Incidence of the JAK2V617F Mutation In Mexican Patients with Myeloproliferative Neoplasms (MPNs)

Abstract Abstract 5180 Introduction: The V617F mutation in JAK2 gene has been described in approximately 50–90% of patients with ET, MF AND PV [essential trombocythaemia, idiopathic myelofibrosis and policithemia vera]; but has also been reported, albeit at a lower frequency, in patients with other myeloid malignancies such as atypical CML, CMML, AML, MDS, JMML and CNL. A single G>T base substitution in exon 12 results in the conversion of valine to a phenylalanine aminoacid at position 617 of the JAK2 gene. The identification in this study were using techniques such as allele-specific PCR, RFLP-PCR and direct sequencing; for to determine the incidence in Mexican patients with MPNs. Patients and Methods: The JAK2V617F mutation was determined in 88 patients and 5 normal blood samples for healthy individuals as controls. About the patients, 60 were cataloged like MPNs and 28 patients with features suggestive of MPNs vs CML. Samples for bone marrow or peripherical blood were taken either at time of diagnosis of MPNs or during treatment with cytoreductive or anti-thrombotic agents. DNA and RNA were extracted using the QIAamp DNA and RNeasy mini kit (Qiagen) and amplified by the three techniques mentioned for JAK2V617F and by nested RT-PCR for BCR/ABL. Results: The five normal blood samples for controls were negative for JAK2V617F mutation and to BCR/ABL. Patients had median age 65 years (47–85 years old), 46% male and 54% female. In de overall patients: 60 patients with MPNs all were BCR/ABL negative and 20 (33%) had JAK2V617F. In the 28 patients with likely MPNs vs CML, 23 were BCR/ABL positive/JAK2 negative, two had the coexistence of both genetics defects [BCR/ABL+ and JAK2V617F+] and 3 BCR/ABL and JAK2 negative. Finally the patiens with JAK2V617F+, were 12 ET, PV 1, MF 2, CML 2, and 5 continued like MPNs. Discussion: The incidence of the JAK2V617F in this study for MPNs patiens were 33% and the incidence varied between MPNs subtype. Less than ten cases of BCR/ABL+ CML with JAK2V617F have been published; we report two patients with the coexistence and we agree with previous reports that screening for JAK2V617F mutation should be considered in any BCR/ABL+ CML patients and the clinical outcome will be define in long period. Disclosures: No relevant conflicts of interest to declare.

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TET2 Mutations in a Cohort of Patients with Myeloproliferative Neoplasms Lacking a Molecular Marker.

Blood ◽

10.1182/blood.v114.22.3912.3912 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3912-3912

Author(s):

Luz Maria Martinez-Aviles ◽

Carlos Besses ◽

Alberto Alvarez-Larran ◽

Aina Pons ◽

Sergi Serrano ◽

...

Keyword(s):

Mutational Analysis ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Receptor Gene ◽

Biological Significance ◽

Jak2v617f Mutation ◽

Missense Mutations ◽

Coding Sequence ◽

Exon 10

Abstract Abstract 3912 Poster Board III-848 Background The myeloproliferative neoplasms (MPNs) include different diseases presenting several mutations in variable frequency. The JAK2V617F mutation is present in 90-95% Polycythemia Vera (PV), 50-60% Essential Thrombocythemia (ET) and 50-60% Primary Myelofibrosis (PMF) patients. In addition, JAK2 exon 12 mutations are observed in 1-3% of PV patients and mutations in the thrombopoietin receptor gene (c-MPL) (S505N, W515K/L) are present in a 5-9% of PMF and in a 1-4% of ET patients. Recently, acquired mutations of the Ten-Eleven Translocation (TET) 2 gene, have been reported in about 14% of sporadic MPNs. TET2 mutations may precede the acquisition of JAK2V617F predisposing to a MPN development. However, the incidence of TET2 mutations in patients lacking the JAK2V617F mutation has not been extensively studied. Aim To analyze the incidence of TET2 mutations in a cohort of MPN patients negative for JAK2V617F, JAK2 exon 12 mutations and c-MPL exon 10 mutations (S505N or W515K/L). Patients and methods From a whole cohort of 241 patients with MPN (93 PV, 16 PMF and 132 ET) we excluded those patients positive for JAK2V617F (determined by quantitative allele-specific PCR), JAK2 exon 12 mutations or c-MPL exon 10 mutations (S505N or W515K/L) (analyzed by direct sequencing). We analyzed the TET2 gene in 5 PV, 5 PMF and 53 ET patients lacking any of the aforementioned molecular markers. The mutational analysis of the coding sequence of TET2 was performed by direct sequencing using cDNA from granulocytes. In 13 patients, DNA from T lymphocytes was obtained to indentify the presence of single nucleotide polymorphisms (SNPs) in germline DNA. Results Sixty-three patients were screened for mutations in the whole coding sequence of the TET2 gene. Only 3 ET patients (4.7%) presented deleterious mutations in the TET2 gene. The three distinct TET2 mutations were: Q706X, S1848X and V1395_R1400delinsR. In 48 out of 63 (76.1%) patients we observed a total of 13 different missense mutations and 2 silent mutations in the coding sequence of the gene. The most frequent missense mutation was the I1762V which was detected in 27 patients. In 13 patients whose matched normal DNA was available, we analyzed the presence of missense mutations being all of them present in the control DNA suggesting that they were SNPs and not acquired mutations. The two nonsense mutations (Q706X and S1848X), were not present in matched normal tissue indicating that these mutations were somatically acquired in myeloid cells. Conclusions TET2 pathogenic mutations are infrequent (<5%) in myeloproliferative neoplasms negative for JAKV617F, JAK2 exon 12 and c-MPL (S505, W515K/L) gene mutations. The role and the biological significance of missense mutations in the coding sequence of TET2 has to be elucidated. Acknowledgments Fellowship FI2008 (AGAUR) to LMM-A, FIS EC07/90791 Disclosures: No relevant conflicts of interest to declare.

