present with only 460 bp-long, digestion-resistant fragments.

Results and Discussion

A total of 144 samples were collected: 52 PV (18 male, mean age 69±11 years), 81 ET (23 male, mean age 63±18 years), and 11 PMF (5 male, mean age 63±10 years). One study had suggested that sex could be a determining factor, as ET was more common in women, while men were predominant in PV (Campbell and Green, 2006). In the present study, women were more frequently found in both PV (65.4%) and ET (71.6%) groups.

Among the PV samples, JAK2 V617F was detected in 46 (88%). Thirty-nine of 81 (47%) ET patients bore the mutation, while 8 (77%) of 11 PMF patients were JAK2 V617-positive. Samples screened positive for JAK2 V617F were further classified according to the pattern of fragments obtained in the PCR-RFLP reactions as “high burden” or “low burden.” These data are summarized in Table 1.

Table 1.

Mutated Allele Burden for JAK2 V617F in Patients with BCR/ABL-Negative Myeloproliferative Neoplasms Followed at the HEMOPE Foundation

Diagnosis	JAK2 V617F high burden	JAK2 V617F low burden	JAK2 V617F negative	Total
PV	17 (32%)	29 (56%)	6 (12%)	52
ET	10 (12%)	29 (35%)	42 (53%)	81
PMF	7 (63%)	1 (14%)	3 (23%)	11

Data are represented as absolute number (percentage of patients in each group).

PV, polycythemia vera; ET, essential thrombocythemia; PMF, primary myelofibrosis.

Our data show that, when present in PV and ET patients, the mutated JAK2 V617F allele burden was more frequently low (63% and 74%, respectively). Higher mutation burden rates were found in both diseases compared with previous reports (25%-30% in PV and 2% in ET) (Baxter et al., 2005; Kralovics et al., 2005; Levine et al., 2005), although still lower than rates found in an Indian population (Sazawal et al., 2010). In the latter study, the mutation was also determined by PCR-RFLP, so a possible explanation for a higher mutation burden in both India and Brazil could be that patients present later in the course of the diseases in these developing countries, as suggested by Sazawal and collaborators.

Table 2 compares the findings of this study with previously published work and highlights that different methods are available to detect JAK2 V617F, for example, allele-specific PCR (AS-PCR), real-time PCR (RT-PCR), multiple direct DNA sequencing methods, including mass spectrometry-based platforms and, as has been the main approach of the current study, PCR-RFLP. Sensitivity among the methods varies and has been well reviewed elsewhere (Steensma, 2006; Cankovic et al., 2009). Briefly, PCR-RFLP has been demonstrated to be able to detect down to 20% of mutated DNA on a wild-type background (Baxter et al., 2005; James et al., 2005), and automated sequencing using autoradiography as commonly used in the past was even less sensitive. Current DNA sequencing methods may prove sensitive down to 5%-10% of mutated DNA depending on the sample type (cell lines or human) and purity, but RT-PCR techniques have increased sensitivity to 2%-4%, and AS-PCR-based studies have reported down to 0.01% of detectable JAK2 V617F-positive DNA (McClure et al., 2006). Nevertheless, variation in mutation prevalence in the compared studies considered appears to rely more on sample number disparity than on differences among methods used, further confirming the utility of PCR-RFLP as a simple and cost-effective method to screen this mutation.

Table 2.

Comparison of the Prevalence and Methods Used for Detection of JAK2 V617F in BCR/ABL-Negative Myeloproliferative Neoplasms Among Different Studies

Study (number of patients with PV, ET or PMF)	Methods	Polycythemia vera	Essential thrombocythemia	Primary myelofibrosis
Silva et al. (present study)		88% (46)	47% (39)	77% (8)
(n=144)	PCR-RFLP	n=52	n=81	n=11
Mahfouz et al. (2011)		100% (13)	68% (28)	NR
(n=54)	RT-PCR	n=13	n=41	NR
Ayad and Nafea (2011)		81% (44)	57% (17)	46% (18)
(n=123)	AS-PCR	n=54	n=30	n=39
Tognon et al. (2011)		83% (10)	NR	NR
(n=49)	RT-PCR	n=12	n=26	n=11
Sazawal et al. (2010)		82% (28)	70% (7)	52% (16)
(n=75)	PCR-RFLP	n=34	n=10	n=31
Zhang et al. (2010)		82% (73)	36% (52)	51% (24)
(n=278)	AS-PCR, DNA seq, MS	n=89	n=142	n=47
Basquiera et al. (2009)		89% (40)	69% (30)	47% (7)
(n=103)	AS-PCR	n=45	n=43	n=15
Monte-Mór et al. (2007)		96% (47)	28% (8)	56% (14)
(n=103)	PCR-RFLP	n=49	n=29	n=25
Levine et al. (2005)		74% (121)	33% (37)	35% (16)
(n=325)	DNA seq, MS	n=164	n=115	n=46
Baxter et al. (2005)		97% (71)	57% (29)	50% (8)
(n=140)	AS-PCR, DNA seq	n=73	n=51	n=16
Kravolics et al. (2005)		65% (83)	23% (21)	57 (13)
(n=244)	DNA seq	n=128	n=93	n=23
James et al. (2005)		89% (40)	43% (9)	43% (3)
(n=73)	DNA seq	n=45	n=21	n=7

