ORIGINAL ARTICLE / ARTICLE ORIGINAL

Prevalence of JAK2 V617F Mutation in West Algerian Populationwith Chronic Myeloproliferative Neoplasms: A Multicenter Study

Prévalence de la mutation JAK2V617F dans la population atteinte de néoplasmesmyéloprolifératifs chroniques dans l’Ouest Algérien : une étude multicentrique

M. Benguella-Benmansour · K. Boucherit · N. Benchikh · N. Mesli · N. Chabni · A. Messaoudi · A. Bekadja ·Z. Boucherit · M.J. Mozziconacci

Received: 6 September 2013; Accepted: 4 October 2013© Springer-Verlag France 2014

Abstract JAK2 V617F point mutation is now well recog-nized in patients with chronic myeloproliferative neoplasms(MPNs). However, its frequency in Algerian patients is stillunknown. We determined the JAK2 V617F mutation inblood samples from 344 patients from five hematologydepartments across West Algeria, using amplification refrac-tory mutation system-polymerase chain reaction (ARMS-PCR). The JAK2 V617F mutation was identified in157/344 patients (45.63%), 80/98 polycythemia verapatients (81.6%), 44/75 essential thrombocythemia patients(58.6%), 6/13 primary myelofibrosis patients (46.2%), 21/87with suspected MPN (12.9%), and 2/32 secondary thrombo-cytosis patients (6.2%), but was not detected in 28 patientswith secondary erythrocytosis. Detection of JAK2 V617Fmutation is an essential molecular tool to establish myelo-

proliferative neoplasm diagnosis, which allowed us to adjustthe diagnosis for 6 patients and should be systematicallyincluded into the initial evaluation of patients suspected ofMPNs in Algeria.

Keywords JAK2 V617F · Mutation · Myeloproliferativeneoplasms · ARMS-PCR

Résumé La mutation ponctuelle JAK2V617F est maintenantbien connue chez les patients présentant des néoplasmesmyéloprolifératifs chroniques (NMPc). Cependant, sa fré-quence chez les patients algériens est encore inconnue. Lamutation JAK2V617F a été recherchée sur des échantillonssanguins prélevés de 344 patients admis aux services d’hé-matologie clinique de différents centres hospitaliers de larégion Ouest d’Algérie. Cette mutation a été détectée parune réaction de polymérisation en chaîne PCR-ARMS(Amplification Refractory Mutation System). La mutationJAK2V617F a été identifiée chez 157/344 patients(45,63 %), dont 80/98 patients atteints de polyglobulie deVaquez (81,6 %), 44/75 patients atteints de thrombocytémieessentielle (58,6 %), 6/13 patients atteints de myélofibroseprimitive (46,2 %), 21/87 patients avec suspicion d’un néo-plasme myéloprolifératif chronique (12,9 %) et 2/32 patientsatteints de thrombocytose secondaire (6,2 %), mais n’a pasété détectée chez 28 patients avec polyglobulie secondaire.La détection de la mutation JAK2 V617F est un outil molé-culaire essentiel pour établir un diagnostic de néoplasmesmyéloprolifératifs, ce qui nous a permis d’ajuster le diagnos-tic de six patients et devrait être incluse systématiquementdans l’évaluation initiale des patients suspectés d’avoir unnéoplasme myéloprolifératif en Algérie.

Mots clés JAK2V617F · Mutation · Néoplasmesmyéloprolifératifs chroniques · PCR-ARMS

M. Benguella-Benmansour (*) · K. Boucherit · Z. BoucheritLaboratoire antibiotiques, antifongiques : physicochimie,synthèse et activité biologique, faculté des sciences,université Abou-Bekr-Belkaïd, Tlemcen, Algériee-mail : b_meriem74@yahoo.fr

N. Benchikh · A. MessaoudiService de biochimie, EHU d’Oran, Algérie

N. MesliService d’oncohématologie,CHU de Tlemcen, Algérie

N. ChabniService d’épidemiologie,CHU de Tlemcen, Algérie

A. BekadjaService d’Hématologie et de thérapie cellulaire,EHU d’Oran, Algérie

M.J. MozziconacciDépartement de Biopathologie, institut Paoli-Calmettes,Marseille, France