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Calr Gene Mutation in Patients with JAK2-Negative Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v124.21.5190.5190 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5190-5190

Author(s):

Xu Jingyan ◽

Rong-Fu Zhou ◽

Bing Chen ◽

Jian Ouyang

Keyword(s):

Mutation Rate ◽

Conflicts Of Interest ◽

Novel Mutation ◽

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Gene Mutations ◽

Jak2v617f Mutation ◽

Positive Rate ◽

Exon 9 ◽

Calr Mutation

Abstract Methods Genomic DNA from bone marrow and peripheral blood cells were extracted from 40 patients with MPD(male 26,female 14),Aged 13 to 76 years old, JAK2V617F mutation and CALR genetic mutations was identified by PCR- direct sequencing. Results The number of MPD(PV15,ET25) 40 cases in patients with JAK2V617F mutation rate was 60%,among them the polycythemia vera ( PV) positive rate was 66. 7% ( 10 /15) ,and essential thrombocythemia ( ET ) positive rate was 56% ( 14 /25) . The number of JAK2V617F -negative MPD 16 cases in patients with CALR mutation rate was 12. 5% ( 2 /16),Two of the 11 patients with ET were CALR mutation positive(18.2%). Two novel mutation in CALR exon 9,c.1099_1150del ( p. chr19F12915572-12915623del) was detected in patient Tao c.1099-1151delinsT( p.chr19:12915572_12915624delinsT) was detected in patient Xu. These mutation was absent in the controls,Two novel mutation in CALR have not been reported so far. Conclusion CALR gene mutations testing helps to JAK2V617F negative MPD diagnosis, makes the MPD early detection and treatment. Disclosures No relevant conflicts of interest to declare.

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Clinical Phenotype of Myeloproliferative Neoplasms with Activating CBL-mutations Resembles Chronic Myelomonocytic Leukemia.

Blood ◽

10.1182/blood.v114.22.3895.3895 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3895-3895

Author(s):

Juliana Popa ◽

Susanne Schnittger ◽

Philipp Erben ◽

Tamara Weiss ◽

Ayalew Tefferi ◽

...

Keyword(s):

