Elevated Leukocyte Alkaline Phosphatase Scores Induced by Jak2 V617F Mutation

Abstract Leukocyte alkaline phosphatase (LAP) enzymatic activity is a marker of the last stages of myeloid differentiation. The level of LAP is quantitated as the LAP score. Estimation of the LAP score has been useful for distinguishing chronic myelogenous leukemia (CML) from BCR-ABL–negative chronic myeloproliferative disorders (MPDs) and neutrophilic reactions in severe infections. CML patients usually have a low LAP score, whereas elevated LAP scores are seen in patients with polycythemia vera (PV), primary myelofibrosis (PMF), and leukocytosis caused by infections. An acquired Jak2 V617F mutation is seen in approximately 95% of patients with PV and in about 50% of patients with essential thrombocythemia or PMF. It has been shown that Jak2 V617F mutation induced constitutive activation of the JAK-STAT signaling pathway. We speculated that an elevated LAP score might be caused due to activation of JAK-STAT signaling through a Jak2 V617F mutation, and conducted this study to address this question. We analyzed the LAP scores in Jak2 V617F-positive and -negative MPD patients. Jak2 V617F-positive MPD patients had higher LAP scores than Jak2 V617F-negative patients. Moreover, patients carrying homozygous mutations had higher LAP scores than patients with heterozygous mutations. AG490, the Jak2 inhibitor, was shown to significantly decrease the LAP expression in neutrophils of Jak2 V617F-positive patients. We lentivirally transfected the acute promyelocytic leukemia cell line NB4 with the Jak2 V617F mutation and wild-type Jak2 V617F. The expression level of Jak2 was not significantly different between the Jak2 V617F mutation and wild-type Jak2 V617F. We then examined the LAP scores of transfected NB4 cells after these cells were differentiated by all-trans retinoic acid and granulocyte colony stimulating factor. It was observed that the Jak2 V617F mutation and not the wild-type Jak2 induced elevated LAP scores. Furthermore, we showed that Jak2 followed the MAP kinase pathway and not the PI3 kinase pathway, as a downstream signaling pathway to elevate the LAP scores using MEK 1/2 (U0126) and PI3 kinase (LY294002) inhibitors. In conclusion, we obtained direct evidence that Jak2 V617F mutation induces elevated LAP scores via the MAP kinase pathway.

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Accuracy of leukocyte alkaline phosphatase score to predict JAK2 V617F mutation

Haematologica ◽

10.3324/haematol.10991 ◽

2007 ◽

Vol 92 (5) ◽

pp. 704-705 ◽

Cited By ~ 5

Author(s):

A. L Basquiera ◽

F. Fassetta ◽

N. Soria ◽

J. M. Barral ◽

B. Ricchi ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Leukocyte Alkaline Phosphatase ◽

V617f Mutation

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JAK2 (V617F) Mutation Status and Neutrophil Apoptotic Resistance in Myelofibrosis with Myeloid Metaplasia (MMM): Correlation and Potential Identification through Phosphorylation Status of STAT3.

Blood ◽

10.1182/blood.v106.11.3503.3503 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3503-3503

Author(s):

Ruben A. Mesa ◽

Ayalew Tefferi ◽

Heather Powell ◽

Terra Lasho ◽

David Loegering ◽

...

Keyword(s):