Data are represented as percentage of patients in each group (absolute number).

NR, not reported; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; RT-PCR, real-time polymerase chain reaction; AS-PCR, allele-specific polymerase chain reaction; DNA seq, DNA sequencing; MS, mass spectrometry-based method.

Despite a multicentric study having demonstrated that the presence of JAK2 V617F in PMF does not determine any specific clinical manifestation, such patients present with poorer survival (Campbell et al., 2006). We found that 77% of the PMF patients were JAK2 V617-positive. Even though our small number of patients (n=11) does not allow to draw any definitive conclusion about the mutation prevalence in this disease in our population, this finding still calls attention to the need for target-specific therapies for this kind of MPN with a worse outcome.

The prevalence of the JAK2 V617F mutation in MPNs in this study was similar to what has been described by other groups around the globe, and confirms that its high prevalence among PV patients allows a more confident diagnosis of this disease. Nonetheless, the diagnosis of all BCR-ABL-negative MPNs, particularly JAK2 V617F-negative PV, should rely on the criteria published in the 2008 WHO classification (Tefferi et al., 2007). A negative screening for JAK2 V617F, found in over a third (51/144) of the patients in this study, should not be considered evidence enough to discard the diagnosis of a MPN. Still, screening for this mutation may help physicians narrow down their diagnostic possibilities when a BCR-ABL-negative MPN is suspected, as positivity for JAK2 V617F is virtually 100% specific for the distinction between PV and other causes of increased hematocrit (James et al., 2006).

Previous studies in Brazil have been performed only in the Southeastern part of the country, in the State of São Paulo (Monte-Mór et al., 2007; Tognon et al., 2011). This is the first study in the Brazilian Northeastern population, and while our data did not differ from other Brazilian or worldwide populations, this suggests that the development of JAK2 V617F-positive MPNs may occur rather independently from an ethnical background. The availability of JAK2 V617F screening may help define better therapeutic strategies for patients with MPNs in our region in the future, especially considering current promising developments in clinical trials with JAK2 inhibitors (Verstovsek et al., 2010; Pardanani et al., 2011). Moreover, this study highlights the need for collaboration in further molecular studies on the pathogenesis of BCR-ABL-negative JAK2 V617F-negative MPNs.

Footnotes

Acknowledgments

This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE).

Author Disclosure Statement

No competing financial interests exist.

References

Ayad

, Nafea

. 2011. Acquired mutation of the tyrosine kinase JAK2V617F in Egyptian patients with myeloid disorders. Genet Test Mol Biomarkers, 15:17-21.

Bang

, Lee

, Ahn

et al. 2009. Vascular events in Korean patients with myeloproliferative neoplasms and their relationship to JAK2 mutation. Thromb Haemost, 101:547-551.

Basquiera

, Soria

, Ryser

et al. 2009. Clinical significance of V617F mutation of the JAK2 gene in patients with chronic myeloproliferative disorders. Hematology, 14:323-330.

Baxter

, Scott

, Campbell

et al. 2005. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet, 365:1054-1061.

Campbell

, Green

. 2006. The myeloproliferative disorders. N Engl J Med, 355:2452-2466.

Campbell

, Griesshammer

, Döhner

et al. 2006. V617F mutation in JAK2 is associated with poorer survival in idiopathic myelofibrosis. Blood, 107:2098-2100.

Cankovic

, Whiteley

, Hawley

et al. 2009. Clinical performance of JAK2 V617F mutation detection assays in a molecular diagnostics laboratory: evaluation of screening and quantitation methods. Am J Clin Pathol, 132:713-721.

Davis

, Dibner

, Battey

. 1986. Basic Methods in Molecular Biology. Elsevier: New York.