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Clinical Phenotype ◽

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Jak2 V617f ◽

Chronic Myelomonocytic Leukemia ◽

A Genome ◽

Length Mutations ◽

Myelomonocytic Leukemia

Abstract Abstract 3895 Poster Board III-831 A genome-wide single nucleotide polymorphism (SNP) screen led to the identification of 11q aUPD in patients diagnosed with various subtypes of myeloproliferative neoplasms (MPN), e.g. chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML) and myelofibrosis (MF) (Grand et al., Blood 2009;113:6182). Further molecular analyses revealed acquired activating point and length mutations in CBL exons 8 and 9 in 10% of CMML, 8% of aCML and 6% of MF cases. Most variants were missense substitutions in the RING or linker domains that abrogated CBL ubiquitin ligase activity and conferred a proliferative advantage to 32D cells overexpressing FLT3. In this study, 160 patients with BCR-ABL and JAK2 V617F negative MPNs were screened for CBL mutations by PCR and direct sequencing. Eighteen known (Y371H, L380P [2x], C381R, C381Y [2x], C384Y, C396Y, H398P, H398Q, W408C, P417H, F418L, R420Q [5x]) and four new (F378L, G397V, I423N, V430M) missense mutations affecting fourteen residues were identified in 20 patients. Two patients harbored two different mutations. The clinical phenotype could be characterized more precisely in 17 patients. Median age was 68 years (range 59–85) with a slight female predominance (f, n=10; m, n=7). Striking hematological features were leukocytosis (14/17; 82%; median 29,000/μl, range 4,500-141,000) with continuously left-shifted granulopoiesis (blasts, promyelocytes, myelocytes, metamyelocytes) in 85% and elevated monocytes (median 2,500/μl, range 630-10,656) >1,000/μL in 88% (15/17) of patients. Eosinophilia (>1,500/μL) was rare (3/17, 18%). Anemia (normal values: f, Hb <12g/dL; m, Hb <14g/dL) was present in all 17 patients (f, median 10g/dL, range 8.7-11.8; m, median 11.2g/dL, range 8.6-12.9). Platelets did not exceed 300,000/μL in any patient while 11/17 (65%) patients presented with thrombocytopenia (median 125,000/μL, range 18,000-271,000). Splenomegaly was present in 11/17 patients (65%) and LDH was elevated (median 304U/L, range 189-729) in 9/17 patients (52%). Bone marrow histology and immunohistochemistry were available from 12 patients. Relevant features were hypercellularity, marked granulopoiesis and microlobulated megakaryocytes without clusters in 11/12 patients (92%), respectively. Increased fibres were seen in 8/12 (67%) patients of whom one showed severe fibrosis. Clinical follow-up was available from 17 patients. Thirteen patients (76%) have died because of progression to secondary acute myeloid leukemia/blast phase (n=7), cytopenia-related complications (n=2) or for unknown reasons (n=4) after a median of 23 months (range 3-60) following diagnosis. In conclusion, point mutations of CBL exons 8 and 9 are present in approximately 6-12% of BCR-ABL and JAK2 V617F negative MPNs. They are associated with a distinct clinical and hematological phenotype presenting with myeloproliferative features allowing diagnosis of a proliferative subtype of CMML rather than aCML or MF in the majority of cases. Patients with left-shifted leukocytosis, monocytosis, anemia and lack of thrombocytosis who are negative for BCR-ABL and point or length mutations of JAK2 should be routinely screened for CBL mutations. Disclosures: No relevant conflicts of interest to declare.

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Is JAK2 Inducible by Cytotoxic Chemotherapy?

Blood ◽

10.1182/blood.v114.22.4979.4979 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4979-4979

Author(s):

Edmond A. Bendaly ◽

Saud S. Rahman ◽

Samiah Zafar ◽

Karen Haglof ◽

Sherif Ibrahim ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Case Reports ◽

Lymphoblastic Leukemia ◽

Myeloproliferative Neoplasm ◽

Cytotoxic Chemotherapy ◽

Jak2v617f Mutation ◽

Platinum Based Chemotherapy ◽

History Of ◽

Increased Susceptibility

Abstract Abstract 4979 Introduction The JAK2V617F mutation accounts for most cases of myeloproliferative neoplasms (MPN). Only a few case reports of MPN following cytotoxic chemotherapy have been reported, and all of them were published prior to the discovery of the JAK2V617F mutation. We report a series of 6 patients who developed a JAK2V617F positive MPN following cytotoxic chemotherapy. Patients From 2006 to 2009, 6 patients with a history of a hematologic or an oncologic malignancy who developed an MPN were identified and their medical records retrospectively reviewed. One patient had acute lymphoblastic leukemia, 1 had Hodgkin lymphoma, 1 had squamous cell carcinoma of the head and neck, 1 had cervical cancer, and 2 had breast cancer. All patients were in remission from their primary malignancies at the time the MPN was diagnosed. Five were females. The median age at diagnosis was 72 years. Median time to development of the myeloproliferative neoplasm was 14 years. Type of chemotherapy exposure, MPN diagnosis and time to MPN in each case is shown in the table below. The JAK2V617F mutation was detected either in the peripheral blood or the bone marrow of all patients. There was no predominance of any specific MPN diagnosis. Patients who received platinum-based chemotherapy developed the MPN sooner than those who received alkylators (6 vs 17.5 years respectively). Treatment consisted of phlebotomy, hydroxyurea, anagrelide, aspirin or a combination as deemed appropriate by the treating hematologist. Conclusion These findings lead us to hypothesize whether the development of JAK2V617F positive MPN may be related to prior exposure to cytotoxic chemotherapy. Exposure to platinum-based chemotherapy may cause the disorder to appear sooner compared with exposure to alkylators. Recently, JAK2V617F positive MPN was found to be strongly associated with a specific constitutional haplotype, 46/11 suggesting increased susceptibility to this mutation. Chromosomal analyses are planned to show whether any of the reported patients exhibit this haplotype. Ref: 1.Jones et al, Nat Genet. 2009 Apr;41(4):446-9. 2009 Mar 15. The authors have no relevant disclosure. Disclosures No relevant conflicts of interest to declare.

