JAK2V617F Mutation In Hematologic Malignancies In Childhood and Myeloproliferative Disorders In Adulthood

Abstract Introduction Mutations in JAK2 have been implicated in polycythemia vera (PV), essential thrombocythemia (ES), primary myelofibrosis (PMF) as wll as other myeloproliferative disorders. Aim In this study we aimed to investigate the frequency of JAK2V617F mutation on 216 patients with hematologic malignancies in childhood and 176 patients with myeloproliferative disorders in adulthood. Materials and method Patient group consist of 164 ALL and 52 AML patients in childhood and 79 PV, 51 ES, 22 chronic myeloid leukemia patients (CML) and 24 PMF patients in adulthood. These patients followed by Cukurova University, Departments of Pediatric and Adult Hematology, are included in this study. Blood samples were collected in these patients group and DNA was isolated using high pure template preparation kit (Roche) and stored -80oC. Gene mutations were studied using TMB (TıbMolBiol) LightMix Kit JAK2V617F genomic and analyzed by Light Cycler 2.0 Roche Diagnostic, GmBh, Germany in both groups. Findings JAK2V617F mutation was found 1 of 164 ALL patients (0,6%), 0 of 52 AML patients (0%) in childhood. Nevertheless, JAK2V617F mutation was also found 71 of 79 PV patients (89,8%), 22 of 51 ET patients (43,1%), 1 of 22 CML patients (4,5%) and 15 of 24 PMF patients (62,5%) in adulthood. Result As a result we found high frequency of JAK2V617F mutation in PV patients than the other myeloproliferative disorders. JAK2V617F mutation was significantly high in myeloproliferative disorders in adulthood comparing with childhood acute leukemias (p<0,01). Also, this mutation was significantly high in PV patients comparing with the other myeloproliferative disorders (P<0,05). We found statistically significance between genotype and allels distribution of JAK2V617F mutation (p<0.001) and determined that T-allels may be risk of disease in terms of allel distrubition (p<0.001). Thsi study is one of the widest series to investigation of JAK2V617F mutation frequency especially in childhood hematologic malignancies and we showed that this mutation is almost all 0% in this age group. On the other hand, we showed that JAK2V617F mutation with using realtime PCR test is very useful test for diagnosing PV and ET desase. Disclosures: No relevant conflicts of interest to declare.

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Analysis of the JAK2 Gene cDNA Full-Length In One Chinese Familial Myeloproliferative Disorders

Blood ◽

10.1182/blood.v116.21.5056.5056 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5056-5056

Author(s):

Ru Feng ◽

Lixia Hao ◽

Yongmin Zhang ◽

Yongqiang Wei ◽

Fen Huang ◽

...

Keyword(s):

