V617F-negative Patients Research Articles

Frequency of JAK2 Exon 12 Mutations in JAK2 Exon 14 V617F-Negative Patients: High Frequency in ET Patients.

The identification of the G to T point (V617F) mutation in exon 14 of the JH2 domain of the Janus kinase 2 gene (JAK2) has recently simplified the diagnosis of many patients affected with a variety of myeloproliferative disorders (MPDs). More recently JAK2 exon 12 mutations have been described in these disorders; although the role of these mutations is as yet unclear. There have been only two investigations which have identified the presence of exon 12 mutations in clinical diagnosis among polycythaemia vera (PV) (Scott et al NEJM 2007, Pardanani et al Leukaemia 2007). A total of 725 patients diagnosed clinically with a variety of MPDs were investigated for both the frequently occurring exon 14 V617F and for the rarer exon 12 mutations.Methods: Peripheral blood samples from all patients were screened for the V617F mutation by gene-scaning on an ABI 3130 fragment analyser. 478 of these patients were further screened for the exon 12 mutant allele by denaturing high performance liquid chromatography (Transgenomic WAVE®) and direct sequencing.Results: Of 725 patients screened for the V617F mutation 204 (28%) were found to be heterozygous (HET) and 30 (4%) homozygously (HM) mutated while 491 (68%) were wildtype (WT). 478 were further tested for the exon 12 mutation and eight were found to carry mutations. Two of the patients had previously described mutations while three novel mutations were described in the remaining six patients. Only one of these was in the PV group while the majority of exon 12 mutations clustered in the essential thrombocythemia (ET) (4/8) and Budd Chiari patients (2/8). All exon 12 mutations were identified at very low level.Conclusions: Combined rapid gene-scanning and WAVE analysis allows for the characterisation of a large cohort of patients diagnosed clinically to have a variety of MPDs. This helps with the correct diagnosis of a frequently difficult group of patients. Detection of exon 12 mutations should be used in patients who are WT for V617F, including patients in the ET and the Budd Chiari syndrome groups. Both V617F and exon 12 mutation analysis tests should be included in primary screening in the routine management of MPD patients.DiagnosisTotalWT for V617F (mutant exon 12)HET for V617F (mutant exon 12)HM for V617F (mutant exon 12)Budd Chiari/PVT6042 (2)16 (0)2 (0)Polycythemia Vera (PV)187112 (1)63 (0)12 (0)Reactive PV2828 (0)0 (none tested)0 (none tested)ET198123 (4)71 (none tested)4 (none tested)MPD12387 (1)29 (none tested)7 (none tested)Inspecified diagnosis12999 (none tested)25 (none tested)5 (none tested)TOTAL725491 (8)204 (0)30 (0)Results of Exon 14 V617F and exon 12 mutation screening

Essential Thrombocythemia in Young Individuals: Frequency and Risk Factors for Vascular Events and Evolution to Myelofibrosis in 126 Patients.

Vascular events and evolution to either myelofibrosis (MF) and acute leukemia (AL) are the main causes of morbidity and mortality in individuals with essential thrombocythemia (ET). However, the frequency of these complications in young ET patients is not well known. The objective of the present study was to assess the frequency of vascular events and the incidence of MF and AL in young patients with ET and to identify the factors associated with the development of such complications. In 126 subjects diagnosed with ET at a median age of 31 years (range: 5–40), overall survival and probability of survival free of either thrombosis, bleeding, MF, AL, and polycythemia vera (PV) were analyzed by the Kaplan-Meier method, followed by the log-rank test. With a median follow-up of eleven years (range: 4–25) three patients have died, being the probability of survival 98% at ten years. A total of 31 thrombotic events were registered in 25 patients; thrombosis-free survival (TFS) was 84% at ten years. Tobacco use was the only factor associated with an increased thrombotic risk, since TFS at 10 years was 72% in smokers versus 90% in non-smokers (p=0.03). Severe hemorrhagic complications were observed in 11 patients, and the estimated probability of bleeding-free survival was 92% at ten years. Evolution to MF was seen in 6 patients, four of whom had never received treatment for ET. MF-free survival was 97% at 10 years, with the risk being higher in patients showing an increased reticulin network in the bone marrow biopsy performed at diagnosis of ET (p=0.005). Transformation to AL was registered in one patient. JAK2 was mutated in 33 out of the 87 assessable patients (38%) and the mutation was associated with higher Hb values at diagnosis (p = 0.001). ET evolved into PV in five patients, being the probability of evolution into PV of 15% in JAK2 V617F positive patients versus 0% in JAK2 V617F negative patients (p=0.01). In conclusion, severe vascular complications are not infrequent in young subjects with ET, whereas transformation to MF or AL is a rare event.