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Disruption of the ASXL1 Gene Is Prevalent In PMF: Analysis of Molecular Genetics and Clinical Phenotypes

Blood ◽

10.1182/blood.v116.21.3074.3074 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3074-3074

Author(s):

Brady L Stein ◽

Donna M Williams ◽

Michael A McDevitt ◽

Christine L. O'Keefe ◽

Ophelia Rogers ◽

...

Keyword(s):

Molecular Genetics ◽

De Novo ◽

Conflicts Of Interest ◽

Single Nucleotide Polymorphism Array ◽

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Snp Array ◽

Uniparental Disomy ◽

Jak2 V617f ◽

Wild Type

Abstract Abstract 3074 Background: The myeloproliferative neoplasms, PV, ET and PMF, share phenotypic features and molecular lesions, yet PMF distinguishes itself by its unfavorable natural history and rate of leukemic evolution. These distinctions may occur as a result of cooperating genomic lesions specific to PMF compared to PV or ET. We performed single nucleotide polymorphism array (SNP-A)-based karyotyping in 210 MPN patients and identified 20q11 deletions in 10% of PMF cases and in none of the PV or ET cases. The 20q11 deletion region spanned 1,662 KB and encompassed 37 genes, of which ASXL1 was included. To test whether ASXL1 contained lesions in the MPN cohort at large, we directly sequenced key regions of the ASXL1 gene in 65 PMF, 11 PV and 14 ET cases, as well as 7 controls from the SNP-array cohort. Genomic DNA from neutrophils and in select cases, purified CD34+ cells was used for both SNP-A and direct sequencing. Clinical parameters were correlated with genomic findings and the quantitative JAK2 V617F neutrophil allele burden Molecular genetics: 26/65 (40%) of PMF cases had abnormalities in ASXL1 (4 deletions, 22 mutations) whereas none of the 32 PV, ET or control cases had such lesions. The majority of ASXL1 sequence variations were nonsense lesions including the previously reported 1934dupG which comprised 30% of all of the mutations. The residual ASXL1 allele in all 20q11 deletion cases containing the ASXL1 gene was intact. In three PMF cases, more than one distinct ASXL1 mutation was identified, and cloning experiments on two of those cases indicated that the lesions were biallelic. Using banked samples, we observed the acquisition of an ASXL1 lesion over time, and established that ASXL1 lesions detected in 2 post ET-MF cases were also detected at low levels in the ET phase of the MPN. Genotype/Phenotype Correlations: ASXL1 deletions and mutations were prevalent in de novo PMF (37%), post PV-PMF (20%) post ET-PMF (62%) and in PMF/AML (33%). ASXL1 mutations did not associate with chemotherapy exposure as the prevalence of hydroxyurea use was similar in patients with and without mutations, and ASXL1 –mutation positive cases were present in patients who had never received any form of chemotherapy. There was no dependence upon JAK2 status as the presence of ASXL1 mutations were identified in JAK2 V617F-negative cases (9/26); JAK2 V617F-heterozygous cases (10/26); and JAK2 V617F-homozygous cases (7/26). Based on results of SNP-A, patients with ASXL1 mutations were equally as likely to have uniparental disomy (involving 9p or other regions) and loss/gain abnormalities (>1MB) compared to those without ASXL1 mutations. There were no differences in sex, age, or disease duration between PMF patients with and without ASXL1 mutations. In the ASXL1-mutant group, there was a trend toward a lower median white blood cell count (8 vs. 12.5 k/cu mm; p=0.3) and hemoglobin (9.7 vs. 11 g/dl; p=0.3) compared to ASXL1-wild-type patients. Furthermore, those PMF patients with ASXL1 mutations were significantly more likely to have received anemia-directed therapy (transfusion, erythropoietin, immunomodulating agents, steroids) compared to those without mutations (15/26 (58%) vs. 11/39 (23%); p=0.02). Post ET-MF patients comprised 31% (8/26) of ASXL1-mutant cases, compared to only 10% (4/39) ASXL1- wild-type cases (p=0.03). However, the presence of an ASXL1 mutation did not associate with an accelerated transition rate from ET to MF; among the 12 post ET-MF cases in the cohort, the median time of transition from ET to MF was 15.5 years in those with ASXL1 mutations compared to 7 years in those with ASXL1 wild-type status (p=0.02). Conclusion: Disruption of the ASXL1 gene occurs in 40% of PMF cases. The association of ASXL1 lesions, due to either mutation or deletion, suggests that ASXL1 haplo-insufficiency is associated with a PMF phenotype in the context of other known and unknown lesions, and that disruption of ASXL1 function may directly contribute to the pathophysiology and clinical complications of primary and secondary myelofibrosis. These data support the concepts that cooperative lesions in addition to JAK2 V617F are critical in generating PMF, that PMF is molecularly more complex than either PV or ET, and that the transition of PV or ET to PMF is associated with the acquisition of genomic lesions, such as ASXL1, that are present in PMF at large. Disclosures: No relevant conflicts of interest to declare.

