Identification of a JAK2 Mutation in Polycythemia Vera

Abstract

The present invention concerns the V617F variant of the protein-tyrosine kinase JAK2, said variant being responsible for Vaquez Polyglobulia. The invention also relates to a first intention diagnostic method for erythrocytosis and thrombocytosis allowing their association with myeloproliferative disorders, or to the detection of the JAK2 V617F variant in myeloproliferative disorders allowing their reclassification in a new nosological group.

Claims

1.-10. (canceled)

21. An isolated nucleic acid comprising a complement of at least 12 consecutive nucleotides of sequence SEQ ID NO: 3 or 4, wherein the isolated nucleic acid comprises a thymine (t) in position 261 in SEQ ID NO: 3 or a thymine (t) at position 50 in SEQ ID NO: 4, and wherein the isolated nucleic acid further comprises a radioactive, fluorescent or enzymatic label.

22. The isolated nucleic acid according to claim 21, wherein said nucleic acid is SEQ ID NO: 11 with a G21T mutation.

23. The isolated nucleic acid according to claim 21, wherein the label is a radioactive label.

24. The isolated nucleic acid according to claim 21, wherein the label is a fluorescent label.

25. The isolated nucleic acid according to claim 21, wherein the label is an enzymatic label.

26. A kit for detecting a G1849T mutation in a human Janus kinase 2 (JAK2) gene in a human tumor, wherein the kit comprises one or more primers or probes comprising a complement of an isolated nucleic acid comprising at least 12 consecutive nucleotides of sequence SEQ ID NO 3 or 4, or having at least 95% homology to at least 17 consecutive nucleotides of sequence SEQ ID NO 3 or 4, wherein the isolated nucleic acid comprises the nucleotide t.sup.261 in SEQ ID NO 3 or t.sup.50 in SEQ ID NO 4, wherein the isolated nucleic further comprises a radioactive, fluorescent or enzymatic label], for the specific detection of the presence or absence of the G1849T mutation in the human JAK2 gene, wherein the G1849T mutation is nucleotide t.sup.261 in SEQ ID NO 3 or t.sup.50 in SEQ ID NO 4.

27. A kit, comprising a first container containing an isolated nucleic acid and a carrier; wherein the isolated nucleic acid comprises a complement of at least 12 consecutive nucleotides of sequence SEQ ID NO 3 or 4, or having at least 95% homology to at least 17 consecutive nucleotides of sequence SEQ ID NO 3 or 4, wherein the isolated nucleic acid comprises the nucleotide t.sup.261 in SEQ ID NO 3 or t.sup.50 in SEQ ID NO 4, wherein the isolated nucleic acid further comprises a radioactive, fluorescent or enzymatic label.

28. The kit according to claim 26, further comprising at least one element selected from a heat resistant polymerase for PCR amplification, one or more solutions for amplification and/or hybridization, and any reagent allowing said specific detection.

29. A method, comprising: a) obtaining and analyzing a nucleic acid sample from a human patient, wherein the analyzing comprises: a1) sequencing a region of the nucleic acid sample comprising position 2343 of a complement of SEQ ID NO: 2, or a2) hybridizing the nucleic acid sample with at least one probe comprising at least 10 consecutive nucleotides of a complement of SEQ ID NO: 2 comprising position 2343 of SEQ ID NO: 2 b) detecting the presence or absence of a thymine (T) in the JAK2 gene at position 2343 of SEQ ID NO: 2 in the nucleic acid sample; c) if T is present at position 2343, treating the patient for polycythemia vera (PV) quantifying red cell mass, quantifying erythroid endogenous colonies or testing bone marrow of the patient; and d) if T is absent at position 2343 of SEQ ID NO 2 quantifying red cell mass, quantifying erythroid endogenous colonies or testing bone marrow of the patient.

