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1.  The C allele of JAK2 rs4495487 is an additional candidate locus that contributes to myeloproliferative neoplasm predisposition in the Japanese population 
BMC Medical Genetics  2012;13:6.
Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are myeloproliferative neoplasms (MPNs) characterized in most cases by a unique somatic mutation, JAK2 V617F. Recent studies revealed that JAK2 V617F occurs more frequently in a specific JAK2 haplotype, named JAK2 46/1 or GGCC haplotype, which is tagged by rs10974944 (C/G) and/or rs12343867 (T/C). This study examined the impact of single nucleotide polymorphisms (SNPs) of the JAK2 locus on MPNs in a Japanese population.
We sequenced 24 JAK2 SNPs in Japanese patients with PV. We then genotyped 138 MPN patients (33 PV, 96 ET, and 9 PMF) with known JAK2 mutational status and 107 controls for a novel SNP, in addition to two SNPs known to be part of the 46/1 haplotype (rs10974944 and rs12343867). Associations with risk of MPN were estimated by odds ratios and their 95% confidence intervals using logistic regression.
A novel locus, rs4495487 (T/C), with a mutated T allele was significantly associated with PV. Similar to rs10974944 and rs12343867, rs4495487 in the JAK2 locus is significantly associated with JAK2-positive MPN. Based on the results of SNP analysis of the three JAK2 locus, we defined the "GCC genotype" as having at least one minor allele in each SNP (G allele in rs10974944, C allele in rs4495487, and C allele in rs12343867). The GCC genotype was associated with increased risk of both JAK2 V617F-positive and JAK2 V617F-negative MPN. In ET patients, leukocyte count and hemoglobin were significantly associated with JAK2 V617F, rather than the GCC genotype. In contrast, none of the JAK2 V617F-negative ET patients without the GCC genotype had thrombosis, and splenomegaly was frequently seen in this subset of ET patients. PV patients without the GCC genotype were significantly associated with high platelet count.
Our results indicate that the C allele of JAK2 rs4495487, in addition to the 46/1 haplotype, contributes significantly to the occurrence of JAK2 V617F-positive and JAK2 V617F-negative MPNs in the Japanese population. Because lack of the GCC genotype represents a distinct clinical-hematological subset of MPN, analyzing JAK2 SNPs and quantifying JAK2 V617F mutations will provide further insights into the molecular pathogenesis of MPN.
PMCID: PMC3277458  PMID: 22251709
JAK2 V617F; SNP; myeloproliferative neoplasms
2.  Self-Renewal of Single Mouse Hematopoietic Stem Cells Is Reduced by JAK2V617F Without Compromising Progenitor Cell Expansion 
PLoS Biology  2013;11(6):e1001576.
In this study, single cell assays and mathematical modeling demonstrate that a single oncogenic point mutation can negatively affect hematopoietic stem cells while leaving progenitor cell expansion intact.
Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F subclinical clonal expansions and the transition to overt MPNs will illuminate the earliest stages of tumor establishment and subclone competition, fundamentally shifting the way we treat and manage cancers.
Author Summary
Recent descriptions of the existence of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumours. In this study, we focus on understanding the stem cell defect that results from a mutation in the JAK2 tyrosine kinase gene, which is present in the majority of patients with myeloproliferative neoplasms (MPNs), a group of clonal bone marrow diseases that are characterised by the overproduction of mature blood cells and increased frequency of leukaemia development. By using single-cell assays and mathematical modeling, followed by the individual assessment of daughter cells from single HSCs, we identify a distinct cellular mechanism that differentially affects stem cell and progenitor cell expansion. Specifically, we show that this single point mutation can negatively affect HSCs while leaving progenitor cell expansion intact. Characterising the mechanisms that link JAK2 mutations with clonal expansions that eventually lead to development of MPNs will inform our understanding of the earliest stages of tumour establishment and of the competition between subclones of proliferating progenitor/stem cells. These findings have direct relevance to all cancers of a suspected stem cell origin.
PMCID: PMC3672217  PMID: 23750118
3.  Development of a highly sensitive method for detection of JAK2V617F 
Ph- myeloproliferative neoplasms (MPNs) represent a heterogeneous group of chronic diseases characterized by increased expansion of hematopoietic cells of the myeloid lineage. JAK2V617F, an activation mutation form of tyrosine kinase JAK2, is found in the majority of patients with MPNs. Studies have demonstrated that JAK2V617F can cause MPNs, and various methods have been developed to detect JAK2V617F for diagnostic purposes. However, a highly sensitive method is still needed for the earliest possible detection and for disease prevention and treatment.
In the present study, we developed a method dubbed restriction fragment nested allele-specific PCR (RFN-AS-PCR). The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the JAK2V617F mutation site, 2) digestion of the PCR products with restriction enzyme BsaXI which only cleaves the wild type allele, and 3) detection of JAK2V617F by allele-specific PCR with nested primers.
We tested the sensitivity of the method by using purified plasmid DNAs and blood cell DNAs containing known proportions of JAK2V617F. We were able to detect JAK2V617F with a sensitivity of 0.001%. We further analyzed blood cell DNA samples from 105 healthy donors with normal blood cell counts and found three JAK2V617F-positive cases, which would have remained undetected using a less sensitive method.
We have developed a highly sensitive method that will allow for detection of JAK2V617F at a very early stage. This method may have major implications in diagnosis and prevention of MPNs and related diseases.