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Correlations Between TET2 Mutation and Clinicohematologic Parameters in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v120.21.1455.1455 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1455-1455

Author(s):

Jung Sook Ha ◽

Jae Hee Lee ◽

Sung Gyun Park ◽

Nam Hee Ryoo ◽

Dong Suk Jeon ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Jak2 V617f ◽

Putative Tumor Suppressor Gene ◽

Allele Burden ◽

Frame Shift ◽

Mutational Status ◽

Tet2 Mutation ◽

Disease Specific

Abstract Abstract 1455 Background: Since the acquired somatic mutation, JAK2 V617F, was discovered as a first molecular marker of myeloproliferative neoplasms (MPN), and it has been detected variably in each MPN subtypes. However, JAK2 V617F does not found in all of MPN cases and not necessarily specific to a particular clinicpathologic entity. Recently, mutation of the putative tumor suppressor gene, Ten-Eleven-Translocation-2(TET2), has been identified in MPN patients. However, the frequency of TET2 mutation or its relationship with JAK2 V617F mutation or pathologic function in MPN has not been concluded, yet. The aim of our study was to evaluate the frequency of TET2 in MPN patients, and whether there is any correlation of TET2 mutation with JAK2V617F mutation or the clinicohematologic parameters. Materials and Methods: Total 99 adult MPN patients (18 PV, 62 ET, 11 PMF and 8 MPN unclassified) whose bone marrow cells had been stored from 2007 to 2010 at point of first diagnosis were included in this study. Hematological diagnoses and subtyping were reconfirmed according to the 2008 WHO classification and clinicohematologic datas were collected from patient records. Direct sequencing for TET2(exon3–11) and JAK2 (exons 12 and 14) were performed using an ABI 3730XL DNA analyzer. The JAK2V617F allele burdens were determined by pyrosequencing for samples available and MPL was analyzed by allele-specific PCR. Results: The overall TET2 mutational frequency was 12.1%, and disease-specific mutational frequencies were 22.2% in PV, 9.7% in ET and 18.2% in PMF. The found mutations included 11 mutations, 7 frame-shift (p.Lys95AsnfsX18, p.Gln967AsnfsX40, p.Lys1022GlufsX4, p.Asp1314MetfsX49, p.Gln1534AlafsX43, p.Tyr1618LeufsX4, p.Leu1609GlufsX45), 1 nonsense (p.Gly1735X), 1 missense (Q599R) and 2 splicing mutations (c.3409+1G>T, c.4044+2insT). Those mutations most frequently involved exon 3(four mutations) and exon 11(four mutaions), and rarely intron 3, intron 8 and exon 7. None of the mutations were associated with a karyotypically apparent 4q24 rearrangement. All patients were also screened for JAK2 V617F, and the overall JAK2 V617F positive rate was 68%(94.4% in PV, 69.4% in ET, 45.5% in PMF and 37.5% in MPN, unclassified). All TET2 mutations occurred in JAK2 V617F positive cases. JAK2 exon12 mutation was not found in all patients. MPL W515L was found in one ET patient who also carried JAK2V617F, but not TET2 mutation. Information on JAK2 V617F allele burden was available in 78 patients. Considering all 99 patients, the patient age, hematologic indexes (leukocyte count, neutrophil fraction, lymphocyte fraction, monocyte fraction, Hb, Hct and platelet count), the frequency of organomegaly, marrow fibrosis or thrombotic/hemorrhagic complications were not different according to carrying TET2 mutation. However, TET2 mutation was more frequently found in JAK2 V617F carriers than non-carriers (P=0.008), but JAK2 V617F allele burden did not correlated with the presence of mutant TET2. When analysis was performed for each PV, ET, and PMF (no TET2 mutation in MPN-unclassifiable patients), correlation between TET2 and JAK2 V617F mutational status was not found in each subtypes (P=0.078 in PV, P=0.099 in ET and P=0.182 in PMF). However, the JAK2 V617F allele burden was significantly higher in PMF harboring TET2 mutation than PMF patients did not (88.0 ± 4.3% vs 19.1 ± 28.7%, P=0.034). In statistical analysis for the correlations of clinicohematologic parameters with TET2 mutation in each PV, ET and PMF patients, only a few statistically significant results were identified. The presence of TET2 mutation was correlated with high Hct in PMF (47.4 ± 5.4 vs 25.5 ± 6.2, P=0.037), and TET2 positive ET patients showed relatively higher frequency of organomegaly compared to ET patients without TET2 mutation (50% vs 19.6%, P=0.018). Conclusions: The overall and disease-specific frequencies of TET2 mutation in our study are similar with previous studies, and frame-shift mutation is the most frequent mutation type. There is no specific relationship between JAK2 V617F and TET2 mutation occurrence, but TET2 mutant PMF has higher JAK2 V617F allele burden than non-mutant. TET2 mutation is also associated with a higher Hct in PMF and higher frequency of organomegaly in ET. Larger scale studies involving more MPN patients are needed. Disclosures: No relevant conflicts of interest to declare.