30. The method according to claim 29, wherein the human patient has erythrocytosis.

31. The method according to claim 30, wherein the human patient has a hematocrit level of higher than 51%.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0107] FIG. 1: Discovery of the key role of JAK2 in PV

[0108] In the basal state, JAK2 is fixed to box 1 in the non-phosphorylated state. The binding to Epo alters the conformation of the receptor and enables transphosphorylation of JAK2 which in return phosphorylates the intracytoplasmic residues of Epo-R thereby recruiting the different positive (->) or negative (−|) effectors of signal transduction.

[0109] FIGS. 2A-2B: Design of a culture model of PV CD34+ progenitors that are erythropoietin-independent.

[0110] FIG. 2A—culture with Epo, SCF and IL-3

[0111] FIG. 2B—culture without Epo

[0112] FIG. 3: Inhibition of JAK-STAT, Pi3-K and Src kinase pathways prevents spontaneous erythroid differentiation.

[0113] FIG. 4: Protocol for inhibiting JAK2 in PV progenitors

[0114] FIGS. 5A-5B: Results of JAK2 inhibition in PV progenitors

[0115] FIG. 5A—Reduction in the cloning capacity of 36+/gpa−.

[0116] Culture D1-D6 in SCF-IL3, electroporation D5, sorting D6 (morpho/36/gpa−).

[0117] Methyl SCF alone. Count on D13 (D7 post-sorting).

[0118] FIG. 5B—Structure of JAK2 with V617F mutation (exon 12).

[0119] FIGS. 6A-6C: Genotyping analyses of SNPs to detect the JAK2 V617F mutation in genomic DNA using LightCycler® and TaqMan® technologies

[0120] FIGS. 6A and 6B: Detection of the mutation by the fusion analysis curve of LightCycler® with FRET hybridisation probes. FIG. 6A: Experiments with various dilutions of HEL DNA in DNA TF-1 are shown. The peak JAK2 V617F (57°) is still detectable at a dilution of 11. FIG. 6B: Results of representative patient samples (#1: homozygous; #2: heterozygous; #3: weak; #4: non mutated).

[0121] FIG. 6C. Detection of the mutation by TaqMan® allele specific amplification. Experiments with dilution of HEL DNA (HEL 100 to 1%: empty squares; TF-1 cells: empty circles) and a few representative patient samples are shown (black crosses). #a: homozygous patients; #b: heterozygous patients; #c: weak patient; #d: non-mutated patients.

[0122] FIG. 7: Proposed diagnostic datasheet to diagnose erythrocytosis (i.e. hematocrit level over 51%).

[0123] The number of patients concerned at each stage of the datasheet is written next to each item (n), only those patients showing all clinical data being listed here (n=81). Detection of JAK2 V617F as a first intention diagnostic test would have prevented 58/81 patients from undergoing other investigations to diagnose a PV type myeloproliferative disorder.

[0124] FIGS. 8 and 9: Expression of V617F Jak2 in HEL cells is reduced 24 hours after treatment with siRNA specific to JAK2 V617F Jak2 (siRNA #1, 3 and 4).

[0125] 0 to 6: HEL cells treated (1 to 6) or non-treated (0) with siRNA V617F Jak2.

[0126] C+: 293HEK cells transfected with the V617F Jak2RV vector

[0127] C−: 293 HEK

[0128] FIG. 10: Level of WT Jak2 expression in K562 cells remains unchanged 24 hours after treatment with siRNA specific to Jak2 V617.

[0129] Je: K562 cells treated with siRNA WT Jak2

[0130] 0 to 6: K562 cells treated with siRNA V617F Jak2

[0131] C−: 293HEK (no expression of JAK2).