Impact of the JAK2 V617F Mutation on Survival in Patients with Catastrophic Intra-Abdominal Thromboses.

Catastrophic intra-abdominal thrombosis can result from a variety of hypercoaguable states, including the chronic myeloproliferative disorders (CMPD), even though the patient's clinical and laboratory findings may not suggest such a diagnosis. Recently a somatic mutation in the tyrosine kinase, JAK2, has been reported in myeloid cells of 65–97% of patients with polycythemia vera and approximately 23–57% of patients with essential thrombocythemia or chronic idiopathic myelofibrosis, but not in normal individuals. To assess the prevalence and clinical implications of this mutation in the setting of intra-abdominal thrombosis, JAK2V617F genotyping was performed in 42 patients who had suffered catastrophic intra-abdominal thromboses that resulted in visceral transplants, as well as in 20 control subjects. The prevalence of V617F was compared to that of other hypercoaguable states for which molecular testing is routinely performed. The V617F mutation was detected in 7 patients (16.7%), who were not distinguishable from the 35 others on the basis of their peripheral blood counts. JAK2 V617F was not detected in any of the control subjects. Six patients were heterozygous (14.3%) for the factor V Leiden mutation and one patient was heterozygous for prothrombin 20210 G>A, all of whom had unmutated JAK2 genes. Evidence for a lupus anticoagulant was found in 7 patients, including one with the V617F mutation. Six of the 7 patients with the V617F mutation died within 36 months post-transplant from the following causes: an aggressive post-transplant lymphoproliferative disorder, sepsis secondary to bone marrow failure, allograft-liver failure, and three cases of sepsis preceded by mental status changes, possibly due to cerebral vascular disease. The median post-transplant survival of V617F-positive patients was 1.5 years compared to 9.8 years for the V617F-negative patients (ratio: 6.6; 95% CI 6.3–7.0). These results highlight the diagnostic utility of JAK2V617F testing in the setting of abdominal thrombosis and underscore the clinical significance of a positive result. The explanations for major thromboses in JAK2 V617F positive patients with clinically silent CMPD and the significantly shorter survival of the JAK2 V617 positive patients in the setting of visceral transplantion are uncertain. The optimal management of these patients also remains to be determined. [Display omitted] Median Peripheral Blood Counts at PresentationPlatelets (109/L)Hemoglobin (g/dl)Hematocrit (%)Leukocytes (109/L)V617F34212.237.26.3WT23911.433.37.2p-value0.220.590.460.89

HFE C282Y Mutation as a Genetic Modifier Influencing Disease Susceptibility for JAK2 V617F Positive Chronic Myeloproliferative Disease.

Common HFE gene point mutations (C282Y, H63D and S65C) are responsible for hemochromatosis type 1 in homozygous or compound heterozygous states. Although HFE-related hemochromatosis is inherited in a recessive manner, heterozygous carriers were shown to have higher iron parameters (serum iron and ferritin levels, transferrin saturation) without progressive iron overload. Heterozygous HFE carriership has been implicated as a genetic modifier of several diseases, where iron homeostasis play an important role in disease susceptibility or disease progression (porphyria cutanea tarda, myelodyslasia). We investigated 336 unrelated patients with chronic myeloproliferative disease (CMPD) [176 patients with polycythemia vera (PV), 131 patients with essential thrombocythemia (ET) and 29 patients with chronic idiopathic myelofibrosis (CIMF)] and 171 first time blood donors as controls for allele frequencies (AFs) of HFE mutations by Light Cycler allele discrimination method. The presence of the acquired JAK2 V617F mutation in CMPD was investigated by allele specific PCR. Laboratory (hemoglobin, white blood cell and platelet count) and clinical features (sex, age at diagnosis, splenomegaly, presence of thrombotic, myelofibrotic or leukemic transformation) were recorded in the patient group. Compared to controls (4.7 +/− 2.3%), decreased HFE C282Y AF was found in patient group (1.8 +/− 1.0%; p=0.02; OR=0.38 [0.17–0.85]). Decreased HFE C282Y AFs were detected in all patient subgroups according to diagnosis (PV, ET, CIMF) and according to JAK2 V617F mutational status, but the difference remained significant only in the patient group with PV or with JAK2 V617F positive CMPD (PV:1.7 +/− 1.4%; p=0.04; JAK2 V617F positive patients: 1.6+/− 1.1%). H63D and S65C mutational status did not differ significantly between control (12.0+/−3.5% and 1.3+/−1.8%) and patient groups (13.1+/−2.6% and 0.5+/−0.5%). JAK2 V617F frequency was 86.9% (153/176) in PV, 60.3% (79/131) in ET, and 72.4% (21/29) in CIMF. We found significantly elevated hemoglobin levels and WBC values (measured at the time of diagnosis) in the V617F-positive PV (and ET patient groups compared to V617F-negative patients. The incidence of vascular complications (arterial and venous thrombosis or bleeding) was higher in JAK2 V617F CMPD. The age of CMPD onset and the rate of different complications were not altered by HFE mutational status. Our data confirm earlier observations that JAK2 V617F-positive ET shares clinical features (elevated hemoglobin) with PV. We found that HFE C282Y mutation may be associated with a protective role against CMPD (also CMPD associated with JAK2 positivity). Chronic iron deficiency or latent anemia may trigger disease susceptibility for CMPD, and HFE C282Y positivity can play a protective role against chronic iron deficiency.

Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study

An acquired V617F mutation in JAK2 occurs in most patients with polycythaemia vera, but is seen in only half those with essential thrombocythaemia and idiopathic myelofibrosis. We aimed to assess whether patients with the mutation are biologically distinct from those without, and why the same mutation is associated with different disease phenotypes. Two sensitive PCR-based methods were used to assess the JAK2 mutation status of 806 patients with essential thrombocythaemia, including 776 from the Medical Research Council's Primary Thrombocythaemia trial (MRC PT-1) and two other prospective studies. Laboratory and clinical features, response to treatment, and clinical events were compared for V617F-positive and V617F-negative patients with essential thrombocythaemia. Mutation-positive patients had multiple features resembling polycythaemia vera, with significantly increased haemoglobin (mean increase 9.6 g/L, 95% CI 7.6-11.6 g/L; p<0.0001), neutrophil counts (1.1x10(9)/L, 0.7-1.5x10(9)/L; p<0.0001), bone marrow erythropoiesis and granulopoiesis, more venous thromboses, and a higher rate of polycythaemic transformation than those without the mutation. Mutation-positive patients had lower serum erythropoietin (mean decrease 13.8 U/L; 95% CI, 10.8-16.9 U/L; p<0.0001) and ferritin (n=182; median 58 vs 91 mug/L; p=0.01) concentrations than did mutation-negative patients. Mutation-negative patients did, nonetheless, show many clinical and laboratory features that were characteristic of a myeloproliferative disorder. V617F-positive individuals were more sensitive to therapy with hydroxyurea, but not anagrelide, than those without the JAK2 mutation. Our results suggest that JAK2 V617F-positive essential thrombocythaemia and polycythaemia vera form a biological continuum, with the degree of erythrocytosis determined by physiological or genetic modifiers.

Molecular Mimicry in the Chronic Myeloproliferative Disorders: Reciprocity between JAK2 V617F Genotype and Mpl Expression.