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Loss of Wild-Type Jak2 Allele Enhances Myeloid Cell Expansion and Accelerates Myelofibrosis in Jak2V617F Knock-in Mice

Blood ◽

10.1182/blood.v120.21.809.809 ◽

2012 ◽

Vol 120 (21) ◽

pp. 809-809

Author(s):

Hajime Akada ◽

Saeko Akada ◽

Dongqing Yan ◽

Robert Hutchison ◽

Golam Mohi

Keyword(s):

Polycythemia Vera ◽

Cell Expansion ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Philadelphia Chromosome ◽

Myeloid Cell ◽

Negative Regulator ◽

Jak2v617f Mutation ◽

Common Mutation ◽

Wild Type

Abstract Abstract 809 The activating JAK2V617F mutation is the most common mutation found in Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs), which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Although a majority of MPN patients carry heterozygous JAK2V617F mutation, loss of heterozygosity (LOH) on chromosome 9p involving JAK2 has been observed in ∼30% of patients with MPNs particularly in PV and PMF. JAK2V617F homozygosity through 9pLOH has been linked to more severe MPN phenotype. However, the contribution of 9pLOH in the pathogenesis of MPNs remains unclear. To investigate the role of wild-type JAK2 in MPNs induced by JAK2V617F, we have utilized conditional Jak2 knock-out and Jak2V617F knock-in alleles and generated heterozygous, hemizygous and homozygous Jak2V617F mice. Whereas heterozygous Jak2V617F expression results in a polycythemia vera-like disease in mice, loss of wild-type Jak2 allele in hemizygous or homozygous Jak2V617F mice results in a significantly greater increase in reticulocytes, white blood cells, neutrophils and platelets in the peripheral blood and larger spleen size. We also have found that hemizygous or homozygous Jak2V617F expression significantly increased megakaryocyte-erythroid progenitors in the bone marrow and spleens and marked infiltration of neutrophils in the liver compared with heterozygous Jak2V617F. More importantly, hemizygous or homozygous Jak2V617F mice show accelerated myelofibrosis compared with heterozygous Jak2V617F-expressing mice. Thus, loss of wild type Jak2 allele increases myeloid cell expansion and enhances the severity of the MPN. Together, these results suggest that wild-type Jak2 serves as a negative regulator of MPN induced by Jak2V617F. Disclosures: No relevant conflicts of interest to declare.

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Latent Myeloproliferative Neoplasm May Underlie Retinal Vein Occlusion In Older Patients

Blood ◽

10.1182/blood.v122.21.5242.5242 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5242-5242

Author(s):

Katerina Zoi ◽

Christine Zoi ◽

Andreas Giannopoulos ◽

Argyri Gialeraki ◽

Kassiani Giannaki ◽

...