Family Members ◽

Gene Mutations ◽

Myeloproliferative Disorders ◽

Full Length ◽

The Other ◽

Jak2v617f Mutation ◽

Chinese Family ◽

Heterozygous Mutation ◽

Homozygous Mutation ◽

The Family

Abstract Abstract 5056 Introduction: JAK2V617F point mutation have been confirmed to be one of the major molecular mechanism of BCR/ABL negative myeloproliferative disorders(MPD). Besides, some other gene mutations such as JAK2 exon12, MPL W515L/K, c-mpl and EPOR have extended the scope of the research in this field. Most of the MPD patients are sporadic and there are seldom reports in Chinese familial MPD. 2008 ASH metting we have reported in a Chinese family of MPD's findings, the two brothers in our hospital diagnosis for MPD (one is a PV, another is ET), then we investigated the 15 members of the family. We discovered that there were three male members carried the JAK2V617F mutation in this family, including the two MPD patients and their father, which affected in two generations. All the family members were confirmed as BCR/ABL, MPL W515L/K, c-mpl, and EPOR negative. Subsequently, in order to understand the existence of family members in addition to the gene JAK2 V617F mutation, the existence of JAK2 gene mutations in other parts of the? if other mutations in existence and the high incidence of family members of MPD? We focus on the cDNA full-length of JAK2 gene to provide some theory basis on the pathogenesis in MPD. Methods: A total of 15 family members were enrolled in our study, including 2 brothers of MPD patients (the older one was thrombocythemia (ET), and another is polycythemia vera (PV)) and the other members in the same family. The mRNA of mononuclear cells from peripheral blood sample was extracted according to the manufacturer's instruction (TAKARA). RT-PCR and DNA sequencing have been used to analyze the cDNA full-length of the JAK2 gene. Results: All of the samples can be analyzed for JAK2 cDNA full-length. 3 members carried the JAK2V617F mutation (1849G®T) in this family, including the two MPD patients and their father. And the older brother was homozygous mutation and the other two were heterozygous mutation. All of the 15 samples were JAK2 exon12 gene mutation negative. 2 persons who were the male ET patient's children had a heterozygous mutation (380G®A) in JAK2 exon 3, caused a glycine-to-asparticacid substitution at position 127. Besides, 13 persons had 489C®T mutation in exon 4 and 14 persons had 2490G→A mutation in exon 17 in this family, But they were both same-sense mutation. Conclusion: It is necessary to do routine analysis of blood and other related inspection for MPD patient's family members, so as to make diagnosis earlier. However, we are not sure that the sequencing results are unique to all the familial MPD and need to be confirmed by more cases. We still do not determine the current discovery point mutations have biological significance, still need to be further explored. Disclosures: No relevant conflicts of interest to declare.

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The JAK2V617F Mutation but NOT Platelet Parameters Predict Thrombotic RISK IN Patients with Philadelphia-Negative MYELOPROLIFERATIVE Disorders (MPDs).

Blood ◽

10.1182/blood.v114.22.4984.4984 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4984-4984

Author(s):

Deepti Wijayasinghe ◽

Stephen Hart ◽

Saida Solkar ◽

Christopher j McNamara

Keyword(s):

Platelet Count ◽

Disease Risk ◽

Conflicts Of Interest ◽

Thrombotic Event ◽

Myeloproliferative Disorders ◽

Jak2v617f Mutation ◽

Polycythaemia Vera ◽

Platelet Volume ◽

Thrombotic Risk ◽

The Absolute

Abstract Abstract 4984 Venous and arterial thromboses are important disease complications of both essential thrombocytopenia (ET) and polycythaemia vera (PV), the commonest MPDs. Predicting and managing this risk is not straightforward as the absolute platelet count does not necessarily correlate with thrombotic risk. We retrospectively studied the platelet parameters, median platelet volume and platelet crit, in addition to the absolute platelet count using a Sysmex XE-2100 in patients with MPDs to determine if there was any relationship between these and subsequent thrombotic risk, as well as the JAK2V617F gene status. This has not been done previously. There were 82 patients (25 PV, 33 ET, 13 chronic idiopathic myelofibrosis, 11 MPD unclassifiable) with a median age of 63 (range 37-94). Thromboses occurred in 32/82 patients (26 venous, 6 arterial) after follow up of 12-108 months. The JAK2V617F mutation was found in 77% of all patients. The median platelet count at diagnosis, prior to any therapy, of those patients who subsequently went on to experience a thrombotic event was not significantly different from those who did not: median platelet count 529 v 563 (p=0.06), median platelet volume 8.6 v 8.3 (p=0.36), platelet crit 0.49 v 0.5 (p=0.079). Thrombosis was more likely to occur in those patients with a JAK2V617F mutation; thrombosis occurred in 43 % of JAK2V617F cases compared with 26% of cases without the mutation. The median platelet count in the JAK2V617F unmutated group was higher than in the group with the mutation (620 v 530 p=0.04); other platelet parameters in the two groups did not show any difference based on the presence of the mutation (median platelet volume 8.2 v 8.6 (p= 0.29), median platelet crit 0.52 v 0.5 (p=0.06)). We conclude that platelet parameters, as assessed by a modern automated haematology analyser, do not predict for thrombotic risk in patients with newly diagnoses MPDs. Other factors such as platelet dysfunction, inherited thrombophilia and underlying vascular disease risk factors may be important. Our findings support the notion proposed by others that the presence of the JAK2V617F is a thrombotic risk factor. Disclosures No relevant conflicts of interest to declare.