PMCID: PMC3207960  PMID: 21985400
Tyrosine kinase; myeloproliferative neoplasms; JAK2; mutation; detection; diagnosis
4.  The implication of identifying JAK2V617F in myeloproliferative neoplasms and myelodysplastic syndromes with bone marrow fibrosis 
Journal of Hematopathology  2008;1(2):111-117.
The myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) occasionally demonstrate overlapping morphological features including hypercellularity, mild/nonspecific dysplastic changes and variable bone marrow fibrosis. Thus, when the associated bone marrow fibrosis results in a suboptimal specimen for morphological evaluation, the descriptive diagnosis “fibrotic marrow with features indeterminate for MDS versus MPN” is often applied. The JAK2V617F mutation was recently shown to be frequently identified in MPN, but it is rarely present in other myeloid disorders. However, the diagnostic utility of JAK2V617F screening in hypercellular bone marrow specimens with fibrosis has not been previously investigated. Using a real-time polymerase chain reaction melting-curve assay capable of detecting JAK2V617F in archived fixed materials, we retrospectively studied JAK2V617F in 45 cases with fibrotic hypercellular bone marrow at initial presentation, including 19 cases initially described as “with features indeterminate for MDS versus MPN”. These 19 cases were reclassified into more specific categories of MDS (n = 14) or MPN (n = 5) based on the availability of subsequent clinical data and/or bone marrow examinations. The JAK2V617F allele was identified in 17 out of 18 BCR/ABL gene-negative MPN cases with marrow fibrosis, whereas only wild-type alleles were identified in the remaining non-MPN cases. Importantly, JAK2V617F alleles were seen in all five cases of “with features indeterminate for MDS versus MPN” at initial presentation that were later determined to be MPN, but they were absent in the 14 cases later determined to be MDS. Our results suggest that JAK2V617F allele evaluation can be a useful ancillary test for discriminating MDS from MPN in specimens with bone marrow fibrosis.
PMCID: PMC2713481  PMID: 19669209
Myeloproliferative neoplasm; Myelodysplastic syndrome; Bone marrow fibrosis; JAK2V617F
5.  JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms 
PLoS ONE  2010;5(8):e12165.
The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs); deletion of the entire exon 14 is rarely detected. In our previous study of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by reverse transcription-PCR (RT-PCR) with direct sequencing, complete deletion of exon 14 (Δexon14) constituted <1% of JAK2 mutations. This appears to be an alternative splicing mutation, not detectable with DNA-based testing.
Methodology/Principal Findings
We investigated the possibility that MPN patients may express the JAK2 Δexon14 at low levels (<15% of total transcript) not routinely detectable by RT-PCR with direct sequencing. Using a sensitive RT-PCR–based fluorescent fragment analysis method to quantify JAK2 Δexon14 mRNA expression relative to wild-type, we tested 61 patients with confirmed MPNs, 183 with suspected MPNs (93 V617F-positive, 90 V617F-negative), and 46 healthy control subjects. The Δexon14 variant was detected in 9 of the 61 (15%) confirmed MPN patients, accounting for 3.96% to 33.85% (mean  = 12.04%) of total JAK2 transcript. This variant was also detected in 51 of the 183 patients with suspected MPNs (27%), including 20 of the 93 (22%) with V617F (mean [range] expression  = 5.41% [2.13%–26.22%]) and 31 of the 90 (34%) without V617F (mean [range] expression  = 3.88% [2.08%–12.22%]). Immunoprecipitation studies demonstrated that patients expressing Δexon14 mRNA expressed a corresponding truncated JAK2 protein. The Δexon14 variant was not detected in the 46 control subjects.
These data suggest that expression of the JAK2 Δexon14 splice variant, leading to a truncated JAK2 protein, is common in patients with MPNs. This alternatively spliced transcript appears to be more frequent in MPN patients without V617F mutation, in whom it might contribute to leukemogenesis. This mutation is missed if DNA rather than RNA is used for testing.
PMCID: PMC2921382  PMID: 20730051
6.  A highly specific q-RT-PCR assay to address the relevance of the JAK2WT and JAK2V617F expression levels and control genes in Ph-negative myeloproliferative neoplasms 
Annals of Hematology  2013;93:609-616.
In Ph− myeloproliferative neoplasms, the quantification of the JAK2V617F transcripts may provide some advantages over the DNA allele burden determination. We developed a q-RT-PCR to assess the JAK2WT and JAK2V617F mRNA expression in 105 cases (23 donors, 13 secondary polycythemia, 22 polycythemia vera (PV), 38 essential thrombocythemia (ET), and 9 primary myelofibrosis (PMF)). Compared with the standard allele-specific oligonucleotide (ASO)-PCR technique, our assay showed a 100 % concordance rate detecting the JAK2V617F mutation in 22/22 PV (100 %), 29/38 (76.3 %) ET, and 5/9 (55.5 %) PMF cases, respectively. The sensitivity of the assay was 0.01 %. Comparing DNA and RNA samples, we found that the JAK2V617F mutational ratios were significantly higher at the RNA level both in PV (p = 0.005) and ET (p = 0.001) samples. In PV patients, JAK2WT expression levels positively correlated with the platelets (PLTs) (p = 0.003) whereas a trend to negative correlation was observed with the Hb levels (p = 0.051). JAK2V617F-positive cases showed the lowest JAK2WT and ABL1 mRNA expression levels. In all the samples, the expression pattern of beta-glucoronidase (GUSB) was more homogeneous than that of ABL1 or β2 microglobulin (B2M). Using GUSB as normalizator gene, a significant increase of the JAK2V617F mRNA levels was seen in two ET patients at time of progression to PV. In conclusion, the proposed q-RT-PCR is a sensitive and accurate method to quantify the JAK2 mutational status that can also show clinical correlations suggesting the impact of the residual amount of the JAK2WT allele on the Ph− MPN disease phenotype. Our observations also preclude the use of ABL1 as a housekeeping gene for these neoplasms.