J. Afr. CancerDOI 10.1007/s12558-014-0304-7

Introduction

Philadelphia-negative chronic myeloproliferative neoplasms(Ph-negative MPNs) as described by World Health Organiza-tion (WHO) 2008 are clonal hematopoietic malignancieswhich arise from transformation of pluripotent stem cells,and lead to an increased production of erythrocytes, leuko-cytes, and/or platelets [1]. The pathogenesis of Ph-negativeMPNs has been partially explained by an acquired pointmutation in exon 14 of JAK2 gene that leads to the substitu-tion of a valine by a phenylalanine in position 617 (V617F) inthe pseudokinase domain of the protein [2,3]. JAK2 is a tyro-sine kinase protein which plays an important role in mediatingcellular signals by membrane receptors of hematopoieticgrowth factors and cytokines. Structural changes in pseudoki-nase JH2 domain that regulates JH1 kinase domain leads to again of function as reflected by the hypersensitivity to and theindependence from cytokines of mutated cells, which pro-motes uncontrolled cell proliferation [4,5].

According to the revised WHO 2008 diagnostic criteria[1], this mutation is helpful to confirm the diagnosis of poly-cythemia vera (PV) as it is found in a great majority ofpatients (95–97%), whereas in essential thrombocytemia(ET) or in primary myelofibrosis (PMF), the JAK2 mutationarises in about 50–60% of cases and serves more as a clonalmarker [6,7].

The frequency of this mutation in Algerian patients hasnever been reported. The aim of our multicenter study was toinvestigate the JAK2 exon 14 mutation in cases classified asmyeloproliferative neoplasms in a cohort of 344 patients.The correlation between JAK2 V617F mutation and theclinicopathologic characteristics of these patients have beenanalyzed.

Patients, design, and methods

Patients and samples

We analyzed 344 samples from patients recruited in hematol-ogy departments of five West Algeria hospitals (CentreHospitalo-Universitaire CHU Oran, EtablissementHospitalo-Universitaire EHU Oran, CHU Tlemcen, CHUSidi Belabbes, and Etablissement Publique Hospitalier EPHMascara) between March 2009 and September 2012. Allpatients were referred for hematological results suggestiveof MPNs. The diagnosis was made on the basis of standardclinicohematological criteria (reported in Table 1). Spleno-megaly was defined as a palpable spleen below the costalmargin with an increased organ size at radiologic scanning.Thrombotic manifestations were defined as confirmed stroke,coronary artery diseases, deep vein thrombosis, and any otherdocumented arterial or venous thrombosis at any time

courses. According to the WHO criteria [8], high levels ofhemoglobin (>18.5 g/dL in men, 16.5 g/dL in women) andplatelets (≥600 × 109/L), splenomegaly, and/or bone marrow(BM) examination allowed us to split up our patients into sixgroups. The first group, 126 patients with polycythemia, wasdivided into two subgroups, primitive (n = 98) and secondary(n = 28) cases, according to clinical and biological analysesand BM cellularity. The second group, 107 patients withthrombocytosis, was similarly subdivided into essential (n =75) and secondary (n = 32) cases. Secondary thrombocytosiswas due to an iron deficiency. It is an inflammatory, infec-tious, or tumoral process as described by the PolycythemiaVera Study Group (PVSG) [9].

The third group, 13 patients with PMF, was based essen-tially on BM fibrosis and splenomegaly.

The fourth group comprised 87 patients with suspectedMPN. This group included all patients presenting withmarked megakaryocytic, granulocytic, and/or erythroidhyperplasia, but who were not clearly classified in oneMPN subtype.

The fifth group was composed of 8 patients diagnosed aschronic myeloid leukemia (CML). Because BCR-ABL1transcript fusion is not routinely available in our centers,clinicians proposed to evaluate JAK2 V617F mutation fora more precise diagnosis in some cases.

The last group was composed of 3 patients with markedthrombocytosis. One patient, suffering from a Budd-Chiarisyndrome (BCS), was addressed by the gastrology depart-ment for high liver enzyme level and abnormal abdominalultrasound examination, and 2 patients with cytopenia anddysplasic BMwith ring sideroblasts suggested a diagnosis ofmyelodysplastic syndrome (MDS).