EXAMPLE 1: IDENTIFICATION OF THE JAK2 V617F MUTATION IN 39/43 PATIENTS

[0132] The design of a cell culture model of pathological progenitors and the use of biochemical inhibitors enabled us to evidence that the JAK2-STAT5, P13 kinase and Src kinase pathways are necessary for Epo-independent differentiation of PV progenitors (Ugo et al 2004). These results reassured our hypothesis that the primary molecular lesion causing PV must be an anomaly of a key protein leading to deregulation of a signalling pathway, like the mutation of a tyrosine kinase imparting a constitutive activity. Nonetheless, it is the study of 43 patients suffering from Vaquez polyglobulia which made it possible to identify the key role played by the JAK2 protein which is the protein located the most upstream in these different signalling pathways, and which is common to the signalling pathways of cytokine receptors for which response anomalies have been described in PV. We examined the involvement of the JAK2 protein kinase in the physiopathology of PV taking three complementary approaches: [0133] a functional approach (inhibition of JAK2 in PV cells by interfering RNA) [0134] a genomic approach (sequencing of the 23 exons of the gene), and [0135] a biochemical approach to search a JAK2 phosphorylation anomaly, the cause of a constitutive activation.

[0136] The biological material used was derived from consenting patients suffering from polyglobulia and corresponds to residues of samples taken for diagnostic purposes and sent to the Hôtel Dieu Central haematology laboratory, or to therapeutic phlebotomy.

1.1. Functional Study

[0137] Study of the JAK2 function in the erythroblasts of patients with Vaquez polyglobulia was conducted by using electroporation to transduce the PV erythroblasts with a siRNA specific to the JAK2 sequence (AMBION, Huntingdon, England) recognizing as target a sequence located on exon 15 of the mRNA of JAK2. We have shown that the inhibition of JAK2 strongly reduces the cloning capacity and “spontaneous” differentiation of PV progenitors in the absence of Epo. Normal erythroblast progenitors transfected with siRNA JAK2 show a reduction in clonogenic potential of 70% compared with the control siRNA, which confirms the efficacy of transfection with siRNA JAK2. In PV, the effects of JAK2 inhibition in the erythroblast progenitors were studied in an Epo-free culture model, making it possible to study the cells of the malignant clone. We compared the clonogenic potential, apoptosis and differentiation of the PV erythroblast progenitors after transfection with a siRNA JAK2 with respect to a control siRNA. Study of the clonogenic potential of PV progenitors cultured without Epo shows a very marked reduction in the number of colonies after transfection of siRNA JAK2 compared with control siRNA. Flow cytometry of the apoptosis of these cells shows a significant increase in the apoptosis of cells transfected with siRNA JAK2 compared with non-relevant siRNA (70 versus 53%). Study of the effects of siRNA JAK2 on differentiation (acquisition of Glycophorin A detected by flow cytometry) only shows a slight difference between the progenitors transfected with siRNA JAK2 versus control siRNA.

[0138] The results of the cell studies therefore showed that JAK2 is necessary for Epo-independent differentiation of PV erythroid progenitors. The initial results of biochemical studies (immunoprecipitation) show more extended phosphorylation of JAK2 after depriving PV erythroid progenitors of cytokines, as compared with normal cells.

1.2 Genomic Study of JAK2

[0139] PCR on the 23 exons was set up on a normal individual using genomic DNA. We then examined 3 patients suffering from PV after extracting the genomic DNA from erythroid cells obtained in vitro after cell culture.

[0140] We identified a point mutation located in exon 12 of JAK2, present in 2 out of the 3 patients tested. This mutation concerns base no 1849 of the encoding sequence ([numbering starting at ATG], GenBank NM_004972) and transforms codon 617 of the JAK2 protein (V617F). [0141] normal 617 codon: gtc code for a Valine (V) [0142] mutated 617 codon: ttc code for a Phenylalanine (F)

[0143] Using the databases published on the Internet we were able to verify that it is not a known polymorphism.

[0144] We then widened the cohort. To date the mutation has been found in 39 patients with PV out of the 43 cases tested. No control (15 cases tested) or patient with secondary polyglobulia (18 cases tested) were found to carry the mutation.