Electronic supplementary material
The online version of this article (doi:10.1007/s00277-013-1920-0) contains supplementary material, which is available to authorized users.
PMCID: PMC3945640  PMID: 24173087
Ph− myeloproliferative neoplasms; JAK2WT level; JAK2V617F level; Housekeeping gene; q-RT-PCR
7.  Single Nucleotide Polymorphism (SNP)-Based Loss of Heterozygosity (LOH) Testing by Real Time PCR in Patients Suspect of Myeloproliferative Disease 
PLoS ONE  2012;7(7):e38362.
During tumor development, loss of heterozygosity (LOH) often occurs. When LOH is preceded by an oncogene activating mutation, the mutant allele may be further potentiated if the wild-type allele is lost or inactivated. In myeloproliferative neoplasms (MPN) somatic acquisition of JAK2V617F may be followed by LOH resulting in loss of the wild type allele. The occurrence of LOH in MPN and other proliferative diseases may lead to a further potentiating the mutant allele and thereby increasing morbidity. A real time PCR based SNP profiling assay was developed and validated for LOH detection of the JAK2 region (JAK2LOH). Blood of a cohort of 12 JAK2V617F-positive patients (n = 6 25–50% and n = 6>50% JAK2V617F) and a cohort of 81 patients suspected of MPN was stored with EDTA and subsequently used for validation. To generate germ-line profiles, non-neoplastic formalin-fixed paraffin-embedded tissue from each patient was analyzed. Results of the SNP assay were compared to those of an established Short Tandem Repeat (STR) assay. Both assays revealed JAK2LOH in 1/6 patients with 25–50% JAK2V617F. In patients with >50% JAK2V617F, JAK2LOH was detected in 6/6 by the SNP assay and 5/6 patients by the STR assay. Of the 81 patients suspected of MPN, 18 patients carried JAK2V617F. Both the SNP and STR assay demonstrated the occurrence of JAK2LOH in 5 of them. In the 63 JAK2V617F-negative patients, no JAK2LOH was observed by SNP and STR analyses. The presented SNP assay reliably detects JAK2LOH and is a fast and easy to perform alternative for STR analyses. We therefore anticipate the SNP approach as a proof of principle for the development of LOH SNP-assays for other clinically relevant LOH loci.
PMCID: PMC3388082  PMID: 22768290
8.  The involvement of Galectins in the modulation of the JAK/STAT pathway in myeloproliferative neoplasia 
In patients with myeloproliferative neoplasia (MPN) the development of fibrosis and increased vessel density correlate with poor prognosis. The JAK2V617F mutation constitutively activates JAK2, which phosphorylates signal transducer activator of transcription (STAT), up-regulating vascular endothelial growth factor (VEGF), which might be responsible for angiogenesis in MPN. Galectins are involved in the development of fibrosis and angiogenesis and might also be involved in activation of the JAK/STAT pathway in MPN.
106 MPN patients, 36 essential thrombocythemia (ET), 25 polycythemia vera (PV) and 45 primary myelofibrosis (PMF), were analyzed for the expression pattern of galectin-1, galectin-3, pSTAT3, pSTAT5 and MVD by immunostaining of bone marrow biopsy sections followed by automated image analysis. The JAK2 mutational status was analysed through real time PCR in blood samples.
The expression of galectin-1 was significantly higher in all MPN patients compared to normal controls. Galectin-3 was expressed more in PV patients. MVD was significantly higher in all MPN patients and correlated with galectin-1 and pSTAT5 expression. pSTAT5 expression showed a trend of higher expression in patients carrying the JAK2V617F mutation as well as in PV patients. PMF patients and all JAK2V617F positive patients showed a significantly higher pSTAT3 expression compared to control and ET patients.
The findings suggest the involvement of galectin-1 in MPN development, regardless of the subtype. Furthermore involvement of galectin-3 in PV development, pSTAT5 in that of PV and JAK2V617F positive patients and angiogenesis, as well as pSTAT3 is involved in the pathogenesis of PMF.
PMCID: PMC3384397  PMID: 22762031
MPN; myeloproliferative neoplasia; galectin; JAK; STAT; angiogenesis; MVD
9.  JAK2 V617F-Dependent Upregulation of PU.1 Expression in the Peripheral Blood of Myeloproliferative Neoplasm Patients 
PLoS ONE  2011;6(7):e22148.