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The Polymorphisms In LNK Gene Correlated To The Clinical Type Of Myeloproliferative Disorders

Blood ◽

10.1182/blood.v122.21.2836.2836 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2836-2836 ◽

Cited By ~ 1

Author(s):

Yan Chen ◽

Yang Hu ◽

Xueqiang Wu ◽

Fang Fang ◽

Qian Liu ◽

...

Keyword(s):

Conflicts Of Interest ◽

Fusion Gene ◽

Adaptor Protein ◽

Myeloproliferative Disorders ◽

Allele Frequencies ◽

Jak2v617f Mutation ◽

Fisher Exact Test ◽

Missense Mutations ◽

Clinical Type ◽

Normal Controls

Abstract Objective LNK (lymphocyte adaptor protein) is an adapter protein negatively regulating JAK/STAT cell signaling pathway. In this study, we observed the effects of variations in LNK gene on the clinical type of myeloproliferative disorders (MPD). Methods A total of 285 MPD cases were recruited, including essential thrombocythemia (ET) 154 cases, polycythemia vera (PV) 76 cases, idiopathic myelofibrosis (IMF) 19 cases, and chronic myeloid leukemia (CML) 36 cases. Ninety-three healthy individuals were used as normal controls. V617F mutation in JAK2 was searched by allele-specific primers method, RT-PCR was used for the detection of BCR/ABL1 fusion gene, and mutations and variations in exons and their flanking sequences of LNK gene were examined by PCR-sequencing. Count data were analyzed by χ2 or Fisher exact test. Results The genotypes of 4 SNPs (rs3184504, rs111340708, rs78894077and rs7973120) could be identified. Missense mutations of A300V, V402M, and R415H in LNK were found in 8 patients, of whom 3 ET patients had homozygous A300V mutation combined with JAK2V617F mutation. The genotypes and allele types of the 3 SNPs (rs3184504, rs111340708 and rs78894077) correlated to the clinical type of MPD. For rs3184504, the allele frequencies of C/T were 0.5/0.5 in normal controls, 0.117/0.883 in ET, 0.132/0.868 in PV, 0.211/0.789 in IMF, and 0.944/0.056 in CML; T allele (p.262Trp) was significantly higher in ET, IMF and PV (P<0.01), and C allele (p.262Arg) was significantly higher in CML (P<0.01), as compared with the controls. For rs78894077, the allele frequencies of C/T were 0.097/0.903 in normal controls, 0.045/0.955 in ET, 0.086/0.914 in PV, 0.053/0.947 in IMF, and 0.167/0.833 in CML; T allele (p.242Ser) was higher in ET (P<0.05) than in normal controls. For rs111340708 which has 2 alleles of repeated sequences (TGGGG x5/TGGGG x4), the allele frequencies of TGGGG x5/TGGGG x4 were 0.462/0.538 in normal controls, 0.719/0.281 in ET, 0.533/0.467 in PV, 0.789/0.211 in IMF, and 0.639/0.361 in CML; TGGGG x4 allele was significantly lower in ET, IMF and CML than in normal controls (P<0.01). However, the allele frequencies of rs3184504 and rs111340708 were unrelated to the presence of JAK2V617F mutation and BCR/ABL1 fusion gene in MPD patients. Conclusion The polymorphisms of SNPs in LNK gene correlated to the clinical type of MPD. 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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A Philadelphia-negatív krónikus myeloproliferativ kórképek epidemiológiai jellemzői Szabolcs-Szatmár-Bereg megyében. 33 év adatainak elemzése