Sequencing Results in Patients

[0145] 39 mutated/43 PV tested (901) [0146] 2/3 heterozygotes [0147] 13/39 “homozygotes” i.e. 30% of cases (same proportion as the loss of heterozygosity at 9p).

Controls

[0148] 0 mutated cases out of 33 controls tested: [0149] including 15 normal individuals [0150] and 18 secondary polyglobulias (no spontaneous colonies).

[0151] The discovery of this anomaly of JAK2 accounts for the hypersensitivity to numerous growth factors involved in PV (Epo, TPO, IL-3, IL-6, GM-CSF, insulin). Indeed, JAK2 is a protein involved in the signalling pathways of the receptors of these cytokines.

[0152] Also, the association of JAK2 with R-Epo is particular in that JAK2 is fixed to E-Epo constitutively: the JAK2/R-Epo association initiated in the Golgi apparatus is necessary for the processing of R-Epo at the membrane of the erythroblasts. A JAK2 anomaly, the cause of modifications to the association of JAK2 with R-Epo, could therefore account for the medullary hyperplasia predominance on the erythroblast line, whereas this protein is involved in numerous signalling pathways. Also, Moliterno et al (Moliterno et al, 1998; Moliterno and Spivak, 1999) have evidenced faulty membrane expression of mpl related to a glycosylation defect. It is possible that JAK2, by analogy with R-Epo, is necessary for the processing of c-mpl. The anomaly of JAK2 could then explain the lack of membrane expression of c-mpl, found in PV.

[0153] JAK2 binds to R-Epo on its proximal domain, at a highly conserved domain, Box2. In the absence of Epo stimulation, JAK2 is constitutively fixed to R-Epo, but in a non-phosphorylated form, hence non-active. After stimulation of the receptor by Epo, the two JAK2 molecules phosphorylate, and then phosphorylate R-Epo enabling the recruitment then the phosphorylation of other proteins involved in signal transduction, such as the proteins STAT5, Grb2, P13K. The JAK2 protein, like all JAKs, has a functional kinase domain (JH1), a pseudo-kinase domain with no tyrosine kinase activity (JH2), and several conserved domains (JH3-JH7), characteristic of members of the JAK family. The JH2 domain appears to be involved in regulating the tyrosine-kinase activity of JAK2. According to available JAK2 protein mapping data (Lindauer, 2001), amino acid 617 is located in this JH2 domain and, following modelling studies, in a region of particular importance for the regulation of kinase activity.

[0154] Over and above the physiopathological interest of this discovery (understanding of the mechanisms of cytokine-independence, breakdown of the different SMPs) the evidencing of this mutation in a patient offers a test for the first time with which it is possible to confirm diagnosis. From a medical diagnosis viewpoint, the search for the mutation of JAK2 can be made on polynuclear neutrophils belonging to the malignant clone.

[0155] The invention also offers the determination of a specific treatment, of kinase inhibiting type specific to the mutated protein, or gene therapy.

EXAMPLE 2: DETECTION OF THE JAK2 V617F MUTANT FOR FIRST INTENTION DIAGNOSIS OF ERYTHROCYTOSIS

2.1 Patients, Materials and Methods

Comparison Between Sequencing and Two Techniques of SNP Genotyping for the Detection of JAK2 V617F.

Patient Cells

[0156] 119 samples of suspected MPD were analysed (i.e. erythrocytosis, thrombocytosis, hyperleukocytosis). 58 samples were taken for perspective analysis and 61 archive samples of bone marrow were analysed retrospectively.

[0157] The peripheral granulocytes were isolated using a density gradient method following the manufacturer's instructions (Eurobio, France). Mononuclear cells were isolated from the bone marrow using Ficoll density gradient centrifugation. The genomic DNA was extracted following standard procedures. To determine the sensitivity of LightCycler® and Taqman® technologies, the DNA derived from a homozygous sample with the allele of minimum residual wild type was diluted in series in normal DNA.