Myeloproliferative neoplasms (MPN) are multiple disease entities characterized by clonal expansion of one or more of the myeloid lineages (i.e. granulocytic, erythroid, megakaryocytic and mast cell). JAK2 mutations, such as the common V617F substitution and the less common exon 12 mutations, are frequently detected in such tumor cells and have been incorporated into the diagnostic criteria published by the World Health Organization since 2008. However, the mechanism by which these mutations contribute to MPN development is poorly understood. We examined gene expression profiles of MPN patients focusing on genes in the JAK–STAT signaling pathway using low-density real-time PCR arrays. We identified the following 2 upregulated genes in MPN patients: a known target of the JAK–STAT axis, SOCS3, and a potentially novel target, SPI1, encoding PU.1. Induction of PU.1 expression by JAK2 V617F in JAK2-wildtype K562 cells and its downregulation by JAK2 siRNA transfection in JAK2 V617F-positive HEL cells supported this possibility. We also found that the ABL1 kinase inhibitor imatinib was very effective in suppressing PU.1 expression in BCR-ABL1-positive K562 cells but not in HEL cells. This suggests that PU.1 expression is regulated by both JAK2 and ABL1. The contribution of the two kinases in driving PU.1 expression was dominant for JAK2 and ABL1 in HEL and K562 cells, respectively. Therefore, PU.1 may be a common transcription factor upregulated in MPN. PU.1 is a transcription factor required for myeloid differentiation and is implicated in erythroid leukemia. Therefore, expression of PU.1 downstream of activated JAK2 may explain why JAK2 mutations are frequently observed in MPN patients.
PMCID: PMC3138766  PMID: 21789226
10.  Effect of NS-018, a selective JAK2V617F inhibitor, in a murine model of myelofibrosis 
Blood Cancer Journal  2014;4(1):e174-.
A single somatic mutation, V617F, in Janus kinase 2 (JAK2) is one of the causes of myeloproliferative neoplasms (MPNs), including primary myelofibrosis, and the JAK2V617F mutant kinase is a therapeutic target in MPN. However, inhibition of wild-type (WT) JAK2 can decrease the erythrocyte or platelet (PLT) count. Our selective JAK2 inhibitor, NS-018, suppressed the growth of Ba/F3 cells harboring JAK2V617F more strongly than that of cells harboring WT JAK2. The 4.3-fold JAK2V617F selectivity of NS-018 is higher than the 1.0- to 2.9-fold selectivity of seven existing JAK2 inhibitors. NS-018 also inhibited erythroid colony formation in JAK2V617F transgenic mice at significantly lower concentrations than in WT mice. In keeping with the above results, in a JAK2V617F bone marrow transplantation mouse model with a myelofibrosis-like disease, NS-018 reduced leukocytosis and splenomegaly, improved bone marrow fibrosis and prolonged survival without decreasing the erythrocyte or PLT count in the peripheral blood. By exploring the X-ray co-crystal structure of NS-018 bound to JAK2, we identified unique hydrogen-bonding interactions between NS-018 and Gly993 as a plausible explanation for its JAK2V617F selectivity. These results suggest that NS-018 will have therapeutic benefit for MPN patients through both its efficacy and its reduced hematologic adverse effects.
PMCID: PMC3913942  PMID: 24413068
JAK2; V617F; myelofibrosis; kinase inhibitor; NS-018
11.  Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms 
Leukemia  2011;26(4):708-715.
The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms (MPNs) and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified 5 different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9 to 33-fold higher EC50 for ruxolitinib compared to native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the ‘gatekeeper’ mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (4-fold increase in EC50). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs.
PMCID: PMC3974504  PMID: 21926964
Myeloid neoplasia; JAK2; drug resistance
12.  Heat shock protein 90 inhibitor is synergistic with JAK2 inhibitor and overcomes resistance to JAK2-TKI in human myeloproliferative neoplasm cells 
We determined the activity of heat shock protein (hsp) 90 inhibitor (HI), and/or JAK2 tyrosine kinase inhibitor (TKI) against JAK2-V617F-expressing cultured mouse (Ba/F3-JAK2-V617F) and human (HEL92.1.7 and UKE1) or primary human CD34+ myeloproliferative neoplasm (MPN) cells.
Experimental Design
Following exposure to the HI AUY922 and/or JAK2-TKI TG101209, the levels of JAK2-V617F, its downstream signaling proteins, as well as apoptosis were determined.
Treatment with AUY922 induced proteasomal degradation and depletion of JAK2-V617F as well as attenuated the signaling proteins downstream of JAK2-V617F, i.e., phospho (p)-STAT5, p-AKT and p-ERK1/2. AUY922 treatment also induced apoptosis of HEL92.1.7, UKE-1 and Ba/F3-hJAK2-V617F cells. Combined treatment with AUY922 and TG101209 caused greater depletion of the signaling proteins than either agent alone, and synergistically induced apoptosis of HEL92.1.7 and UKE-1 cells. Co-treatment with AUY922 and TG101209 also induced significantly more apoptosis of human CD34+ MPN versus normal hematopoietic progenitor cells. As compared to the sensitive controls, JAK2-TKI-resistant HEL/TGR and UKE1/TGR cells exhibited significantly higher IC50 values for JAK2-TKI (p <0.001), which was associated with higher expression of p-JAK2, p-STAT5, p-AKT and Bcl-xL, but reduced levels of BIM. Unlike the sensitive controls, HEL/TGR and UKE/TGR cells were collaterally sensitive to the HIs AUY922 and 17-AAG; accompanied by marked reduction in p-JAK2, p-STAT5, p-AKT and Bcl-xL, with concomitant induction of BIM.
Findings presented here demonstrate that co-treatment with HI and JAK2-TKI exerts synergistic activity against cultured and primary MPN cells. Additionally, treatment with HI may overcome resistance to JAK2-TKI in human MPN cells.