Orvosi Hetilap ◽

10.1556/650.2017.30701 ◽

2017 ◽

Vol 158 (15) ◽

pp. 572-578

Author(s):

János Jakó ◽

László Szerafin

Keyword(s):

Hematological Malignancies ◽

Primary Myelofibrosis ◽

Myeloproliferative Disorders ◽

Hematologic Malignancies ◽

Female Dominance ◽

Polycythaemia Vera ◽

Essential Thrombocythaemia ◽

Chronic Myeloproliferative Disorders ◽

The Mean ◽

Epidemiologic Features

Abstract: Introduction: In their previous work, the authors reported findings from 30 years on the incidence of hematological malignancies in Szabolcs-Szatmár-Bereg county, Hungary. Until now there are no other studies on this topic available in Hungary. Aim: Detailed analysis of epidemiologic features of patients with Philadelphia-negative chronic myeloproliferative disorders was carried out. Method: During a 33-year period (between January 1, 1983 and December 31, 2015) 4523 adult patients with hematologic malignancies were recorded in the leukaemia/lymphoma registry of Szabolcs-Szatmár-Bereg county. Among them, 255 patients with polycytaemia vera, 102 with primary myelofibrosis, and 331 with essential thrombocytaemia were registered. Results: The incidence of polycythaemia vera and essential thrombocythaemia in Szabolcs-Szatmár-Bereg county showed an increasing tendency, with an overall incidence rate of 1.35 and 1.75/100 000 inhabitants/year, respectively; while the incidence of primary myelofibrosis decreased in the course of years (0.54/100 000 inhabitants/year). In cases of polycythaemia vera and primary myelofibrosis the male:female ratio was found to be equal, however essential thrombocythaemia showed a female dominance. The mean age of patients with polycythaemia vera was 65 (21–95) years, similar to essential thrombocythaemia with 65 (19–85) years, and to primary myelofibrosis with 65.5 (33–84) years. There were only two villages found in this county where the occurrence of patients with Philadelphia-negative chronic myeloproliferative disorders per one thousand inhabitants was significantly higher, than the average (1.22). In every familial cases of these, the manifestation of the disease in the second and the third generations became earlier than in the first genetration. The perceived average degree of the anteposition (anticipation) was found to be 22 years. Conclusion: The epidemiologic features of Philadelphia-negative chronic myeloproliferative disorders in Szabolcs-Szatmár-Bereg county are essentially similar to data published in the literature. Orv. Hetil., 2017, 158(15), 572–578.

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The Analysis of JAK2 and MPL Mutation as Well as JAK2 SNPs in MPN Patients by MassARRAY Assay.

Blood ◽

10.1182/blood.v114.22.4989.4989 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4989-4989

Author(s):

Su-Jiang Zhang ◽

Hongxia Qiu ◽

Jianyong Li

Keyword(s):

Conflicts Of Interest ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Myeloproliferative Disorders ◽