Cell Lines

[0158] Serial solutions of DNA were used (1, 0.5, 0.1, 0.05, 0.01) from the human erythroleukaemia cell line (HEL) mutated homozygous fashion (JAK2 V617F) in DNA of TF-1 cell line (non-mutated) as standard positive controls. The cells lines grew in MEM-alpha medium (Invitrogen) enriched with foetal calf serum.

Detection of the Mutation by Analysis of the Fusion Curve of LightCycler® with FRET Hybridisation Probes.

[0159] Primers and probes were designed to amplify and hybridise to the target sequence of exon 12 of JAK2. The position of the mutation site (1849G/T) was covered with a donor capture probe labelled with fluoresceine at 3′, and the adjacent acceptor anchor probe labelled with LightCycler® Red 640 (LCRed640) at its 5′ end; its 3′ end was phosphorylated to avoid extension. Amplification and analysis of the fusion curve were performed on the LightCycler® instrument (Roche Diagnostics, Meylan, France). The final reaction volume was 20 μl using 10 ng DNA, 14 μl LightCycler FastStart DNA Master mixture, 3 mM MgCl.sub.2, 0.2 μM primers, 0.075 μM of each probe. In brief, the samples were heated to 95° C. for 10 minutes and PCR amplification of 45 cycles (10 seconds at 95° C., 10 seconds at 53° C., 15 seconds at 72° C.) was followed by a denaturing step at 95° C. for 10 seconds, two hybridisation steps at 63° C. and 45° C. for 30 seconds each and a fusion curve located in the domain lying between 45 and 70° C. (0.1° C./sec). Analysis on the LightCycler® programme was performed by plotting the curve of the fluorescence derivative with respect to temperature [2(dF12/F11)/dT) versus T]. The mutated peaks and WT were observed at 57 and 63° C. respectively.

[0160] Detection of the Mutation by Specific Amplification of an Allele Using TaqMan®.

[0161] Two primers were designed to amplify a product of 92 bp encompassing SNP at position 1849. Two fluorogenic MGB probes were designed with different fluorescent colourings, one targeted towards the WT allele, and one targeted towards the mutated allele. Genotyping was conducted in 96-well plates using the method based on Taqman® PCR. The final reaction volume was 12 μl using 10 ng genomic DNA, 6.25 μl TaqMan® Universal Master Mix and 0.31 μl 40× Assays-on-Demand SNP Genotyping Assay Mix (Applied Biosystems). The plate was heated to 95° C. for 10 minutes followed by 40 denaturation cycles at 92° C. for 15 seconds and matching/extension at 60° C. for 1 minute. Thermocycling was conducted on the 7500 Real Time PCR System (Applied Biosystems). Analysis was made using the SDS programme version 1.3. Genotyping of end point allele discrimination was performed by visual inspection of a fluorescence curve (Rn) derived from the WT probe against the Rn of the mutated JAK2 generated from post-PCR fluorescence reading.

Patients with Erythrocytosis and Sample Collection

[0162] We evaluated 88 patients with hematocrit levels of more than 51%, at the time of diagnosis, before any form of treatment, and we studied the presence of WHO and PVSG criteria. The value of 51% was chosen for the upper end of the normal range for hematocrit level (Pearson T C et al, Polycythemia Vera Updated: Diagnosis, Pathobiology and Treatment. Hematology (AM. Soc. Hematol. Educ. Program.) 2000: 51 to 68). Bone marrow cells were collected for clonogenic assays and excess cells were collected for DNA extraction. Serum erythropoietin (Epo) was measured in different laboratories and it is therefore reported as being low when below the lower value of the normal domain in each laboratory, normal or high. The peripheral granulocytes derived from the same patients were purified as described previously, the blood samples of each time being available. The samples of bone marrow and blood were collected after receiving informed consent.