Keyword(s):

Retinal Vein Occlusion ◽

Older Patients ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Significant Risk ◽

Jak2v617f Mutation ◽

Thrombotic Complication ◽

Allele Burden ◽

Vein Occlusion ◽

Increased Risk

Abstract Background Myeloproliferative neoplasms (MPNs) have been associated with a high incidence of thrombosis and bleeding episodes, which significantly contribute to disease-related morbidity and mortality. Clinical data indicate an association of the JAK2V617F mutation, seen in nearly all polycythemia vera (PV) cases and almost 60% of those with essential thrombocythemia (ET) and myelofibrosis (MF). The mutation is also seen in 37% of patients with in splachnic vein thrombosis (SVT) and its presence was associated with an increased risk for SVT. However, the prevalence of JAK2V617 seems to be low in patients with other thromboembolic events in unusual sites such as cerebral sinus, upper limb deep venous thrombosis (DVT). Additionally, activating mutations of MPL gene, seen in 3% of ET and 5% of MF patients, are considered as a significant risk factor for microvessel disturbances and have been associated with an increased risk of arterial thrombosis. Retinal vein occlusion (RVO) is a thrombotic complication in an uncommon site that may result in sight threatening disease. In this study we investigated the prevalence of JAK2V617F and MPLW515L/K mutations in a prospectively assembled cohort of patients with RVO, hypothesizing that some cases may be associated with an underlying undiagnosed MPN. Patients and Methods We studied 52 (23 males and 29 females) consecutive patients with no evidence of an underlying MPN who had been diagnosed with RVO confirmed with fluorangiography from January 2007 to September 2011. The mean age was 70 years (range: 49-85) Twenty eight patients (53.8%) presented with central RVO and 24 patients with branched RVO (46.5%). DNA was extracted from peripheral blood samples by standard procedures. The JAK2V617F mutation was detected using a tetra-primer amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) assay with a sensitivity of 1% and the allele burden was estimated with a semi-quantitative method. MPLW515L/K were detected using allele-specific PCR (AS-PCR) assays with a sensitivity of 1%. Results Overall, MPN associated mutations were detected in 5/52 cases. JAK2V617F was detected in 2/52 cases (3.8%; 95%CI-1.4%-9%), while MPL exon 10 mutations were detected in 3/52 (5.7%; 95%CI-0.6%-12%). The JAK2V617F allele burden in the two positive patients was 45% and 52% respectively. Both patients who carried the JAK2V617F mutation were female. The first patient had been already diagnosed with ET according to the WHO criteria at the time of RVO screening. She was receiving hydroxyurea and aspirin and her platelet count was normal. The second patient who also carried the JAK2V617F mutation had a PLT count of 850.000/μl at the time of screening and was diagnosed with ET within the 3 following months. The patients with MPL mutations presented with normal blood counts. Conclusions Our findings indicate that a latent MPN could underlie RVO even in the absence of conventional diagnostic criteria. Our results represent the first report that MPL mutations could underlie RVO cases and suggest that routine screening of RVO cases for MPN mutations may be useful, especially in older patients. Disclosures: No relevant conflicts of interest to declare.

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Screening for MPL mutations in Essential Thrombocythemia and Primary Myelofibrosis: Normal Mpl Expression and Absence of Constitutive STAT3 and STAT5 Activation in MPLW515L-Positive Platelets.

Blood ◽

10.1182/blood.v114.22.3910.3910 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3910-3910

Author(s):

Laura Korin ◽

Ana C Glembotsky ◽

Paola R Lev ◽

Carlos D Chazarreta ◽

Rosana F Marta ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Low Frequency ◽