Haferlach

, Bacher

, Kern

et al. 2008. The diagnosis of BCR-ABL-negative chronic myeloproliferative diseases (CMPD): a comprehensive approach based on morphology, cytogenetics, and molecular markers. Ann Hematol, 87:1-10.

10.

Hermouet

, Dobo

, Lippert

et al. 2007. Comparison of whole blood vs purified blood granulocytes for the detection and quantitation of JAK2(V617F) Leukemia, 21:1128-1130.

11.

James

, Delhommeau

, Marzac

et al. 2006. Detection of JAK2 V617F as a first intention diagnostic test for erythrocytosis. Leukemia, 20:350-353.

12.

James

, Ugo

, Le Couedic

et al. 2005. A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera. Nature, 434:1144-1148.

13.

Kravolics

, Passamonti

, Buser

et al. 2005. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med, 352:1779-1790.

14.

Levine

, Wadleigh

, Cools

et al. 2005. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell, 7:387-397.

15.

Levine

, Wernig

. 2006. Role of JAK-STAT signaling in the pathogenesis of myeloproliferative disorders. Hematol Am Soc Hematol Educ Program, 233-239510.

16.

Lippert

, Boissinot

, Kralovics

et al. 2006. The JAK2-V617F mutation is frequently present at diagnosis in patients with essential thrombocythemia and polycythemia vera. Blood, 108:1865-1867.

17.

Lussana

, Caberlon

, Pagani

et al. 2009. Association of V617F Jak2 mutation with the risk of thrombosis among patients with essential thrombocythaemia or idiopathic myelofibrosis: a systematic review. Thromb Res, 124:409-417.

18.

Mahfouz

, Hoteit

, Salem

et al. 2011. JAK2 V617F gene mutation in the laboratory work-up of myeloproliferative disorders: experience of a major referral center in Lebanon. Genet Test Mol Biomarkers, 15:263-265.

19.

McClure

, Mai

, Lasho

. 2006. Validation of two clinically useful assays for evaluation of JAK2 V617F mutation in chronic myeloproliferative disorders. Leukemia, 20:168-171.

20.

Monte-Mór

BCR

, Cunha

, Pagnano

KBB

et al. 2007. JAK2 V617F prevalence in Brazilian patients with polycythemia idiopathic myelofibrosis and essential thrombocythemia. Genet Mol Biol, 30:336-338.

21.

Pardanani

, Gotlib

, Jamieson

et al. 2011. Safety and efficacy of TG101348, a selective JAK2 inhibitor, in myelofibrosis. J Clin Oncol, 29:789-796.

22.

Parganas

, Wang

, Stravopodis

et al. 1998. Jak2 is essential for signaling through a variety of cytokine receptors. Cell, 93:385-395.

23.

Sazawal

, Bajaj

, Chikkara

et al. 2010. Prevalence of JAK2 V617F mutation in Indian patients with chronic myeloproliferative disorders. Indian J Med Res, 132:423-427.

24.

Scott

, Campbell

, Baxter

et al. 2009. The V617F JAK2 mutation is uncommom in cancers and in myeloid malignancies other than classic myeloproliferative disorders. Blood, 106:2920-2921.

25.

Steensma

. 2006. JAK2 V617F in myeloid disorders: molecular diagnostic techniques and their clinical utility: a paper from the 2005 William Beaumont Hospital Symposium on Molecular Pathology. J Mol Diagn, 8:397-411quiz 526.

26.

Tefferi

, Thiele

, Orazi

et al. 2007. Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel. Blood, 110:1092-1097.

27.

Tognon

, Gasparotto

, Leroy

et al. 2011. Differential expression of apoptosis-related genes from death receptor pathway in chronic myeloproliferative diseases. J Clin Pathol, 64:75-82.

28.

Vainchenker

, Delhommeau

, Constantinescu

, Bernard

. 2011. New mutations and pathogenesis of myeloproliferative neoplasms. Blood, 118:1723-1735.

29.

Verstovsek

, Kantarjian

, Mesa

et al. 2010. Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Engl J Med, 363:1117-1127.

30.

Ward

, Touw

, Yoshimura

. 2000. The Jak-Stat pathway in normal and perturbed hematopoiesis. Blood, 95:19-29.

31.

Wilks

. 1989. Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction. Proc Natl Acad Sci U S A, 86:1603-1607.

32.

Zhang

, Qiu

, Li

et al. 2010. The analysis of JAK2 and MPL mutations and JAK2 single nucleotide polymorphisms in MPN patients by MassARRAY assay. Int J Lab Hematol, 32:381-386.