All patients were treated with standard protocols, includ-ing aspirin, hydroxyurea, or bleeding, according to eachpathology.

All patients signed an informed consent and the study wasapproved by local research ethic committees.

DNA extraction

Peripheral blood samples were collected from patients andprocessed immediately or conserved at −20°C. GenomicDNA was extracted using salt method as previouslydescribed [10]. After extraction, DNA was quantified usingspectrophotometeric measurements (NanoDrop Technolo-gies Inc) and stored in 25 ng/μl aliquots before PCR analysis.

Amplification refractory mutationsystem-polymerase chain reaction

The detection of the JAK2 V617F mutation was performedusing amplification refractory mutation system-polymerase

2 J. Afr. Cancer

Tab

le1

Clin

icalandhematologicalcharacteristicsof

patients.

Group1

Polycythem

ia(n

=12

6)

Group2

Thrombocytosis

(n=10

7)

Group3

(n=13

)

Group4

(n=87

)

Group5

(n=8)

Group6

(n=3)

PV

(n=98

)

Secon

dary

(n=28

)

ET

(n=75

)

Secon

dary

(n=32

)

PMF

Suspected

MPN

CML

MDS

BCS

Num

ber

9828

7532

1387

82

1

Medianage

57.81

(24–

89)

47.73

(19–

79)

52.88

(11–

89)

48.32

(22–

76)

55.25

(20–

78)

50.56

(8–80

)

56.17

(20–

80)

50 (30–

70)

43

Sex

ratio(M

/F)

59/39

19/09

31/44

15/17

10/03

44/43

03/05

2/0

1/0

MeanHb(g/dl)

19.2

(17.2–

21.5)

17.3

(16.7–

19)

16.8

(10.6–

18.3)

14.5

(6–15

.2)

10.1

(5.8–14

.1)

16.1

(13–

20.4)

8.6

(7.4–20

.6)

10.0

(7.0–13

.1)

12.5

MeanHt(%

)59

.48

(50.6–

67)

48.8

(48.2–

56.5)

49.75

(30.1–

53.4)

42.8

(18–

45.6)

31.62

(17.4–

43.1)

49.51

(39–

62.1)

34.0

(21–

61.3)

3.27

(21.6–

38.4)

20.7

MeanWBC

coun

t

(×10

9/L)

10.82

(3.9–21

.7)

8.89

(4.6–10

.1)

12.36

(6.6–15

.2)

9.77

(8.6–15

.8)

7.08

(2.1–12

.4)

11.71

(5.4–16

.0)

32.5

(2.2–35

0)

11.7

(5.9–17

.8)

4.7

Meanplatelet

coun

t

(×10

9/L)

463.09

(329

–58

5)

329.38

(121

–44

8)

911.85

(673

–14

36)

522.66

(480

–87

3)

332.61

(40–

502)

565.77

(452

–82

5)

359.1

(32–

490)

892

(701

–10

83)

695

Splenom

egaly(%

)42

.90

57.1

3010

020

.587

.50

–

Hepatom

egaly(%

)8.33

00

011.53

00

010

0

BM

fibrosis

(%)

32.82

ND

38.5

ND

100

16ND

0ND

Treatments

Hyd

roxy

urea

(%)

26–

61–

61.5

3737

.510

0–

Bleeding(%

)10

054

––

––

––

–

Aspirin

(%)

7345

83–

–12

––

–

Allcontinuous

values

areexpressedas

median(range).

Hb:

hemoglobin;

Ht:hematocrit;WBC:w

hitebloodcell;

BM:b

onemarrow;PV:p

olycythemiavera;E

T:essentialthrombocytemia;PMF:prim

arymyelofibrosis;MPN:m

yelopro-

liferativeneoplasm

;CML:chronicmyeloid

leukem

ia;ND:notdeterm

ined.

J. Afr. Cancer 3

chain reaction (ARMS-PCR) as previously described [11].Primers were purchased from Sigma-Aldrich. Briefly, ampli-fications were performed for 40 cycles with HotStarTaq poly-merase (Qiagen, Spain) at an annealing temperature of 58°C.PCR products were resolved on 2% agarose gel for 60minutesat 80V and visualized after staining with ethidium bromide.The positive human erythroleukemia (HEL) cell line genomicDNA and negative controls were kindly provided by Ipsogensociety (France). This assay has the ability to distinguish het-erozygote from homozygotes cases. A 229-bp product isexpected for wild type DNA [2,11].