Polycythemia vera (PV), idiopathic myelofibrosis (IMF) and essential thrombocytosis (ET) are clonal myeloproliferative disorders (MPD) that share in common increased blood production, megakaryocyte dysplasia, growth factor-independent colony formation and similar cytogenetic defects. Until recently, a dearth of molecular markers hindered analysis of the interrelationship of PV, IMF and ET. Mpl protein expression is decreased in most PV patients and the degree of impairment correlated with disease duration and extent. Impaired Mpl expression was also present in many IMF and ET patients. The recent discovery of the JAK2 V6217F mutation in PV recapitulated this behavior since JAK2 V617F was present in many classical IMF and ET patients. Importantly, JAK2 is an obligate Mpl chaperone required for Mpl surface expression (JBC 280: 27251, 2005). Since JAK2 V617F is constitutively active, we hypothesized that both its presence and gene dosage could influence Mpl expression. Therefore, we examined the concordance of the JAK2 V617F genotype and platelet Mpl protein expression in 78 PV, 13 IMF and 82 ET patients, using allele-specific, real-time PCR screening of granulocyte DNA for JAK2 V617F and quantitative densitometry for platelet Mpl with a cutoff of ≤ 60% for reduced and ≥ 120% for overexpression. JAK2 V617F prevalence was 91% in PV (40% homozygous), 46% in IMF (66% homozygous) and 41% in ET (none homozygous). JAK2 V617F-negative patients (7 PV, 7 IMF and 58 ET) were indistinguishable clinically from JAK2 V617F-positive patients. Mean disease duration was similar for homozygous JAK2 V617F (11 years, range 1–25) and heterozygous JAK2 V617F (8 years, range 1–22) PV patients. Mean disease duration was also similar for JAK2 V617F-positive (7.5 years, range 1–18) and JAK2 V617F-negative (6 years, range 1–22) ET patients. Mean Mpl expression was 31% in PV patients (range 1–227, reduced in 89%), 7% in IMF (range 1–22, reduced in 100%) and 72% in ET (range 1–246, reduced in 52%). Of the JAK2 V617F-positive patients, mean platelet Mpl was 27% in PV (range 1–86%, reduced in 92%), 5% in IMF (range 1–11%, reduced in 100%) and 38% in ET (range 1–127%, reduced in 81%). JAK2 V617F genotype significantly inversely correlated (p<0.004) with platelet Mpl expression: in JAK2 V617F homozygotes, mean platelet Mpl expression was 12% (range 1–44, 100% reduced); in JAK2 V617F heterozygotes, mean Mpl expression was 35% (range 1–127, 86% reduced), while in JAK2 V617F-negative patients, mean Mpl was 82% (range 1–246%, 44% reduced). JAK2 V617F-negative PV and IMF patients had either markedly reduced Mpl (8 patients, mean 6%) or markedly elevated Mpl (2 patients, mean 200%). In ET, JAK2 V617F-positive patients had reduced platelet Mpl (38% of normal, range 1–127%) compared to JAK2 V617F-negative patients (92% of normal, range 1–246%) (p=0.004). However, JAK2 V617F-negative ET patients were heterogeneous; 42% had reduced, 24% normal and 33% elevated platelet Mpl expression. These data indicate that JAK2 V617F expression and gene dosage directly influence platelet Mpl expression in PV, IMF and ET. Less commonly however, impaired Mpl expression, often marked, also occurred in the JAK2 V617F-negative setting, suggesting that other genetic or epigenetic lesions can create the same molecular abnormality. We conclude that Mpl protein expression abnormalities, whether associated with JAK2 V617F or not, provide a common denominator for the MPD phenotype. JAK2 V617F genotype does not necessarily predict MPD phenotype, but the degree of Mpl reduction does.

JAK2 V617F Mutation Identifies a Biologically Distinct Subtype of Essential Thrombocythemia Which Resembles Polycythemia Vera.

An acquired V617F mutation in JAK2 occurs in most patients with polycythemia vera (PV) but only half of those with essential thrombocythemia (ET) and idiopathic myelofibrosis. It is not known whether mutation-bearing patients are biologically distinct from those lacking the mutation, or why the same mutation is associated with different phenotypes. Two sensitive PCR-based methods were used to assess the JAK2 mutation status of 806 patients with ET, including 776 from the MRC PT-1 trial and two other prospective studies. The combined cohort provides a unique resource for studying ET, particularly in view of its large size, centralized review of end-points and comprehensive follow-up. The involvement of a large number of secondary and tertiary centers, together with the inclusion of patients in all risk categories, suggest the results are of general relevance.JAK2 mutation status divided the cohort into two distinct subgroups. Mutation-positive patients (53.4%) displayed multiple features resembling PV, with significantly increased hemoglobin levels (p<0.0001), neutrophil counts (p<0.0001), bone marrow erythropoiesis (p=0.001) and granulopoiesis (p=0.005). They suffered more venous thromboses in the year before diagnosis (p=0.04) and during follow-up (p=0.06), together with a higher incidence of polycythemic transformation (p=0.01). To explore the resemblance between V617F-positive ET and PV further, we analysed factors that might constrain erythropoiesis. Compared to mutation-negative patients with ET, mutation-positive patients had lower serum epo (p<0.0001), lower ferritin (p=0.01), and were more likely to be microcytic (p<0.0001). V617F-positive patients were more sensitive to hydroxyurea, requiring lower doses to control platelet count than V617F-negative patients (p<0.0001), a pattern not seen with anagrelide. Despite lower doses of hydroxyurea, V617F-positive patients had greater reductions in hemoglobin, platelet and white cell counts than V617F-negative patients, with no analogous differences noted between V617F-positive and negative patients randomized to anagrelide (p=0.004, p=0.04, p=0.0003 for platelet count, Hb and WCC). Mutation-negative patients did exhibit many clinical and laboratory features characteristic of a myeloproliferative disorder, including cytogenetic abnormalities, hypercellular bone marrow, abnormal megakaryocyte morphology, PRV1 over-expression, growth of epo-independent erythroid colonies, and a risk of myelofibrotic or leukemic transformation.JAK2 mutation status defines two biologically distinct subgroups of ET with differences in presentation, outcome and response to therapy. Our results suggest a model in which V617F-positive ET and PV form a continuum, with the degree of erythrocytosis determined by physiological or genetic modifiers, including iron stores, epo homeostasis, gender and V617F-homozygosity. This concept has major implications for the classification, diagnosis and management of MPDs.