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Neutrophil Apoptosis ◽

Wild Type ◽

Jak2 Mutation ◽

Myeloid Metaplasia ◽

Mutation Status ◽

Stat3 Phosphorylation ◽

V617f Mutation ◽

Myelofibrosis With Myeloid Metaplasia

Abstract Background: We have previously described a resistance to the normal process of apoptosis in neutrophils of patients with myelofibrosis with myeloid metaplasia (MMM) (Blood2003;102:11). Most recently, an activating mutation of JAK2 (V617F) has been described in approximately half of the patients with MMM as well as in variable proportion of patients with other myeloproliferative disorders (MPD). In the current study, we investigated the correlation between JAK2 V617F mutation status and neutrophil apoptosis in MMM. Methods: Neutrophils were isolated by density centrifugation from patients with MMM, other MPDs, and normal controls and assessed for apoptosis at baseline and after 24 hours in culture (IMDM with 20% sterilized fetal calf serum to simulate spontaneous apoptosis). Apoptosis was quantified using three-color flow cytometry using CD45 (to confirm leukocyte presence), annexin V (AN) (marker of apoptosis; detects aberrant externalization of phosphatidylserine during apoptosis), and propidium iodide (PI) (marker of dead cells). Mutation analysis for JAK2 V617F was performed in DNA derived from the isolated neutrophils using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Apoptotic rates after 24 hours in culture were correlated between patients and controls for both JAK2 mutation status and clinical parameters. Immunoblotting was performed on a subset of patients for correlation of JAK2 mutation status and downstream phosphorylation of the JAK2 target, STAT3, which transcriptionally activates several antiapoptotic genes. Results: Spontaneous neutrophil apoptosis was significantly decreased in MMM patients (n=50; median % apoptotic cells at 41%) compared to both healthy volunteers (n=9; 66%) and patients with other MPD (n=11; 53%) (p=0.002). Resistance to apoptosis in MMM correlated with both anemia (p=0.01) and the presence of the JAK2 V617F mutation (p=0.01). Furthermore, the specific abnormality was more pronounced in patients with homozygous JAK2 V617F; median % apoptotic cells of 47% for patients with wild-type allele (n=22) vs. 39% for heterozygotes (n=23) vs. 22% for homozygotes (n=5; p=0.008). The JAK2 mutation status did not appear dependent on other peripheral blood or clinical features. Neutrophils from 14 MMM patients were assessed simultaneously for both JAK2 mutation and STAT3 phosphorylation status by immunoblotting. Strong expression of phosphorylation of STAT3 was seen in all 3 homozygotes and 4 of 5 heterozygotes, but only 1 of 6 with wild-type allele (p=0.026). Conclusions: Impaired neutrophil apoptosis in patients with MMM correlates with the functional presence of JAK2 V617F in an allele-dose dependent manner and STAT3 phosphorylation. The current observation supports a pathogenetic role for the specific mutation in sustaining clonal myeloproliferation.

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Detection of the JAK2 V617F Mutation by Real Time PCR in Granulocytes and Platelets of ET Patients.

Blood ◽

10.1182/blood.v108.11.4877.4877 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4877-4877

Author(s):

Beatriz Bellosillo ◽

Eva Gimeno ◽

Raquel Longaron ◽

Lourdes Florensa ◽

Antonio Salar ◽

...

Keyword(s):

Real Time ◽

Direct Sequencing ◽

Specific Treatment ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Wild Type ◽

Rt Pcr ◽

Jak2 Mutation ◽

Allele Specific ◽

V617f Mutation

Abstract Introduction. The JAK2 V617F mutation has been detected in 23%–57% of ET patients by direct sequencing or allele-specific (AS) PCR. It remains unknown, however, if the mutation detected in the granulocyte population, may be equally detected in platelets from these patients. Objective. To compare the detection of the JAK2V617F mutation in granulocytes and platelets from ET patients by real time AS RT-PCR. Patients and methods. Platelets and granulocytes from 50 ET patients from a single institution were studied. Patients were diagnosed according to the WHO criteria. At the time when JAK2 mutation was analyzed 16/50 patients were receiving platelet-lowering therapy ± ASA, 14/50 patients only received ASA and 20/50 received no specific treatment. JAK2 mutation was analyzed by real-time AS RT-PCR with probes specific for the mutated and the wild type form. Results. The V617F JAK2 mutation was detected in 18 out of 50 patients in both granulocytes and platelets by real time AS RT-PCR, and was negative in both cell populations in the remaining 32 patients. In the V617F JAK2 positive cases, the mean Ct(V617FJAK2)/Ct(wild type JAK2) ratio was 1.074±0.062 for granulocytes and 1.038±0.039 for platelets (p=0.048). These values corresponded to a 17.79 ±7.4% of mutated population when granulocytes were analyzed, whereas, a significantly higher percentage of mutated population was observed, 23.45±7.78 %, when platelets were analyzed (p=0.032). Conclusions. The results of V617FJAK2 mutation detection by AS RT-PCR were the same in granulocytes and platelets (either positive or negative). The percentage of clonal population detected in ET patients was significantly higher in platelets than in granulocytes.