PMCID: PMC3743080  PMID: 21976548
JAK2-V617F; JAK2 inhibitor; hsp90 inhibitors; myelofibrosis
13.  mTOR Inhibitors Alone and in Combination with JAK2 Inhibitors Effectively Inhibit Cells of Myeloproliferative Neoplasms 
PLoS ONE  2013;8(1):e54826.
Dysregulated signaling of the JAK/STAT pathway is a common feature of chronic myeloproliferative neoplasms (MPN), usually associated with JAK2V617F mutation. Recent clinical trials with JAK2 inhibitors showed significant improvements in splenomegaly and constitutional symptoms in patients with myelofibrosis but meaningful molecular responses were not documented. Accordingly, there remains a need for exploring new treatment strategies of MPN. A potential additional target for treatment is represented by the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway that has been found constitutively activated in MPN cells; proof-of-evidence of efficacy of the mTOR inhibitor RAD001 has been obtained recently in a Phase I/II trial in patients with myelofibrosis. The aim of the study was to characterize the effects in vitro of mTOR inhibitors, used alone and in combination with JAK2 inhibitors, against MPN cells.
Mouse and human JAK2V617F mutated cell lines and primary hematopoietic progenitors from MPN patients were challenged with an allosteric (RAD001) and an ATP-competitive (PP242) mTOR inhibitor and two JAK2 inhibitors (AZD1480 and ruxolitinib). mTOR inhibitors effectively reduced proliferation and colony formation of cell lines through a slowed cell division mediated by changes in cell cycle transition to the S-phase. mTOR inhibitors also impaired the proliferation and prevented colony formation from MPN hematopoietic progenitors at doses significantly lower than healthy controls. JAK2 inhibitors produced similar antiproliferative effects in MPN cell lines and primary cells but were more potent inducers of apoptosis, as also supported by differential effects on cyclinD1, PIM1 and BcLxL expression levels. Co-treatment of mTOR inhibitor with JAK2 inhibitor resulted in synergistic activity against the proliferation of JAK2V617F mutated cell lines and significantly reduced erythropoietin-independent colony growth in patients with polycythemia vera.
These findings support mTOR inhibitors as novel potential drugs for the treatment of MPN and advocate for clinical trials exploiting the combination of mTOR and JAK2 inhibitor.
PMCID: PMC3561413  PMID: 23382981
14.  Relevance of JAK2V617F positivity to hematological diseases - survey of samples from a clinical genetics laboratory 
JAK2V617F is found in the majority of patients with Ph- myeloproliferative neoplasms (MPNs) and has become a valuable marker for diagnosis of MPNs. However, it has also been found in many other hematological diseases, and some studies even detected the presence of JAK2V617F in normal blood samples. This casts doubt on the primary role of JAK2V617F in the pathogenesis of MPNs and its diagnostic value.
In the present study, we analyzed JAK2V617F positivity with 232 normal blood samples and 2663 patient blood, bone marrow, and amniotic fluid specimens obtained from a clinical genetics laboratory by using a simple DNA extraction method and a sensitive nested allele-specific PCR strategy.
We found JAK2V617F present in the majority (78%) of MPN patients and in a small fraction (1.8-8.7%) of patients with other specific hematological diseases but not at all in normal healthy donors or patients with non-hematological diseases. We also revealed associations of JAK2V617F with novel as well as known chromosomal abnormalities.
Our study suggests that JAK2V617F positivity is associated with specific hematological malignancies and is an excellent diagnostic marker for MPNs. The data also indicate that the nested allele-specific PCR method provides clinically relevant information and should be conducted for all cases suspected of having MPNs as well as for other related diseases.
PMCID: PMC3032761  PMID: 21235771
15.  JAK2 V617F mutation in myelodysplastic syndrome, myelodysplastic syndrome/myeloproliferative neoplasm, unclassifiable, refractory anemia with ring sideroblasts with thrombocytosis, and acute myeloid leukemia 
The JAK2 V617F mutation has been noted in the cases of polycythemia vera, essential thrombocythemia, and primary myelofibrosis patients. This mutation occurs less frequently in acute myeloid leukemia (AML) and other hematologic diseases, such as myelodysplastic syndrome (MDS); myelodysplatic syndrome/myeloproliferative neoplasm, unclassifiable (MDS/MPN-U); and refractory anemia with ring sideroblasts with thrombocytosis (RARS-T).
Patients diagnosed with hematologic diseases other than MPN who visited Seoul St Mary's Hospital from January 2007 to February 2010 were selected. A total of 43 patients were enrolled in this study: 12 MDS, 9 MDS/MPN-U, 7 RARS-T, and 15 AML patients. The diseases were diagnosed according to the 2008 WHO classification criteria. Data obtained from JAK2 V617F mutation analysis and cytogenetic study as well as complete blood count and clinical data were analyzed.
Of the 43 patients, 6 (13.9%) harbored the JAK2 V617F mutation. The incidence of the JAK2 V617F mutation in each patient group was as follows: 8.3% (1/12), MDS; 22.2% (2/9), MDS/MPN-U; 14.3% (1/7), RARS-T; and 13.3%, (2/15) AML. The platelet count was higher than 450×109/L in 3 of the 6 patients (50%) harboring the JAK2 V617F mutation, and it was in the normal range in the remaining 3 patients. Among the 6 patients, 1 MDS and 1 MDS/MPN-U patients had the 46,XX,del(20)(q11.2) karyotype.
The JAK2 V617F mutation is associated with an increased platelet count in MDS, MDS/MPN-U, RARS-T, and AML patients. Cytogenetic abnormalities of del(20)(q11.2) occurred in 1/3 of patients with the JAK2 V617F mutation but further studies are required to confirm this association.