Molecular Pathogenesis ◽

Significant Difference ◽

Positive Rate ◽

Allele Specific ◽

Polymerase Chain ◽

Distribution Frequency

Abstract Abstract 4989 Introduction Recent studies have shown that JAK2 V617F, MPL W515L/K and JAK2 exon 12 mutations underlie the major molecular pathogenesis of myeloproliferative disorders (MPN). Methods To ascertain the real prevalence of these mutations and the influence of genetic susceptibility in Chinese MPN patients, we applied Allele-Specific Polymerase Chain Reaction (AS-PCR), directly sequencing and MassARRAY assay into our study. Results The positive rate of JAK2 V617F in polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) was 82.0%, 36.6% and 51.1% individually. We also found one ET patient, two PMF patients harboring MPL W515L mutation, and three PV patients harboring JAK2 exon 12 mutations. All of these patients were confirmed as JAK2 V617F negative. Moreover, clinical data demonstrated that PV patients with JAK2 exon 12 mutations had higher hemoglobin and lower age as well as WBC than PV patients with JAK2 V617F. In addition, through analysis of 4 polymorphic loci of JAK2 gene, no significant difference of distribution frequency was found among PV, ET and PMF patients. Distribution frequency of haplotype was not found to have significant difference among PV, ET and PMF patients either. Conclusion We conclude that JAK2 V617F is major molecular pathogenesis in Chinese MPN patients. MPL W515L mutation and JAK2 exon 12 mutations can also be found in JAK2 V617F negative MPN patients. Disclosures No relevant conflicts of interest to declare.

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Double-Unit Cord Blood (CB) Transplantation Combined With Haplo-Identical CD34+ Cell-Selected PBSC Results In 100% CB Engraftment With Enhanced Myeloid Recovery

Blood ◽

10.1182/blood.v122.21.298.298 ◽

2013 ◽

Vol 122 (21) ◽

pp. 298-298 ◽

Cited By ~ 4

Author(s):

Juliet N Barker ◽

Doris M Ponce ◽

Parastoo B Dahi ◽

Sean Devlin ◽

Katherine L Evans ◽

...

Keyword(s):

Conflicts Of Interest ◽

Hematologic Malignancies ◽

T Cell Subsets ◽

Acute Leukemias ◽

Platelet Recovery ◽

Neutrophil Recovery ◽

Cd34 Cells ◽

Grade Ii ◽

Initial Hospitalization ◽

The Cost

Abstract Background Double-unit CB transplantation (DCBT) has provided high rates of sustained donor engraftment in patients with hematologic malignancies. However, delayed engraftment is frequent with a median neutrophil & platelet recovery of 25 & 48 days, respectively, in adult DCBT recipients at our center. This delay is associated with increased transplant-related mortality (TRM). It is also associated with prolonged hospitalization with a median discharge time of +42 days (range 25-76) in recent adult myeloablative DCBT recipients. Methods We investigated the combined transplantation of a 4-6/6 HLA-A,-B antigen, -DRB1 allele matched double-unit CB allograft (infused on day 0) with peripheral blood stem cell derived Miltenyi column selected haplo-identical CD34+ cells (haplo-CD34+, infused on day 0 or +1) to speed myeloid recovery. We used DCB grafts to facilitate comparison with historic/concurrent DCB controls transplanted without haplo-CD34+. Results Of 23 protocol eligible patients, 6/23 (26%) underwent DCBT only due to the lack of any suitable haplo-identical donor. Thus, 17 patients [median 39 years (range 16-69), median 78 kg (range 63-133)] were transplanted 9/2012-6/2013 with DCB plus haplo-CD34+ cells for high-risk hematologic malignancies. Diagnoses included 12 acute leukemias & 5 lymphomas. Conditioning was myeloablative with CSA/MMF immune suppression & no ATG. Median infused CB TNC x 107/kg was 2.29 (larger unit, range 1.73-2.95) & 1.82 (smaller unit, range 1.26-2.48). Haplo-identical donors (median 37 years, range 19-71) had a median donor-recipient HLA-match of 5/10 (range 5-7). 15 patients received the targeted infused haplo-CD34+ cell dose of 3 x 106/kg whereas 2 each received haplo-CD34+ cell doses of 1 x 106/kg. The median infused haplo-CD3+ dose was 0.6 x 103/kg (range 0.3-1.6). One patient died on day 14. Of 16 remaining evaluable patients, all (100%) engrafted with a median neutrophil recovery of 13.5 days (range 11-31) in 14 patients who received 3 x 106/kg haplo-CD34+ cells, and 26 and 18 days in the 2 patients who received 1 x 106/kg haplo-CD34+ cells. Platelet recovery ≥ 20 × 109/l has occurred in 12/15 patients (median 27 days, range 18-46) to date. Serial chimerism results demonstrating the contribution of haplo-CD34+ cells & each CB unit to date is shown (Table). While myeloid recovery on day 14 was predominantly haplo-CD34+ cell mediated, one CB unit dominated by day 28 in both neutrophil & T-cell subsets. The median total donor chimerism was 100% the dominant CB unit by day 100. With a median follow-up of survivors of 5 months (range 1-10), to date 9 of 15 evaluable patients have developed grade II-IV aGVHD by day 100 (7 grade II, 1 grade III, 1 grade IV). One patient with refractory leukemia transplanted with disease has relapsed, & 4 have died of TRM (2 organ failure, 1 grade IV aGVHD, 1 CMV infection). Excluding early deaths, of patients who were discharged in the first 100 days (n = 13), the median day of discharge was day +33 (range 21-60). Conclusions Double-unit CBT supplemented by haplo-CD34+ cells is safe. The incidence of neutrophil engraftment is high & the speed of neutrophil recovery is enhanced compared with recent DCBT controls. A shorter time of initial hospitalization (9 days) has offset the cost of the addition of haplo-CD34+ cells. It is intriguing that the dominant CB unit can rapidly reject the haplo-identical donor. This may be facilitated by omission of ATG, and the determinants of the speed of haplo-donor rejection are under investigation. Whether the same results could be achieved with a single CB unit plus haplo-CD34+ cells requires investigation. Addition of haplo-CD34+ cells is also an alternative to expansion, although expansion remains an important strategy to augmenting myeloid recovery given some patients do not have any suitable haplo-identical donors. Disclosures: No relevant conflicts of interest to declare.