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Ratio of Mutant JAK2-V617F to Wild Type Jak2 Determines the MPD Phenotypes in Transgenic Mice.

Blood ◽

10.1182/blood.v110.11.674.674 ◽

2007 ◽

Vol 110 (11) ◽

pp. 674-674

Author(s):

Ralph Tiedt ◽

Hui Hao-Shen ◽

Marta A. Sobas ◽

Renate Looser ◽

Stephan Dirnhofer ◽

...

Keyword(s):

Mouse Model ◽

Transgenic Mice ◽

Bone Marrow Cells ◽

Jak2 V617f ◽

Artificial Chromosome ◽

Cre Recombinase ◽

Jak2 V617f Mutation ◽

Wild Type ◽

Retroviral Transduction ◽

V617f Mutation

Abstract The reason why the JAK2-V617F mutation is associated with several phenotypic manifestations of human myeloproliferative disorders (MPD), i.e. polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), is currently unknown. We established an inducible transgenic mouse model for MPD using a bacterial artificial chromosome (BAC) containing the human JAK2-V617F gene under the control of the JAK2 promoter. The sequences encoding the kinase domain were placed in the inverse orientation and flanked with antiparallel loxP sites to make the construct inducible by Cre-recombinase. A transgenic strain (FF1) containing 9 copies of the JAK2-V617F transgene was analyzed in detail. In this strain, Cre activity can lead to activation and/or excision of the multiple transgene copies. Depending on the number of actively rearranged transgene copies, we observed graded levels of expression of the JAK2-V617F mRNA and different MPD phenotypes. Crossing FF1 mice with transgenic mice expressing Cre-recombinase under the control of the hematopoiesis specific Vav promoter (VavCre) led to reduction of the FF1 copy number and low levels of JAK2-V617F expression (approximately 40% of endogenous wild type Jak2). These FF1;VavCre mice developed a phenotype resembling ET with strongly elevated platelet counts and moderate neutrophilia (Figure 1A). In contrast, induction of the JAK2-V617F transgene with the interferon-inducible MxCre resulted in less excision and higher JAK2-V617F transgene expression (approximately equal to wild type Jak2). These MxCre;FF1 mice displayed a PV phenotype with increased hemoglobin, thrombocytosis and neutrophilia (Figure 1B). The highest expression levels of JAK2-V617F were achieved by retroviral transduction (approximately 300% of wild type Jak2). Transplantation of these bone marrow cells into irradiated recipients caused a PV-like phenotype without thrombocytosis. Thus, the phenotype correlated with the ratio of mutant to wild type JAK2 mRNA. In patients with MPD, we found a similar correlation between the ratio of mutant to wild type JAK2 mRNA and the ET, PV and PMF phenotypes. In contrast to our transgenic mice, which display graded levels of JAK2-V617F with wild type JAK2 being present in every cell, each individual blood cell from patients with MPD can only be homozygous or heterozygous for the mutation, or normal. Therefore, the molecular mechanism determining the phenotype in humans may be more complex than in our mouse model and appears to be linked to the transition of the JAK2-V617F mutation to homozygosity. Figure Figure

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Homologous Recombination of Wild TYPE JAK2, A NOVEL EARLY STEP In the DEVELOPMENT of Myeloproliferative Neoplasm

Blood ◽

10.1182/blood.v118.21.2805.2805 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2805-2805

Author(s):

Mathias Vilaine ◽

Damla Olcaydu ◽

Ashot Harutyunyan ◽

Jonathan Bergeman ◽

Tiab Mourad ◽

...