PMCID: PMC2983014  PMID: 21120162
16.  Advantages of the quenching probe method over other PCR-based methods for detection of the JAK2 V617F mutation 
Oncology Letters  2012;4(2):205-208.
The detection of a V617F mutation (G to T exchange at nucleotide 1,849) in the JAK2 gene is crucial for the diagnosis of myeloproliferative neoplasms (MPN) such as polycythemia vera. Although sequence analysis is the standard method for detection, it is not suitable for clinical examinations due to the requirement of expensive equipment. In this study, we evaluated the efficiencies of four PCR-based methods to detect JAK2 V617F: allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), high-resolution melting analysis (HRM) and the quenching probe method (QP). The HEL cell line, which harbors a homozygous JAK2 V617F mutation, as well as bone marrow samples from 16 MPN patients and normal control samples, were used in this assessment. The sensitivity of the detection limit of all four methods was also examined using samples of HEL cells mixed in a variety of ratios with cells containing wild-type JAK2. The results of all four methods were found to be concordant. AS-PCR was shown to be the most sensitive; however, it produced false positive results. Although PCR-RFLP demonstrated high specificity, it was time consuming. By contrast, results were obtained using HRM and QP in only 2 h. It was easier to recognize the curves derived from the mutant allele obtained using QP. QP is also suitable for the rough estimation of allele burden. JAK2 V617F assays are mainly used for diagnosis at presentation in clinical settings. We therefore conclude that in situations where high sensitivity is not required, QP is the preferable method for the detection of JAK2 V617F. To the best of our knowledge, this is the first report to demonstrate the efficiency of the QP method for the detection of JAK2 V617F using a standard thermal cycler.
PMCID: PMC3402723  PMID: 22844354
janus kinase 2; myeloproliferative neoplasms; allele-specific polymerase chain reaction; PCR-restriction fragment length polymorphism; high-resolution melting analysis; quenching probe
17.  MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia  
PLoS Medicine  2006;3(7):e270.
The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR).
Methods and Findings
DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9–4.0 × 10 12/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis.
Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.
Editors' Summary
Myelofibrosis with myeloid metaplasia (MF) is one of a group of chronic blood disorders, known as chronic myeloproliferative disorders. These disorders sometimes turn into acute leukemia. The main abnormality in myelofibrosis is for the bone marrow to become filled with fibrous (scar) tissue (hence the name myelofibrosis), which stops it from producing normal blood cells efficiently. In addition, the white blood cells that remain are abnormal (that is, metaplastic). The clinical effect of these abnormalities are that patients are anemic (they have low numbers of red cells), are more likely to get infections because of the abnormal white cells which cannot fight infections normally, and may bleed more easily because of a lack of the platelets that help the blood to clot. Scientists who study this disorder believe that the disease starts from just one abnormal cell, which divides to replace all the other cells—that is, all the abnormal cells are part of one clone.
Why Was This Study Done?
In two similar diseases, polycythemia vera (in which the bone marrow produces too many red blood cells) and essential thrombocytosis (in which the bone marrow produces too many platelets), and in some patients with MF, scientists have found genetic changes which seem to trigger these diseases. However, there are some patients with MF in which no abnormal gene has been found. The scientists here wanted to look at other genes to see if they could find any changes that might trigger MF.
What Did the Researchers Do and Find?
They decoded the DNA sequence of three genes that are known to be involved in how blood cells develop for 45 patients with MF. They looked at DNA from white blood cells, and also from normal cheek cells for comparison. They found that in four of the 45 patients the DNA in the bone marrow, but not the cheek, carried a mutation in a gene for the thrombopoietin receptor (also called MPL). This gene is necessary for the cells that make platelets to grow correctly. The mutation was not present in any samples from patients with diseases related to MF, nor in 270 normal samples. The mutation that was identified was at position 515 in the MPL gene sequence, hence the name MPLW515L—the W and the L are the shorthand way of indicating exactly which change occurred. The change meant that the gene became abnormally active. The researchers tested the effect of the abnormal gene by putting it into cells grown in culture in the laboratory; they found that it made the cells grow more than was normal. In addition, when cells with the abnormal gene were put into mice, the mice developed a blood disorder similar to that seen in humans with MF.
What Do These Findings Mean?
It seems likely that the genetic change that has been identified here is responsible for the MF that develops in some patients. The MPL gene is known to be part of a pathway of genes that control how certain blood cells develop. However, it is not yet clear exactly how the genetic change found here causes the blood cells to grow abnormally, or how it causes the other clinical effects of MF. Further work will also need to be done to see if it is possible to develop drugs that can act on this gene mutation, or on the other genes that it affects so as to return the cells to normal.
Additional Information.
Please access these Web sites via the online version of this summary at
• MedlinePlus, a Web site of the US National Library of Health, has pages of information on myelofibrosis and related diseases
• The National Cancer Institute, which funds research into many cancers, has information for patients on myelofibrosis, including information on clinical trials
• The MPD Foundation has information for patients with myelofibrosis and related diseases
Activation of JAK-STAT signaling via a mutation - MPLW515L- in the thrombopoietin receptor seems to have a role in the pathogenesis of some patients with myelofibrosis.
PMCID: PMC1502153  PMID: 16834459
18.  Allelic Expression Imbalance of JAK2 V617F Mutation in BCR-ABL Negative Myeloproliferative Neoplasms 
PLoS ONE  2013;8(1):e52518.