EEC Assays

[0163] In vitro assays of erythroid Epo-response were all performed in the same laboratory (Hotel Dieu, Paris) using a plasma-clot culture technique as described previously (Casadevall N, Dupuy E, Molho-Sabatier P, Tobelem G, Varet B, Mayeux P. Autoantibodies against erythropoietin in a patient with pure red-cell aplasia. N. Engl. J. Med. 1996; 334: 630 to 633).

Statistical Analysis

[0164] Correlation of the markers was made using the Spearman rank correlation coefficient (R).

2.2 Results

Feasibility and Sensitivity of Genotyping Techniques Based on PCR for Detection of the Mutation JAK2 V617F.

[0165] To assess the efficacy of sequencing, LightCycler® and Taqman® technologies for detection of the JAK2 V617F mutation, we searched its presence in 119 samples taken from patients suspected of having a MPD, using the 3 techniques in parallel. The JAK2 V617F mutation was efficiently detected in 83/119 samples, and 35 samples did not show the mutation with any of the 3 techniques. In only one sample, sequencing failed to detect the mutation revealed by the two technologies LightCycler® and Taqman® thereby suggesting that the latter may be more sensitive.

[0166] To assess the sensitivity of the technique, we used two different methods: we tested serial dilutions of DNA of the HEL cell line with homozygous mutation in the DNA of the non-mutated TF-1 cell line, and serial dilutions of the genomic DNA derived from a homozygous patient for the mutation JAK2 V617F in normal DNA. Sequencing failed to detect the mutated allele with 5% DNA of the HEL cell line diluted in the DNA of the TF-1 cell line, and with 10% of the DNA from the patient with homozygous mutation diluted in normal DNA. The sensitivity of the LightCycler® and Taqman® techniques was equivalent, slightly better than sequencing, reaching 0.5 to 1% of the DNA from the HEL cell line diluted in the DNA of the TF-1 cell line (FIG. 6) and 2 to 4% of the DNA from a patient with homozygous mutation diluted in normal DNA.

Characteristics of Patients with Erythrocytosis at the Time of Diagnosis

[0167] The chief characteristics of 88 patients with hematocrit levels of more than 51% at the time of diagnosis are summarized in Table I.

TABLE-US-00005 TABLE 1 Patient Characteristics WHO criterion PVSG criterion WHO and PVSC criteria idiopathic idiopathic Secondary Hct >50% PV erythrocytosis PV erythrocytosis erythrocytosis but no AE n = 61 n = 11 n = 45 n = 21 n = 5 n = 3 Sex ratio 38/23 11/0  28/17 18/3  4/1 3/0 (male/female) Mean age 61 (23 to 92) 57 (24 to 81) 53 (23 to 92) 60 (53 to 31) 65 (55 to 77) 48.6 (domain) mean Ht  59 ± 4.6 54.6 ± 1.44 59.2 ± 4.5  57.8 ± 4.2  55.8 ± 3.1  53.3 ± 0.8  (%) ± σ Mean Hb 19.2 ± 1.39 18.3 ± 0.34 19.3 ± 1.41  19 ± 1.0 18.9 ± 0.8  18.6 ± 0.5  (g/dL) ± σ Mean WBC 12.2 ± 4.4  7.0 ± 2.5 13.5 ± 4.9  8.2 ± 2.5 3.8 ± 1.9 6.6 ± 0.4 (×/10.sup.9) ± σ Mean 463 ± 148 212 ± 38  503 ± 149  245 ± 60.4 212 ± 29  175 ± 19  platelet count (×/10.sup.9) ± σ Splenomegalia 16/55  0/11 14/39  0/21 0/5 0/3 EEC presence 59/60  1/11 43/44 11/21 0/5 0/3 Low Epo level 39/47 2/8 27/33 10/17 0/3 1/1 Normal Epo  8/47 6/8  6/33  7/17 3/3 0/1 level Cytogenetic  7/32 0/3  6/23 0/7 nd 0/1 anomalies Positive 57/61  0/11 43/45  8/21 0/5 0/3 JAK2V617F