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Changing Concepts of Diagnostic Criteria of Myeloproliferative Disorders and the Molecular Etiology and Classification of Myeloproliferative Neoplasms: From Dameshek 1950 to Vainchenker 2005 and Beyond

Acta Haematologica ◽

10.1159/000358580 ◽

2014 ◽

Vol 133 (1) ◽

pp. 36-51 ◽

Cited By ~ 18

Author(s):

Jan Jacques Michiels ◽

Zwi Berneman ◽

Wilfried Schroyens ◽

Hendrik De Raeve

Keyword(s):

Bone Marrow ◽

Polycythemia Vera ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Myeloproliferative Disorders ◽

Jak2v617f Mutation ◽

Wild Type ◽

Mutation Load ◽

Molecular Etiology ◽

Calr Mutations

The Polycythemia Vera Study Group (PVSG) and WHO classifications distinguished the Philadelphia (Ph1) chromosome-positive chronic myeloid leukemia from the Ph1-negative myeloproliferative neoplasms (MPN) essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (MF) or primary megakaryocytic granulocytic myeloproliferation (PMGM). Half of PVSG/WHO-defined ET patients show low serum erythropoietin levels and carry the JAK2V617F mutation, indicating prodromal PV. The positive predictive value of a JAK2V617F PCR test is 95% for the diagnosis of PV, and about 50% for ET and MF. The WHO-defined JAK2V617F-positive ET comprises three ET phenotypes at clinical and bone marrow level when the integrated WHO and European Clinical, Molecular and Pathological (ECMP) criteria are applied: normocellular ET (WHO-ET), hypercellular ET due to increased erythropoiesis (prodromal PV) and hypercellular ET associated with megakaryocytic granulocytic myeloproliferation (EMGM). Four main molecular types of clonal MPN can be distinguished: JAK2V617F-positive ET and PV; JAK2 wild-type ET carrying the MPL515; mutations in the calreticulin (CALR) gene in JAK2/MPL wild-type ET and MF, and a small proportion of JAK2/MPL/CALR wild-type ET and MF patients. The JAK2V617F mutation load is low in heterozygous normocellular WHO-ET. The JAK2V617F mutation load in hetero-/homozygous PV and EMGM is clearly related to MPN disease burden in terms of splenomegaly, constitutional symptoms and fibrosis. The JAK2 wild-type ET carrying the MPL515 mutation is featured by clustered small and giant megakaryocytes with hyperlobulated stag-horn-like nuclei, in a normocellular bone marrow (WHO-ET), and lacks features of PV. JAK2/MPL wild-type, CALR mutated hypercellular ET associated with PMGM is featured by dense clustered large immature dysmorphic megakaryocytes and bulky (cloud-like) hyperchromatic nuclei, which are never seen in WHO-ECMP-defined JAK2V617F mutated ET, EMGM and PV, and neither in JAK2 wild-type ET carrying the MPL515 mutation. Two thirds of JAK2/MPL wild-type ET and MF patients carry one of the CALR mutations as the cause of the third distinct MPN entity. WHO-ECMP criteria are recommended to diagnose, classify and stage the broad spectrum of MPN of various molecular etiologies.