Keyword(s):

Homologous Recombination ◽

Snp Array ◽

Myeloproliferative Neoplasm ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Wild Type ◽

Jak2 Mutation ◽

Chromosome 9P ◽

V617f Mutation ◽

Array Analyses

Abstract Abstract 2805 Background: Adequate expression and function of Jak2 in hematopoietic progenitors is critical for normal myelopoiesis. The JAK2 46/1 (GGCC) haplotype, a congenital particularity, predisposes to myeloproliferative neoplasm (MPN) both independently and through mutation of the JAK2 gene. The JAK2 V617F mutation and acquired homozygous status for JAK2 V617F are frequent in MPN. JAK2 V617F homozygosity is currently explained acquisition of the JAK2 V617F mutation followed by mitotic homologous recombination (HR) of JAK2 occurred between wild-type and mutant alleles, leading to uniparental disomy (UPD) of chromosome 9p (9pUPD). Here we report the cases of 2 PV patients (Na1061 and Na1253) with acquired homozygous status for the JAK2 46/1 haplotype yet their granulocytes carried <20% JAK2 V617F. Aim: To determine whether HR of JAK2 can precede the V617F mutation in MPN. Methods: Granulocyte DNA and CD3+ lymphocyte DNA were examined in parallel with qPCR assays specific for the wild type and 46/1 haplotypes using rs12343867, a JAK2 intron 14 marker, as well as 4 other single nucleotide polymorphisms (SNP) on chromosome 9p. 9pUPD clonality and length were determined using SNP array studies. Results: For both patients, lymphocytes were heterozygous for the 46/1 haplotype, confirming that granulocyte 46/1 homozygosity was acquired. Direct sequencing of the JAK2 and GNE genes and SNP array analyses revealed homologous recombination of part of the JAK2 gene (exons 6–19, patient Na1061) and of the complete 46/1 JAK2 haplotype (patient Na1253). Furthermore, for both patients, full length sequencing of JAK2 cDNA revealed no additional mutation. In both cases, HR of wild-type JAK2 was associated with growth advantage and high expression of recombined JAK2. For both patients, further SNP array analyses revealed partial 9pUPD concerning <30% cells, which correlated with %JAK2 V617F and was consistent with 9pUPD having occurred after JAK2 V617F (Figure 1). The distortion of SNP allelic differences was higher at the telomeric end than in the centromeric region of chr. 9p. This indicated 2 distinct partial 9pUPDs for Na1061 and 1 partial 9pUPD for Na1253. Conclusion: Homologous recombination involving wild type JAK2 can precede JAK2 mutation and 9pUPD in MPN. Thus multiple paths and diverse alterations of the JAK2 gene can lead to MPN in individuals carrying the JAK2 GGCC haplotype. We propose a new model with JAK2 HR as early event, followed or not by JAK2 mutation, or/and JAK2 mutation(s) facilitating subsequent recombination resulting in 9pUPD and JAK2 V617F homozygosity. Disclosures: No relevant conflicts of interest to declare.

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Response to high osmotic conditions and elevated temperature in Saccharomyces cerevisiae is controlled by intracellular glycerol and involves coordinate activity of MAP kinase pathways

Microbiology ◽

10.1099/mic.0.26110-0 ◽

2003 ◽

Vol 149 (5) ◽

pp. 1193-1204 ◽

Cited By ~ 73

Author(s):