The discovery of a single point mutation in the JAK2 gene in patients with BCR/ABL-negative myeloproliferative neoplasms (MPNs) has not only brought new insights and pathogenesis, but also has made the diagnosis of MPNs much easier. Although, to date, several mechanisms for the contribution of single JAK2V617F point mutation to phenotypic diversity of MPNs have been suggested in multiple studies, but it is not clear how a unique mutation can cause the phenotypic diversity of MPNs. In this study, our results show that allelic expression imbalance of JAK2 V617F mutant frequently occurs and contributes to phenotypic diversity of BCR-ABL-negative MPNs. The proportion of JAK2 V617F mutant allele was significantly augmented in RNA levels as compared with genomic DNA differently by distinct MPNs subtypes. In detail, preferential expression of JAK2 mutant allele showed threefold increase from the cDNA compared with the genomic DNA from patients with essential thrombocythemia and twofold increase in polycythemia vera. In conclusion, allelic expression imbalance of JAK2 V617F mutant proposes another plausible mechanism for the contribution of single JAK2 point mutation to phenotypic diversity of MPNs.
PMCID: PMC3551963  PMID: 23349688
19.  The Constitutive Activation of Jak2-V617F is Mediated by a π Stacking Mechanism Involving Phe 595 and Phe 617 
Biochemistry  2010;49(46):9972-9984.
Somatic mutations in the Jak2 allele that lead to constitutive kinase activation of the protein have been identified in human disease conditions such as the myeloproliferative neoplasms (MPNs). The most common mutation in these patients is a V617F substitution mutation, which is believed to play a causative role in the MPN pathogenesis. As such, identifying the molecular basis for the constitutive activation of Jak2-V617F is important for understanding its clinical implications and potential treatment. Here, we hypothesized that conversion of residue 617 from Val to Phe resulted in the formation of novel π stacking interactions with neighboring Phe residues. To test this, we first examined the Jak2 structure via molecular modeling and identified a potential π stacking interaction between F594, F595 and F617. Disruption of this interaction through site directed mutagenesis impaired Jak2 auto-phosphorylation, Jak2 dependent gene transcription and in vitro kinase activity of the Jak2-V617F protein. Further, substitution of F594 and F595 with Trp did not affect Jak2 function significantly, but replacement with charged residues did, showing the conservation of aromaticity and hydropathy index at these positions. Using molecular dynamics (MD) simulations, we found that the π stacking interaction between residues 595 and 617 in the Jak2-V617F protein was of much greater energy and conformed to the properties of π stacking, relative to the Jak2-WT or Jak2-V617F/F594A/F595A. In summary, we have identified a π stacking interaction between F595 and F617 that is specific to and is critical for the constitutive activation of Jak2-V617F.
PMCID: PMC2982877  PMID: 20958061
Janus kinase 2 (Jak2); Pi stacking; Constitutive Activation
20.  Correlations between Janus Kinase 2 V617F Allele Burdens and Clinicohematologic Parameters in Myeloproliferative Neoplasms 
Annals of Laboratory Medicine  2012;32(6):385-391.
This study evaluated potential correlations between the allele burden of the Janus kinase 2 (JAK2) V617F mutation and clinicohematologic characteristics in patients with myeloproliferative neoplasms (MPN).
Clinical and hematologic features were reviewed for 103 MPN patients, including patients with polycythemia vera (PV, 22 patients), essential thrombocythemia (ET, 64 patients), and primary myelofibrosis (PMF, 17 patients). JAK2 V617F allele status and allele burdens were measured by allele-specific PCR and pyrosequencing, respectively.
The JAK2 V617F mutation was detected in 95.5%, 68.8%, and 52.9% of PV, ET, and PMF patients, respectively. JAK2 V617F-positive ET patients were significantly older and exhibited higher neutrophil fractions, a higher frequency of thrombotic events, and a higher myelofibrosis rate than JAK2 V617F-negative patients (P <0.05). PV patients carried the highest mean T allele burden (66.0%±24.9%) compared with ET (40.5%±25.2%) and PMF patients (31.5%±37.0%) (P =0.00). No significant correlations were detected between V617F allele burden and patient age, white blood cell count, Hb, Hct, or the platelet count for PV, ET, or PMF patients. ET patients with organomegaly had a higher JAK2 V617F allele burden (53.4%±23.7%) than patients without organomegaly (35.6%±24.3%) (P =0.03).
The JAK2 V617F mutational status and its allele burden correlate with the clinicohematologic phenotypes of ET patients, including older age, higher neutrophil count, and greater rates of organomegaly, thrombotic events, and myelofibrosis. For PV and PMF patients, larger-scale studies involving more MPN patients are needed.
PMCID: PMC3486931  PMID: 23130336
Janus kinase 2; Allele; Myeloproliferative neoplasm
21.  Genetic resistance to JAK2 enzymatic inhibitors is overcome by HSP90 inhibition 
Hsp90 inhibition in B cell acute lymphoblastic leukemia overcomes resistance to JAK2 inhibitors.
Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of myeloproliferative neoplasms (MPNs), B cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit cytokine receptor–like factor 2 (CRLF2), and other tumors with constitutive JAK2 signaling. In this study, we identify G935R, Y931C, and E864K mutations within the JAK2 kinase domain that confer resistance across a panel of JAK inhibitors, whether present in cis with JAK2 V617F (observed in MPNs) or JAK2 R683G (observed in B-ALL). G935R, Y931C, and E864K do not reduce the sensitivity of JAK2-dependent cells to inhibitors of heat shock protein 90 (HSP90), which promote the degradation of both wild-type and mutant JAK2. HSP90 inhibitors were 100–1,000-fold more potent against CRLF2-rearranged B-ALL cells, which correlated with JAK2 degradation and more extensive blockade of JAK2/STAT5, MAP kinase, and AKT signaling. In addition, the HSP90 inhibitor AUY922 prolonged survival of mice xenografted with primary human CRLF2-rearranged B-ALL further than an enzymatic JAK2 inhibitor. Thus, HSP90 is a promising therapeutic target in JAK2-driven cancers, including those with genetic resistance to JAK enzymatic inhibitors.
PMCID: PMC3280877  PMID: 22271575
22.  Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in Essential Thrombocythemia and Myelofibrosis 
Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34+ hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters.
By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34+ cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34+ cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-XL and BCLW. In contrast, pro-apoptotic BID and BIMEL expression were downregulated in ET leukocytes. Increased BCL-XL protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly.
Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.
PMCID: PMC3298534  PMID: 22300941
Chronic Myeloproliferative Neoplasms; Apoptosis; JAK2 V617F allele burden; PRV1 ; BCL2 family members
23.  Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells 
Cancer cell  2010;17(6):584-596.
We report a Jak2V617F knock-in mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild type HSCs. In contrast, myeloid progenitor populations are expanded and skewed towards the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F positive MPN.
The JAK2V617F mutation is a promising candidate for molecularly targeted therapy in MPN. Early data from JAK2 inhibitor clinical trials have called into question the capacity of these compounds to alter the natural history of JAK2V617F mediated MPN. Determining the effect of JAK2 inhibitors on the disease-initiating population requires a model in which the JAK2V617F allele is expressed at physiological levels in hematopoietic stem and progenitor cells, as it is in humans. Our model demonstrates that JAK2V617F causes expansion of erythroid progenitors but that only the HSC compartment can initiate disease in a transplanted mouse. We further demonstrate that the HSC compartment, the definitive target for curative therapy of JAK2V617F mediated MPN, is resistant to treatment with a JAK2 inhibitor.
PMCID: PMC2909585  PMID: 20541703
24.  Myeloproliferative Neoplasm Animal Models 
Myeloproliferative neoplasm (MPN) animal models accurately re-capitulate human disease in mice and have been an important tool for the study of MPN biology and therapy. Transplantation of BCR-ABL transduced bone marrow cells into irradiated syngeneic mice established the field of MPN animal modeling and the retroviral bone marrow transplantation (BMT) assay has been used extensively since. Genetically engineered MPN animal models have enabled detailed characterization of the effects of specific MPN associated genetic abnormalities on the hematopoietic stem and progenitor cell (HSPC) compartment and xenograft models have allowed the study of primary human MPN-propagating cells in vivo. All models have facilitated the pre-clinical development of MPN therapies. JAK2V617F, the most common molecular abnormality in BCR-ABL negative MPN, has been extensively studied using retroviral, transgenic, knock-in and xenograft models. MPN animal models have also been used to investigate additional genetic lesions found in human MPN and to evaluate the bone marrow microenvironment in these diseases. Finally, several genetic lesions, although not common, somatically mutated drivers of MPN in humans induce a MPN phenotype in mice. Future uses for MPN animal models will include modeling compound genetic lesions in MPN and studying myelofibrotic transformation.
PMCID: PMC3459181  PMID: 23009938
Myeloproliferative neoplasms; preclinical murine models; BCR-ABL; JAK2V617F; hematopoietic stem cells; bone marrow microenvironment; myelofibrosis; oncogenes
25.  Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms 
PLoS ONE  2014;9(1):e86401.
Most cases of BCR-ABL1-negative myeloproliferative neoplasms (MPNs), essential thrombocythemia, polycythemia vera and primary myelofibrosis are associated with JAK2V617F mutations. The outcomes of these cases are critically influenced by the transition from JAK2V617F heterozygosity to homozygosity. Therefore, a technique providing an unbiased assessment of the critical allele burden, 50% JAK2V617F, is highly desirable. In this study, we present an approach to assess the JAK2V617F burden from genomic DNA (gDNA) and complementary DNA (cDNA) using one-plus-one template references for allele-specific quantitative-real-time-PCR (qPCR). Plasmidic gDNA and cDNA constructs encompassing one PCR template for JAK2V617F spaced from one template for JAK2Wild Type were constructed by multiple fusion PCR amplifications. Repeated assessments of the 50% JAK2V617F burden within the dynamic range of serial dilutions of gDNA and cDNA constructs resulted in 52.53±4.2% and 51.46±4.21%, respectively. The mutation-positive cutoff was estimated to be 3.65% (mean +2 standard deviation) using 20 samples from a healthy population. This qPCR approach was compared with the qualitative ARMS-PCR technique and with two standard methods based on qPCR, and highly significant correlations were obtained in all cases. qPCR assays were performed on paired gDNA/cDNA samples from 20 MPN patients, and the JAK2V617F expression showed a significant correlation with the allele burden. Our data demonstrate that the qPCR method using one-plus-one template references provides an improved assessment of the clinically relevant transition of JAK2V617F from heterozygosity to homozygosity.
PMCID: PMC3903535  PMID: 24475114

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