Statistical analysis

Statistical analyses were performed by using the Excel pro-gram (Microsoft, Redmond, WA) and EPI InfoTM software[version 3.5.3, January 26, 2011 CDC, Atlanta, Georgia,USA]. Differences between groups of samples were assessedby using ANOVA t tests, assuming unequal variances, thechi-square test, and Fisher’s test. The values of p less than0.05 (2 tailed) were considered significantly different.

Results

Over all the population study, there were 185 men (53.8%)and 159 women (46.2%). The mean age at the time of diag-nosis was 52.8 (range 8–89 years). The median follow-upperiod was 42 months (Table 1).

Among the patients, 98 were diagnosed for PV, 75 for ET,13 for PMF, 28 for secondary polycythemia, 32 for second-ary thrombocytosis, 8 for CML, and 87 for suspected MPN.Two patients had a MDS and one patient a BCS.

The JAK2 V617F mutation was detected in 157 patients(45.63%), including PV (81.6%), ET (58.7%), and PMF(46.1%). We also found it in the following cases: 2 patientswith secondary thrombocytosis, 21 of 87 cases with sus-pected MPN, 1 CML, 2 MDS, and 1 BCS (Table 2).

Comparing the age of patients, those with JAK2 V617Fwere older than those with wild type alleles (p = 0.04).Mutated patients displayed a significantly higher hemoglo-bin, hematocrit, and white blood cell (WBC) counts (p =0.031; p = 0.029; p = 0.017 respectively). As well, the inci-dence of splenomegaly and hepatomegaly was higher amongour mutated patients (p = 0.007 and p = 0.001 respectively).

Conversely, no significant difference in platelet count wasfound between our mutated and non-mutated groups in spiteof slightly higher value inmutation-positive patients (Table 3).

The JAK2 V617F mutation can be heterozygous orhomozygous. In our study, homozygosity was significantlymore frequent in PV (about 20%) than in ET (about 9%)(Fig. 1).

Discussion

In this multicenter study, 344 patients were recruited forJAK2 V617F mutation screening to confirm or establish adiagnosis or discard any ambiguity.

In support of previous reports, we observed that the JAK2V617F mutation was present in 157 out of 344 (45.63%)patients.

The frequency of JAK2 V617F in our PV (81.6%) patientgroup was slightly low compared to previous data where themutation was found in 95–97% of PV patients in Westerncountries [7,12,13]. This might be explained by some inac-curate diagnosis in our centers. Perhaps some cases of sec-ondary polycythemia were included and considered asessential polycythemia. This ambiguous diagnosis betweenthe two groups could be explained by the lack of determina-tion of serum erythropoietin (EPO) levels and of formationof endogenous erythroid colonies (ECC) in routine tests forPV diagnosis in our centers despite the WHO recommenda-tions. No case of secondary polycythemia was positive forthe mutant allele for JAK2 in our series.

The frequency of JAK2 V617F mutation in our ET cases(58.7%) was comparable to those previously reported inWestern countries [2,3]. However, 2 patients out of 32 diag-nosed as secondary thrombocytosis were JAK2 V617Fpositive.

These results could be explained by an error of diagnosisas they presented with platelet levels (480 × 109/L and 510 ×109/L) exceeding the WHO criteria. Indeed, the plateletcount allowing the diagnosis of ET has been lowered in2008 from 600 × 109/L to 450 × 109/L [14,15].

The rate of mutant allele for JAK2 V617F in PMF cases isconsistent with literature results [16].

In cases with suspected MPN, determination of the muta-tion allowed to assert the diagnosis of MPN.

Table 2 Mutational JAK2 V617F status by pathology.