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Conditional Expression of Heterozygous or Homozygous Jak2V617F From Its Endogenous Promoter Induces a Polycythemia Vera-Like Disease.

Blood ◽

10.1182/blood.v114.22.437.437 ◽

2009 ◽

Vol 114 (22) ◽

pp. 437-437

Author(s):

Hajime Akada ◽

Dongqing Yan ◽

Haiying Zou ◽

Robert E. Hutchison ◽

M. Golam Mohi

Keyword(s):

Polycythemia Vera ◽

Conflicts Of Interest ◽

Gene Dosage ◽

Primary Myelofibrosis ◽

Myeloproliferative Disorders ◽

Serum Levels ◽

Severe Form ◽

Downstream Signaling ◽

Conditional Expression

Abstract Abstract 437 A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in a majority of patients with polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). However, the contribution of JAK2V617F in these three clinically distinct myeloproliferative disorders (MPDs) remained unclear. To investigate the actual role of JAK2V617F in the pathogenesis of MPDs, we generated an inducible Jak2V617F knock-in mouse, in which the expression of Jak2V617F is under control of the endogenous Jak2 promoter. Expression of heterozygous Jak2V617F evoked all major features of human PV, which included marked increase in hemoglobin and hematocrit, increased red blood cells, leukocytosis, thrombocytosis, splenomegaly, reduced serum levels of erythropoietin (Epo) and Epo-independent erythroid colonies. Homozygous Jak2V617F expression resulted in a more severe form of PV associated with markedly elevated leukocytosis, neutrophilia and thrombocytosis, and a majority of these mice succumbed due to massive cardiac thrombosis. Activation of Stat5, Akt and Erk was significantly enhanced in erythroblast cells expressing homozygous Jak2V617F compared to heterozygous Jak2V617F, suggesting that the degree of activation of downstream signaling pathways would be affected by the Jak2V617F gene dosage. This may also partly explain the severe phenotype in mice expressing homozygous Jak2V617F. We conclude that heterozygous Jak2V617F is sufficient to cause PV, whereas homozygous Jak2V617F increases the severity of PV disease and the risk of thrombosis. Our results also provide strong evidence that Jak2V617F gene dosage does not play a defining role in determining PV versus ET phenotype in this mouse model of MPD. Disclosures: No relevant conflicts of interest to declare.

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Study of Megakaryopoiesis and Proplatelet Formation In Myeloproliferative Disorders

Blood ◽

10.1182/blood.v116.21.3075.3075 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3075-3075

Author(s):

Stefania Badalucco ◽

Maria Teresa Pugliano ◽

Vittorio Rosti ◽

Marco Cattaneo ◽

Giovanni Barosi ◽

...