Iwona Wojda ◽

Rebeca Alonso-Monge ◽

Jan-Paul Bebelman ◽

Willem H. Mager ◽

Marco Siderius

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Osmotic Stress ◽

Map Kinase ◽

Growth Temperature ◽

Wild Type ◽

Hog Pathway ◽

Map Kinase Pathway ◽

Kinase Pathway ◽

Intracellular Glycerol

In the yeast Saccharomyces cerevisiae, response to an increase in external osmolarity is mediated by the HOG (high osmolarity glycerol) MAP kinase pathway. HOG pathway mutant strains display osmosensitive phenotypes. Recently evidence has been obtained that the osmosensitivity of HOG pathway mutants is reduced during growth at elevated temperature (37 °C). A notable exception is the ste11ssk2ssk22 mutant, which displays hypersensitivity to osmotic stress at 37 °C. This paper reports that overexpression of FPS1 or GPD1 (encoding the glycerol transport facilitator and glycerol-3-phosphate dehydrogenase, respectively, and both affecting intracellular glycerol levels) reduces the hypersensitivity to osmotic stress of ste11ssk2ssk22 at 37 °C. Although in this particular HOG pathway mutant a correlation between suppression of the phenotype and glycerol content could be demonstrated, the absolute level of intracellular glycerol per se does not determine whether a strain is osmosensitive or not. Rather, evidence was obtained that the glycerol level may have an indirect effect, viz. by influencing signalling through the PKC (protein kinase C) MAP kinase pathway, which plays an important role in maintenance of cellular integrity. In order to validate the data obtained with a HOG pathway mutant strain for wild-type yeast cells, MAP kinase signalling under different growth conditions was examined in wild-type strains. PKC pathway signalling, which is manifest at elevated growth temperature by phosphorylation of MAP kinase Mpk1p, is rapidly lost when cells are shifted to high external osmolarity conditions. Expression of bck1-20 or overexpression of WSC3 in wild-type cells resulted in restoration of PKC signalling. Both PKC and HOG signalling, cell wall phenotypes and high osmotic stress responses in wild-type cells were found to be influenced by the growth temperature. The data taken together indicate the intricate interdependence of growth temperature, intracellular glycerol, cell wall structure and MAP kinase signalling in the hyperosmotic stress response of yeast.

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Incidence and Clinical Profile of JAK2 V617F Mutation in Myelofibrosis with Myeloid Metaplasia.

Blood ◽

10.1182/blood.v106.11.256.256 ◽

2005 ◽

Vol 106 (11) ◽

pp. 256-256

Author(s):

Giovanni Barosi ◽

Monia Marchetti ◽

Margherita Massa ◽

Vittorio Rosti ◽

Alessandro Vannucchi ◽

...

Keyword(s):

Tyrosine Kinase ◽

Severity Score ◽

Jak2 V617f ◽

Mutant Gene ◽

Jak2 V617f Mutation ◽

Homozygous Mutation ◽

Wild Type ◽

Myeloid Metaplasia ◽

V617f Mutation ◽

Myelofibrosis With Myeloid Metaplasia

Abstract An aberrant tyrosine kinase signalling due to non-receptor tyrosine kinase JAK2 V617F mutation has been highlighted in the pathogenesis of polycythemia vera (PV). In myelofibrosis with myeloid metaplasia (MMM) this mutation has been reported to be present in approximately 50% of the patients and its pathogenetic role is not elucidated. Here we report the results of JAK2 V617F mutation in a retrospective analysis of blood samples from 170 patients with MMM. Search for JAK2 mutation was performed by an allele specific PCR from DNA purified from granulocytes. To evaluate whether the mutation was carried in the homozygous or heterozygous state, digestion of PCR products with BsaXI restriction enzyme was performed. The overall frequency of JAK2 V617F mutation was 60% and homozygosity for the mutation was found in 39.2% of mutant samples. Disease duration was similar in JAK2 mutated and wild type patients. Patients who harboured an homozygous mutation had an higher myeloproliferative severity score (that indexed leukocytosis, thrombocytosis and splenomegaly) than patients who had a wild type or heterozygous genotype. In post-PV MMM, the mutant gene was present in 22/22 (100%) of patients, and the frequency of homozygosity was 59% of the mutated cases. In post-ET MMM (n=13), the mutant gene was present in 46% of the patients, and the frequency of homozygosity was 16% of the mutant samples. In idiopathic MMM (n=135), the incidence of the mutational state was 54.8%, with 35.1% of homozygote mutation. Patients who had received a diagnosis of prefibrotic myelofibrosis (WHO classification) had an incidence of the mutation significantly lower than patients who were diagnosed in the fibrotic stage (6/18, 33% vs 68/112, 60.7%; P=0.001). By considering only patients not receiving cytoreductive or disease modifying agents, patients who had an heterozygote mutation, had a mean Hb value higher than wild type patients (9.4 g/dL vs. 11.4 g/dL; P=0.004). Moreover, patients who had an homozygote mutation had the myeloproliferative severity score higher than both heterozygote and wild type patients (2.26 vs 1.59, P=0.008, and 2.26 vs.1.52, P=0.001, respectively). We conclude that JAK2 V617F mutation is significantly represented in MMM patients. It is a necessary event in the transformation from PV to MMM while not in the transformation from ET to MMM. Patients who harboured an heterozygote state maintained higher Hb values than patients with a wild type genotype, while the homozygote mutation was associated with leukocytosis, thrombocytosis and splenomegaly.