Pathology Number of mutated

patients/total number

of patients

(%)

Polycythemia vera 80/98 81.6

Essential thrombocytemia 44/75 58.7

Primary myelofibrosis 6/13 46.2

Secondary erythrocytosis 0/28 0

Secondary thrombocytosis 2/32 6.2

Suspected MPN 21/87 12.9

Chronic myeloid leukemia 1/8 12.8

Myelodysplasic syndrome 2/2 100

Budd-Chiari syndrome 1/1 100

4 J. Afr. Cancer

In our CML cases, 1 patient was positive for JAK2V617F mutation. Previous data showed that JAK2 V617Fmutation and BCR-ABL1 transcript are generally mutuallyexclusive [17], but rare cases of concomitant JAK2 V617Fmutation and BCR-ABL1 rearrangement have been reported

[18,19]. Actually, our patient aged 63 presented with highWBC (73 × 109/L), high myelemia (31%), high hemoglobin(20.6 g/dL), normal platelet (270 × 109/L) levels, and amarked BM hypercellularity mainly for granulocytic lineagevery evocative of CML. The diagnosis was strengthened by

Table 3 Comparison of clinical characteristics and laboratory data between JAK2 mutation positive and negative groups.

All patients V617F Positive V617F Negative p-value

Number 344 157 (45.63%) 187 (54.36%) –

Median age 52.83

(8–89)

57.34

(22–86)

48.33

(8–89)

0.043

Sex ratio (M/F) 185/159 85/77 100/82 0.398

Median Hb (g/dl) (range) 14.03

(5.8–21.5)

16.32

(10.7–21.5)

13.45

(5.8–19.2)

0.031

Median Ht (%) (range) 42.24

(17.4–64.8)

49.04

(32.2–64.5)

40.34

(17.4–57.5)

0.029

Mean WBC count (×109/L) (range) 10.67

(2.1–21.7)

11.78

(4.2–19.4)

9.32

(2.1–21.7)

0.017

Median platelet (×109/L) 537.1

(32–1436)

611.06

(74–1436)

513.34

(32–1266)

0.149

Splenomegaly 49% 62% 38% 0.007

Hepatomegaly 43.8% 59% 32.7% 0.001

BM fibrosis 33% 46.7% 18% 0.000

Hb: hemoglobin; Ht: hematocrit; WBC: white blood cell; BM: bone marrow.

Fig. 1 ARMS-PCR assay for detection of JAK2 V617F mutation; lane 1 is 100-base pair (bp) markers; lanes 2, 3, 4, 5, 6, 7, 8, and 9 are

samples patients

J. Afr. Cancer 5

the positivity of the JAK2 V617Fmutation which allowed usto reclassify the case from CML to MPN (probably PV).

In West Algeria, the detection of BCR-ABL1 transcript isnot done in routine diagnosis, because it requires a molecularequipment which is not available in our laboratories.

In agreement with other studies, we found the JAK2V617F mutation in PV, ET, and PMF [4,7]. It has also beenreported in other hematologic diseases like chronic myelomo-nocytic leukemia (CMML), acute myeloid leukemia (AML),and myelodysplastic syndromes (MDS) [20,21].

In our study, the mutation was detected in 2 cases withMDS. For these patients, the diagnosis was based mainly onBM analysis, and no complementary cytogenetic analysiswas performed. The positivity of mutant allele for JAK2V617F and the high thrombocytosis (689 × 109/L and 817× 109/L) led to hypothesize that these patients were RARS-T(refractory anemia with ring sideroblasts and thrombocyto-sis) cases, a frontier form betweenMPN andMDS accordingto WHO 2008 criteria [1]. In literature, the incidence ofJAK2 V617F is about 7% and 53% in MDS and RARS-Trespectively [22,23].

Finally, our BCS case was also mutated. Approximately59% of BCS patients have an underlying MPN, usuallymasked by the development of portal hypertension after theoccurrence of the thrombosis [24]. Our patient had a highplatelet count (695 × 109/L) which allowed us to considerthis case as an ET more than a BCS.

As reported in the literature, JAK2 V617F mutation wascorrelated with old age [25]. This may be related to the accu-mulation of molecular abnormalities and a progressivelyincreasing genomic instability [26]. These patients also dis-played a higher hemoglobin, hematocrit, and WBC count.However, as previously described, no significant differencein platelet count was found between our mutated and non-mutated groups like in previous studies [26].

The JAK2 V617F can be heterozygous or homozygous.In our study, homozygosity for V617F was more frequent inPV than in ET. These results are in accordance with literature[27,28].