Keyword(s):

Conflicts Of Interest ◽

Primary Myelofibrosis ◽

Myeloproliferative Disorders ◽

Molecular Defect ◽

Intrinsic Defects ◽

Alpha Tubulin ◽

Platelet Production ◽

Cytokines And Chemokines ◽

Proplatelet Formation

Abstract Abstract 3075 Introduction. Myeloproliferative neoplsms (MPNs) are clonal disorders that include primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocytemia (ET). Several abnormalities that could explain the myeloproliferation have been reported, and most important is a striking involvement of the megakaryocyte (Mk) lineage, with hyperplasia and dysplasia resulting in an excessive production of several cytokines and chemokines. Whereas molecular defect(s) associated with the development of MPNs have been described, their pathogenesis is not thoroughly elucidated. Hypothesis. The general objective of this research was to study the process of megakaryopoiesis in MPNs in order to understand whether the pathological mechanisms underlying MPNs are intrinsic defects of Mk function or depend on an altered regulation by the bone marrow microenvironment. Patients and Methods. We analyzed in vitro the Mk differentiation and proplatelet formation in 25 PMF patients, 13 ET/PV patients, and 19 healthy controls (HCs). Mks were differentiated from peripheral blood CD34+ or CD45+ cells in the presence of TPO for 14 days. Mature Mks were, then, grown in suspension or plated onto glass coverslips coated with collagen I or fibrinogen for additional 16 hours. Mk differentiation-maturation and proplatelet formation were evaluated by phase contrast and fluorescence microscopy upon cell staining with anti alpha-tubulin and CD41 antibodies. Measurement of Mk diameters was performed on acquired images, at least one hundred Mks were analyzed for each sample. Mk ploidy was evaluated by flow cytometry after staining with propidium iodide. Controls were analyzed in parallel with each patient sample. Results. Mk output was higher in patients with MPNs than HCs. Patients with PMF and PV had the highest Mk output, and in PMF the number of Mks was strictly associated with the V617F JAK2 genotype. PMF Mks presented smaller size and increased nuclear/cytoplasm ratio with respect to the other MPNs and HCs. Further, the measurement of Mk diameters revealed that PMF-derived Mks rarely reached the diameter of 40 μm, while HCs and ET/PV presented larger Mks. Finally, PMF produced decreased numbers of polyploid Mks (>8N) with respect to HCs. In order to explore if defects in Mk development were associated to altered Mk function, we investigated the competence to generate proplatelet by MPN-derived and HC-derived Mks. In HC samples, a 7.5% median of the total Mks formed proplatelets, while a significant defect in extending proplatelets was observed in PMF-derived Mks with respect to other MPNs and HCs. On the contrary, patients with PV or ET showed increased numbers of Mks forming proplatelets with respect to PMF and HCs. No differences were observed between pre-fibrotic and fibrotic PMF, and pre-fibrotic PMF-derived Mks formed a decreased number of proplatelet as compared to both ET/PV and HCs. Most importantly, platelet counts correlated with Mk ability of extending proplatelets of different MPN patients. Finally, we found that the proplatelets extended by patients with PMF presented peculiar morphological features with respect to the normal counterpart and patients with PV and ET. Their more evident alteration was a significantly reduced number of bifurcations in secondary processes despite a normal length of the individual proplatelet shafts, thus indicating a defect in proplatelet branching. On the opposite, ET- and PV-derived proplatelets presented an enormous increase of bifurcations with respect to PMF and HCs. Summary and Conclusions. Mks from patients affected by MPNs presented high proliferative capacity and defects in proplatelet formation. Thus, platelet production in MPNs may not depend only on an increased number of Mks, but also on intrinsic defects in Mk function. Interestingly, our results also showed that Mk of pre-fibrotic and fully fibrotic PMF present similar biological characteristics and different from those of ET, providing a rationale for considering pre-fibrotic as a different entity from ET. In conclusion, this study proposes important new elements in the understanding of the biology of Mk and proplatelet formation in MPNs, and open a new perspective into the understanding of the pathophysiology of defects of platelet production in MPNs. Disclosures: No relevant conflicts of interest to declare.

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