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Novel Link between PI 3′ Kinase/p38 Map Kinase Pathway in Dasatinib (BMS354825) Mediated Effect in BCR-ABL Expressing Cells.

Blood ◽

10.1182/blood.v110.11.4538.4538 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4538-4538

Author(s):

Crystal Lumby ◽

VeerPal Singh ◽

Shrisha Reddy ◽

Sue Sivess-Franks ◽

Jonathan Dowell ◽

...

Keyword(s):

Map Kinase ◽

Cell Lines ◽

P38 Map Kinase ◽

Pi3 Kinase ◽

Wild Type ◽

Inhibitory Effects ◽

Growth Inhibitory ◽

Map Kinase Pathway ◽

Pi3 Kinase Pathway ◽

Kinase Pathway

Abstract Background: Dasatinib (BMS354825), a dual Src/Abl tyrosine kinase inhibitor, exhibits potent antileukemic effects in vitro and in vivo. Despite the well-established role of BMS354825 in the treatment of imatinib-resistant chronic myelogenous leukemia (CML), the molecular mechanisms that result in generation of antileukemic responses remain unknown. Methods: BCR/ABL (wild type and those carrying mutations: E255K, H396P, Y253F, M351T and T315I) expressing murine BAF3 cell lines were exposed to varying concentrations of BMS354825 for variable times to evaluate for effect on phosphorylation/activation of p38 MAP Kinase and PI3′ Kinase pathway in presence or absence of pharmacologic inhibitors of p38 MAP Kinase and mTOR respectively. Results: In the present study we provide evidence that BMS354825 induces phosphorylation of the p38 MAP Kinase, and activation of its kinase domain, in BCR-ABL expressing cell lines except for those carrying T315I mutation. We also identify the kinases MapKapK-2 which is upregulated in response to BMS354825 as shown by increased phosphorylation of hsp27. Importantly, pharmacological inhibition of p38 MAP Kinase by SB203580 reverses the growth inhibitory effects of BMS354825 on primary leukemic CFU-GM progenitors from patients with CML (see figure). Moreover, SB203580 leads to impairment of the BMS354825 mediated antiproliferative effects in both wild type and mutated CML lines except for those carrying T315I. On the other hand, BMS354825 leads to dephosphorylation of p70S6 Kinase and its downstream effector pathway including down regulation of ribosomal S6. We also report that the pharmacological inhibition of mTOR by Rapamycin augments the growth inhibitory effects of BMS354825 on primary leukemic CFU-GM progenitors from CML patients (see figure). Furthermore, pharmacologic inhibition of p38 MAP Kinase by SB203580 led to reversal of the BMS354825 mediated dephosphorylation of p70 S6 Kinase demonstrating that it maybe downstream of p38 MAP Kinase activation. Altogether, our data establish that activation of the p38 MAP Kinase signaling cascade plays an important role in the generation of the effects of BMS354825 on BCR-ABL expressing cells. Conclusion: We have identified a novel crosstalk mechanism between the p38 MAPK and the PI3′ Kinase pathway which is unique to the effect of BMS354825 in CML. p38 MAP Kinase pathway may play an important role in developing of resistance to BMS354825 in CML. mTOR inhibition may augment effect of BMS354825 in CML and its role in combination with BMS354825 should be explored in resistant disease. Figure Figure