Primary Myelofibrosis ◽

Receptor Activation ◽

Experimental Models ◽

Platelet Surface ◽

Specific Pcr ◽

Exon 10

Abstract Abstract 3910 Poster Board III-846 The identification of exon 10 MPL mutations has begun to unravel the pathogenesis of JAK2V617F-negative essential thrombocythemia (ET) and primary myelofibrosis (PMF). Overall frequency of MPL mutations ranges from 1 to 4% for ET and 5 to 11% for PMF patients. Overexpression of these mutant alleles in cell lines and animal models lead to constitutive receptor activation, activation of downstream signaling pathways and hypersensitivity to thrombopoietin (TPO). However, the precise functional effects of these mutations in signaling and TPO-response in patient samples have not been investigated. Decreased Mpl expression is a molecular hallmark of myeloproliferative neoplasms (MPN). This defect is more frequent in patients positive for JAK2V617F, suggesting a biologic relationship between Mpl expression and the underlying molecular pathogenesis. The pattern of Mpl expression in patients with exon 10 MPL mutations has not been explored. The aim of this study was to analyze the frequency of MPLW515L, MPLW515K and MPLS505N mutations in a cohort of patients with ET and PMF and to determine whether MPLW515L leads to impaired Mpl expression, constitutive STAT3 and STAT5 activation and enhanced response to thrombopoietin (TPO) in patient samples. One in one hundred (1%) patients with ET and 1 in 11 with PMF were positive for MPLW515L by allele-specific PCR and sequencing in platelet and/or leukocyte samples, while none harboured the MPLW515K and MPLS505N mutations; both MPL515L-positive patients were JAK2V617F-negative. Platelet surface Mpl expression in the MPLW515L-positive ET patient by flow cytometry did not differ from a normal control, Mpl/isotype ratio was 3.6 vs 3.2, respectively, and normal total Mpl content was found by Western blot, Mpl/B3 integrin ratio was 99% of controls (n=5), while plasma TPO levels were mildly elevated by ELISA, 45.8 pg/mL vs 0 (0-32) pg/mL in controls (n=20). MPL transcripts by real-time RT-PCR in platelets from both MPLW515L-positive patients were similar to values found for this ET cohort (n=20), which did not differ significantly from normal controls (n=10), MPL/GAPDH ratio was 0.25 and 0.26, for MPL-positive patients, 0.24 (0.12-0.97) for MPL-negative patients, and 0.39 (0.21-0.78) for controls, p= 0.1. Constitutive STAT3 and STAT5 phosphorylation was not detected by immunoblotting and phosphorylation in response to increasing concentrations of TPO did not differ from controls. The low frequency of MPL mutations in this cohort is in agreement with previous studies, highlighting the need for identifying additional molecular defects in JAK2V617F-negative patients. The finding of normal Mpl levels in MPLW515L-positive platelets indicates this mutation does not lead to dysregulated Mpl expression, as frequently shown for MPN patients. Therefore, although impaired Mpl expression can arise from a molecular mechanism different from JAK2V617F, this phenotypic abnormality seems not to be linked to MPLW515L. The lack of spontaneous STAT3 and STAT5 activation and the normal response to TPO is unexpected as MPLW515L leads to constitutive receptor activation and hypersensitivity to TPO in experimental models. Disclosures: No relevant conflicts of interest to declare.

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46/1 Haplotype Permits to Follow JAK2 Homologous Recombination: Modeling JAK2V617F clonal Architecture in PV Patients

Blood ◽

10.1182/blood.v120.21.1757.1757 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1757-1757

Author(s):

Salma Hasan ◽

Jean Pierre Le Couedic ◽

Fabrizia Favale ◽

Barbara Monte-Mor ◽

Catherine Lacout ◽

...

Keyword(s):

Homologous Recombination ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Clonal Structure ◽

Hematopoietic Stem ◽

Clonal Architecture ◽

Specific Pcr ◽

Allele Specific ◽

Proliferative Advantage

Abstract Abstract 1757 Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC) disorders characterized by excess proliferation of one or several myeloid lineages. More than 95% polycythemia vera (PV) and 50–60% essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients harbor a somatic 1849 G>T mutation in JAK2 gene. Moreover about 30% of PV patients are homozygous for this mutation due to a loss of heterozygosity after a mitotic homologous recombination (HR). Among 92 haplotypes of chromosome 9p, 46/1 haplotype is strongly associated with the cis-aquisition of JAK2V617F mutation. The purpose of this study was to estimate the clonal frequency of WT, JAK2V617F/+ and JAK2V617F/V617F in progenitors compartments. Here, we have modeled the JAK2V617F clonal architecture in 9 PV patients heterozygous for the 46/1 haplotype by using the level of JAK2 and the 46/1 haplotype as a marker to follow HR. First we measured the global JAK2V617F and 46/1 allele burden in CD34+ cells either by allele-specific PCR or by Ion Torrent sequencing in order to calculate the expected WT, JAK2V617F/+ and JAK2V617F/V617F clones. Next, we compared the results with the experimental clonal frequency of WT, JAK2V617F/+ and JAK2V617F/V617F cells in individual colonies derived from the CD34+CD38+ compartment. In majority of patients, the observed values corresponded to the expected values suggesting that JAK2 46/1 haplotype can be used to estimate JAK2V617F clonal structure in PV patients. In three JAK2 46/1 heterozygous hemochromatosis patients used as controls, no JAK2 46/1 homozygous clone was observed showing that 46/1 haplotype itself was not responsible for HR. Furthermore, we have studied the proliferative advantage of the mutated clones in patients. No proliferative advantage of JAK2V617F clone has been observed in between CD34+CD38− and CD34+CD38+ progenitors stages whereas strong amplification of JAK2V617F clone was found in terminally differentiated polynuclear neutrophils (PNN). Moreover, during evolution of MPN in one patient, we observed an amplification of the JAK2V617F/V617F clone in both the CD34+CD38− and CD34+CD38+cell compartments suggesting acquisition of a proliferative advantage of the homozygous clone over time. This simple modeling could help to understand the effect of treatments on the JAK2V617F clonal structure without working at the unicellular level. Disclosures: No relevant conflicts of interest to declare.

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