[0168] 88 patients with hematocrit levels of over 51 h were diagnosed in accordance with PVSG and WHO criteria into four groups: Vaquez disease (PV), idiopathic erythrocytosis, secondary erythrocytosis and “no absolute erythrocytosis” (no AE) when measured red cell mass had not increased. 8 patients could not have any definite diagnosis since some clinical data were not available. Hct: hematocrit; Hb: haemoglobin; WBC: white blood cells; EEC: endogenous erythroid colonies; Epo: erythropoietin; σ: standard deviation. The patients could be divided into 4 main groups in accordance with WHO criteria (Pierre R et al, editors, World Health Organization Classification of Tumours; Pathology and Genetics of tumours of hematopoietic and lymphoid tissues. Lyon; IARC Press: 2001: 32 to 34) and PVSG criteria (Pearson T C, Messinezy M. The diagnostic criteria of polycythaemia rubra vera. Leuk Lymphoma 1996; 22 Suppl 1:87 to 93): 61 and 45 patients with PV diagnosis, 5 with secondary erythrocytosis, 11 and 21 with idiopathic erythrocytosis and 3 with no absolute erythrocytosis (normal red cell mass). The clinical data were incomplete for 7 patients, accounting for the fact that PV diagnosis could not be confirmed either with WHO criteria or with PVSG criteria. On account of the difference between the A1 criteria of the two classifications, 6 patients who had no red cell mass measurement could be classified in the WHO classification but not in the PVSG classification. One patient showed both hypoxia and EEC formation, thereby making diagnosis difficult. Cytogenetic analysis was performed in 35 patients; among 32 PV patients (WHO criteria) 7 showed cytogenetic anomalies: 5 with trisomy 9, 1 with 7q- and 1 with additional material on chromosome 18.

The Presence of JAK2 V617F Corresponds to PVSG and WHO Criteria for PV

[0169] JAK2 V617F was present in 43/45 (96%) of patients diagnosed with PV in accordance with PVSG criteria and in 57/61 (93%) of patients diagnosed using WHO criteria (Table I). Nonetheless, 8/29 patients classified as non-PV according to PVSG criteria showed the mutation, but none of the 19 WHO non-PV patients; these 8 patients were considered IE with PVSG criteria and PV with WHO criteria. None of the patients diagnosed with SE nor the patient with normal red cell mass (“no AE”) had the mutation. The presence or absence of JAK2 V617F therefore corresponds to positive PV diagnosis in 76/80 patients (95′% R=0.879, p<0.0001) with WHO criteria, and in 64/74 patients (86.5%, R=0.717, p<0.0001) with PVSG criteria. In addition, since none of the patients diagnosed as non-PV according to WHO criteria showed the mutation, the detection of JAK2 V617F has a 100% predictive value in the context of absolute erythrocytosis.

[0170] Some authors (Mossuz P et al, Diagnostic value of serum erythropoietin level in patients with absolute erythrocytosis. Haematologica 2004; 89: 1194 to 1198) consider the measurement of serum erythropoietin level as a first intention diagnostic test for patients with absolute erythrocytosis, with a specificity of 97%, and a predictive value of 97.8% for diagnosis of PV if the Epo level is below the lower value of the normal range. In our study, the correlation between the Epo level and PV diagnosis according to WHO and PVSG criteria was respectively observed in 50/61 (82%, R=0.488, p=0.0002) and 39/56 (70s, R=0.358, p=0.0067) patients. We then compared the serum Epo level in the presence or absence of V617F JAK2 and it was found that 52/68 patients (76%, R=0.416, p=0.0004) showed adequate correlation.

[0171] The presence of the JAK2 V617F mutation corresponds to the capacity for forming EECs.