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The Relationship Between Wild Type and Mutant-Positive Platelets in Essential Thrombocythemia Patients with a JAK2 V617F Mutation

Blood ◽

10.1182/blood.v112.11.3728.3728 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3728-3728

Author(s):

Jonathan R Lambert ◽

Rosemary Gale ◽

David C. Linch

Keyword(s):

Platelet Count ◽

Essential Thrombocythemia ◽

Jak2 V617f ◽

Absolute Number ◽

Jak2 V617f Mutation ◽

Secondary Process ◽

Wild Type ◽

Significant Difference ◽

V617f Mutation ◽

The Absolute

Abstract The myeloproliferative condition essential thrombocythemia (ET) is characterized by a persistent thrombocytosis in the absence of a recognizable cause. Approximately 50% of patients carry the acquired mutation JAK2 V617F, but it is unclear whether this is the initiating event in the overproduction of platelets or a secondary process arising in a situation where the thrombopoietic drive is already increased. In vitro studies have shown that mutant-positive erythroid cells have a proliferative advantage compared to wild-type (WT) cells. However, in JAK2 V617F-positive ET patients, the mutation is only found in a proportion of neutrophils, with the mutant level remaining stable over many years, and the JAK2 WT neutrophils are polyclonal by X-chromosome inactivation analysis. This may not be true of platelets as expansion of the mutant-positive cells may be restricted to the megakaryocytic lineage. We therefore quantified the mutant level in neutrophils and platelets purified from 10 JAK2 V617F-positive ET patients prior to the initiation of cytoreductive therapy using PCR with a fluorescently-labeled reverse primer and a mismatch forward primer that allowed discrimination between WT and JAK2 V617F alleles following AflIII digestion. There was no significant difference in the mutant levels determined using neutrophil DNA and RNA (median 15% [range, 11%–27%] and 21% [12%–31%] respectively). Mutant levels in platelet RNA were significantly higher than those in neutrophil RNA (median 27% [range, 20%–39%] versus 21% [12%–31%] respectively; P = 0.002), but still indicated that the JAK2 mutant was present in only a subpopulation of platelets. Assuming that all cells were heterozygous for the mutation, the data indicate that a median of only 54% (range 40%–78%) of the platelets were mutant-positive. We then calculated the absolute number of JAK2 WT and mutant-positive platelets for each patient from the quantified proportion of mutant alleles in platelet RNA and the total platelet count at the time of testing. The absolute number of JAK2 mutant-positive platelets in the patients varied between 263 and 798 × 109/L, and strongly correlated with the percentage of JAK2 mutant alleles (r2 = 0.81, P = 0.0004). The WT platelet count varied between 225 and 426 × 109/L, and there was a significant negative correlation between the absolute number of WT and mutant-positive platelets (r2 = 0.70, P = 0.002). However, when the absolute number of WT platelets was plotted against the total platelet count, there was no relationship between them (r2 = 0.003, P = 0.87). These data suggest that the negative feedback from the total platelet mass on normal (JAK2 WT) thrombopoiesis was incomplete; in no case was the WT platelet count below the lower limit of normal. This may relate to the observation that in ET, levels of thrombopoietin, the lineage-specific cytokine which drives platelet production, are often normal or even increased, unlike the situation in polycythemia vera where erythropoietin levels are reduced and normal red cell production is suppressed. Furthermore, extrapolation of the data from the WT platelet counts and JAK2 V617F mutant levels raises the possibility that when the mutation was first acquired and mutant levels were very low, the WT platelet count was at the upper limit of normality or elevated. This suggests that the JAK2 V617F mutation could have arisen on a background of increased thrombopoiesis, and was not the initiating event in the development of the disorder.

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Peptide Nucleic Acid (PNA)-Clamping Competitive PCR: A New Tool for the High Sensitive Detection of JAK2V617F Mutation in Myeloproliferative Neoplasms (MPNs)

Blood ◽

10.1182/blood.v118.21.5168.5168 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5168-5168

Author(s):

Ferdinando Frigeri ◽

Raffaele Di Francia ◽

Luigi Petraccone ◽

Concetta Giancola ◽

Annunziata Cummaro ◽

...