In our study, 187 patients did not have the JAK2 V617Fmutation. In these patients (18 PV and 31 ET), an activatingJAK2 exon 12 or MPLW515L/K mutation might be found inPV and ET ones respectively [29,30]. These abnormalitiessign the clonality of the proliferation and eliminate furtherconsideration of a reactive process [1,8] but these investiga-tions are not yet performed in West Algeria.

In conclusion, rates of V617F mutation obtained in ourET and PMF cases strongly correlate with literature data butwe found lower rates in PV cases.

Through this multicenter study undergone in WestAlgeria, screening for JAK2 V617F mutation in patientswith MPNs appeared to be an essential tool for diagnosisas recommended by WHO criteria. Interestingly, it allowed

us to reclassify 6 cases: move 2 patients from secondary toessential thrombocytosis and change diagnosis for oneCML, two MDS, and one BCS cases to MPNs. Clearly, thediagnosis of MPNs can be difficult due the heterogeneity ofthese disorders. Using only hematological and clinical anal-yses can led to an inefficient strategy for classifying MPNs.

Moreover, although anti-JAK2 molecules are still in ther-apy assays, determination of this mutation in routine testswill be of great interest for adaptation of treatment and sur-vey of these pathogenesis in the future.

Acknowledgments The authors thank Ipsogen Society(Marseille, France) that kindly provided positive and nega-tive controls for JAK2 V617F mutation. We are very gratefulto Dr. Jérôme Rey for his helpful discussion and advice.

Conflict of interest: M. Benguella-Benmansour, K. Bou-cherit, N. Benchikh, N. Mesli, N. Chabni, A. Messaoudi,A. Bekadja, Z. Boucherit, and MJ. Mozziconacci have noconflict of interest to report.

References

1. Vardiman JW, Thiele J, Arber DA, et al (2009) The 2008 revisionof the World Health Organisation (WHO) classification of mye-loid neoplasm and acute leukemia: Rationale and importantchanges. Blood 114:937–51

2. Jones AV, Kreil S, Zoi K, et al (2005) Widespread occurrence ofthe JAK2 V617F mutation in chronic myeloproliferative disor-ders. Blood 106:2162–8

3. Baxter EJ, Scott LM, Campbell PJ, et al (2005) Acquired muta-tion of the tyrosine kinase JAK2 in human myeloproliferativedisorders. Lancet 365:1054–61

4. Steensma DP (2006) JAK2 V617F in myeloid disorders: molecu-lar diagnostic techniques and their clinical utility. A paper fromthe 2005 William Beaumont Hospital Symposium on MolecularPathology. J Mol Diagn 8:397–411

5. Hsu HC (2007) The pathogenic role of JAK2V617F mutation inthe chronic myeloproliferative disorders. J Chin Med Assoc70:89–93

6. Tefferi A, Vardiman JW (2008) Classification and diagnosis ofmyeloproliferative neoplasms: The 2008 World Health Organiza-tion criteria and point-of-care diagnostic algorithms. Leukemia22:14–22. doi:10.1038/sj.leu.2404955

7. Kiladjian JJ (2012) The spectrum of JAK2-positive myeloprolif-erative neoplasms. Hematology 1:561–6

8. Vardiman JW, Brunning RD (2002) The World Health Organisa-tion (WHO) classification of the myeloid neoplasms. Blood100:2292–302

9. Murphy S, Petreson P, Iland H, Laszlo J (1997) Experience of thePolycythemia Vera Study Group with essential thrombocythemia:a final report on diagnostic criteria, survival, and leukemic transi-tion by treatment. Semin Hematol 34:29–39

10. Miller SA, Dykes DD, Polesky HP (1988) A simple salting outprocedure for extracting DNA from human nucleated cells.Nucleic Acids Res 16:1213–5

11. Chen Q, Lu P, Jones AV, et al (2007) Amplification refractorymutation system, a highly sensitive and simple polymerase

6 J. Afr. Cancer

chain reaction assay, for the detection of JAK2 V617F mutationin chronic myeloproliferative disorders. J Mol Diagn 9:272–6