[0172] Three different teams have shown that Epo-dependent cell lines transfected with JAK2 V617F were Epo-independent and Epo-hypersensitive for their growth, thereby mimicking the independence and hypersensitivity of the erythroid progenitors described in PV. Therefore, we have put forward the hypothesis that patients carrying JAK2 V617F also showed EEC formation. Among the 20 patients with erythrocytosis with no EEC formation, one showed the mutation, raising the query of the positive predictive value of JAK2 V617F detection; however, even if this patient showed no EEC, he/she met the numerous WHO and PVSG positive criteria allowing the patient's classification as PV in both classifications. This patient should therefore be considered a “false-negative to EEC” rather than a “false-positive for JAK2”. Among the 67 patients who had EEC formation, 62 carried the JAK2 V617F mutation, 5 patients not being mutated using detection-sensitive techniques. Among these 5 patients, 4/5 and 2/5 could be classified in the PV group according to WHO and PVSG criteria respectively. Overall, out of the 87 analysed patients, the presence or absence of the JAK2 V617F mutation corresponded to the capacity or incapacity to form EECs in 81/87 patients (93.1%, R=0.824, p<0.0001).

[0173] The presence of the JAK2 V617F mutation in bone marrow mononuclear cells (BMMC) corresponds to its presence in the peripheral granulocytes.

[0174] To examine whether the use of granulocytes of peripheral blood to detect JAK2 V617F mutation at the time of diagnosis could avoid the assay of bone marrow cells, we compared the results obtained by each of the methods: sequencing, LightCycler® and TaqMan®, in 50 patients (including 35 PV, 8 SE and 8 suspected MPD) for whom both bone marrow samples and peripheral blood samples were available at the time of diagnosis. In all cases (34 mutated, 16 non-mutated) mutation was identically detected.

III—Conclusion

[0175] We therefore propose a new PV diagnosis datasheet in which the detection of JAK2 V617F in the granulocytes using sensitive techniques is the first step in the diagnosis of erythrocytosis, except in the case of obvious secondary erythrocytosis (FIG. 7). This approach has several advantages: it avoids having to conduct measurement of isotopic red blood cells, which is not always available and whose result is sometimes subject to debate. It can also avoid bone marrow procedure and EEC assays which are time-consuming and are not well standardized. It drastically reduces the cost of positive PV diagnosis, since only those patients with hematocrit levels of over 51% and who are JAK2 V617F negative need to undergo all the investigations which are actually carried to characterize an erythrocytosis. Even if the detection alone of JAK2 V617F in an erythrocytosis context will support PV diagnosis, performing a bone marrow biopsy may still be useful since it may reveal signs of myelofibrosis or the presence of blastic cells, thereby confirming the leukaemic transformation of PV. Nonetheless, we feel that a bone marrow biopsy should be performed for optional study with cytogenetic analysis.

[0176] JAK2 V617F was also detected in 30% of ET, 50% of IMF and a few rare non-characterized MPDs, thereby defining a new MPD group with different clinical signs. The reasons for these differences remain unknown and it is still too early to group these diseases into a single myeloproliferative entity with a common physiopathological cause and different phenotypes. Subsequent precise clinical studies would characterize more specifically the common signs between PV, ET, IMF and other rare MPDs carrying JAK2 V617F, especially in terms of absolute erythrocytosis, Epo level, myelofibrosis and cytogenetic anomalies. It is therefore contemplated to use the detection of JAK2 V617F as an initial tool for the diagnosis of chronic hyperleukocytosis, thrombocytosis and erythrocytosis. The presence of JAK2 V617F will not only allow a new definition of a MPD group, but it will also most certainly form the basis for developing specific targeted therapies.

EXAMPLE 3: SIRNAS SPECIFIC TO THE V617F JAK2 MUTATION INHIBIT V617F JAK2 BUT NOT JAK2 WT

[0177] The siRNAs 1, 3 and 4 corresponding to sequences SEQ ID No 25 to 27 inhibit the mutated protein V617F JAK2 expressed by the HEL line without inhibiting the wild-type JAK2 protein expressed by the K562 line. The results are shown FIGS. 8, 9 and 10.

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