Keyword(s):

Genomic Dna ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Energetic Cost ◽

Wild Type ◽

Competitive Pcr ◽

Dna Duplex ◽

Pcr Assay ◽

V617f Mutation

Abstract Abstract 5168 Background: Myeloproliferative neoplasms (MPNs) are a group of diseases characterized by clonal expansion of single or multiple lineages of myeloid subset (i.e. granulocytic, erythroid, megakaryocytic and mast cell). Since 2008, the WHO included the detection of JAK2 mutations (the common V617F and the less common exon 12 mutations), into the diagnostic criteria of MPNs. The specific pathogenic implication of JAK2 mutations in MPNs is still under investigation. Preliminary data emerging from the treatment of Idiopathic Myelofibrosis patients with JAK2 inhibitors have shown only clinically significant benefits (improvement of splenomegaly and constitutional symptoms) but not a clear evidence of disease-modifying activity. Several techniques (e.g. ASO-PCR, ARMS-PCR, Direct sequencing, HRM, DHPLC, etc) have been used to detect the JAK2 V617F mutation, but all of them were low sensitive and time-consuming. We developed a PNA-clamping competitive PCR assay able to detect JAK2 V617F mutation with a very high level of sensitivity and specificity. Methods: In order to promote the selective amplification of the JAK2 V617F mutant allele, a specific PNA oligonucleotide, full matching with the wild-type (wt) sequence of the portion of JAK2 gene containing the V617F mutation, was designed to compete with the reverse primer used in the PCR assay. It was experimentally demonstrated that a PNA concentration of 6 μmol/L occurred for the complete clamping of 100 ng of wt genomic DNA used as template for the PCR reaction. The sensitivity of the assay was determined by a serial dilution (100% through 0.01%) of genomic DNA containing JAK2 V617F mutation, obtained from HEL cell line, with wt DNA obtained from healthy donors. The specificity of PCR products was assessed by sequencing in both forward and reverse directions. The thermal dissociation profile of PNA/DNA and DNA/DNA duplexes was studied by monitoring the Enthalpy as function of the temperature (range 20–100°C), with an heating/cooling rate of 1°C/min. Results: PNA-clamping competitive PCR was able to detect JAK2 V617F mutation with a sensitivity of 0.01%. Thermodynamic studies clearly showed that the melting temperature (Tm) of fully matched PNA/DNA duplex is always higher than Tm of the corresponding DNA/DNA duplex. The enthalpy values for the hybrid PNA/DNA are always greater than the corresponding DNA/DNA duplex and a single mismatch has an energetic cost higher in a PNA/DNA than in DNA/DNA duplex. This energetic cost is more evident in the melting temperature with a ΔTm of 16°C and 7°C for the PNA/DNA and DNA/DNA duplex, respectively. In the PCR assay the PNA complementary to the JAK2 wild type sequence strongly compete with the primer resulting in a complete knock out of the wild type allele. In a blind screening of samples obtained from 308 MPN patients, PNA clamping competitive PCR was able to detect JAK2 V617F mutation in 61.4% cases of Polycythemia Vera, 54.4% of Essential Thrombocythemia, 57.9% of Idiopathic Myelofibrosis and in 21.4% of Ph negative-MPNs, respectively. In 40 unselected patients, PNA clamping PCR results were confirmed by other techniques such as ASO-PCR, ARMS-PCR and direct sequencing. Conclusions: The PNA-clamping competitive PCR assay could be used as convenient, high-sensitive and reliable diagnostic test for detection of JAK2 V617F mutation both on genomic DNA and cDNA samples. As suggested from the thermodynamic studies, a similar technique could be developed for the detection of any known single point mutation, widely contributing to the improvement of molecular diagnostic tests usable in clinical practice. Disclosures: No relevant conflicts of interest to declare.

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