12. James C, Ugo V, Le Couedic JP, et al (2005) A unique clonalJAK2 mutation leading to constitutive signalling causes polycy-thaemia vera. Nature 434:1144–8

13. Delhommeau F, Jeziorowska D, Marzac C, et al (2010) Molecu-lar aspects of myeloproliferative neoplasms. Int J Hemato191:165–73

14. Palandri F, Ottaviani E, Salmi F, et al (2009) Jak2 V617F muta-tion in essential thrombocytemia: correlation with clinical charac-teristics, response to therapy and long term outcome in a cohortof 275 patients. Leuk Lymphoma 50:247–53

15. Sacchi S, Vinci G, Gugliotta L, et al (2000) Diagnosis of essen-tial thrombocytemia at platelet counts between 400 and 600 x109/L. Gruppo Italiano Malattie Mieloproliferative Croniche(GIMMC). Haematologica 85:492–5

16. Campbell PG, Griesshammer M, Dohner K, et al (2006) V617Fmutation in JAPK2 is associated with poorer survival in idio-pathic myelofibrosis. Blood 107:2098–100

17. Cambier N, Renneville A, Cazaentre T, et al (2008) JAK2V617F-positive polycythemia vera and Philadelphia chromosome-positive chronic myeloid leukemia: one patient with two distinctmyeloproliferative disorders. Leukemia 22:1454–5

18. Pieri L, Spolverini A, Scappini B, et al (2011) Concomitant occur-rence of BCR-ABL and JAK2V617F mutation. Blood 118:3445–6

19. Makishima H, Jankowska AM, McDevitt MA, et al (2011) CBL,CBLB, TET2, ASXL1, and IDH1/2 mutations and additionalchromosomal aberrations constitute molecular events in chronicmyelogenous leukemia. Blood 117:198–206

20. Levine RL, Wadleigh M, Cools J, et al (2005) Activating muta-tion in the tyrosine kinase JAK2 in polycythemia vera, essentialthrombocythemia, and myeloid metaplasia with myelofibrosis.Cancer Cell 7:7387–97

21. Nielsen C, Birgens HS, Nordestgaard BG, et al (2011) The JAK2V617F somatic mutation, mortality and cancer risk in the generalpopulation. Haematologica 96:450–3

22. Schmitt-Graef AH, Teo SS, Olschewski M, et al (2008)JAK2V617F mutation status identifies subtypes of refractory ane-mia with ringed sideroblasts associated with marked thrombocy-tosis. Haematologica 93:34–40

23. Malcovati L, Porta MG, Pietra D, et al (2009) Molecular andclinical features of refractory anemia with ringed sideroblastsassociated with marked thrombocytosis. Blood 114:3538–45

24. Smalberg JH, Arends LR, Valla DC, et al (2012) Myeloprolifera-tive neoplasms in Budd-Chiari syndrome and portal vein throm-bosis: a meta-analysis. Blood 120:4921–8

25. Vannucchi AM, Antonioli E, Guglielmelli P, et al (2008) Clinicalcorrelates of JAK2V617F presence or allele burden in myeloprolif-erative neoplasms: a critical reappraisal. Leukemia 22:1299–307

26. Hsiao HH, Yang MY, Liu YC, et al (2007) The associationof JAK2V617F mutation and leukocytosis with thromboticevents in essential thrombocythemia. Exp Hematol 35:1704–7.doi:10.1016/j.exphem.2007.08.011

27. Godfrey AL, Chen E, Pagano F, et al (2012) JAK2V617F-homozygosity arises commonly and recurrently in PV and ET,but PV is characterized by expansion of a dominant homozygoussubclone. Blood 120:2704–7

28. Scott LM, Scott MA, Campbell PJ, Green AR (2006) Progenitorshomozygous for theV617F mutation occur in most patients withpolycythemia vera, but not essential thrombocythemia. Blood108:2435–7

29. Pardanani A, Lasho TL, Finke C, et al (2007) Prevalence andclinicopathologic correlates of JAK2 exon 12 mutations inJAK2V617F-negative polycythemia vera. Leukemia 21:1960–3

30. Vainchenker W, Delhommeau F, Constantinescu SN, Bernard OA(2011) New mutations and pathogenesis of myeloproliferativeneoplasms. Blood 118:1723–35

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