Detect JAK2 V617F Research Articles

Observation of a higher JAK2 V617F homozygous mutated clone in polycythemia vera compared to essential thrombocythemia

Single-tube bi-directional allele specific amplification (SB-ASA) and real-time quantitative polymerase chain reaction (RQ-PCR) assays were developed and performed for JAK2V617F detection on 40 polycythemia vera (PV) samples, 31 essential thrombocythemia (ET) samples, 40 acute leukemia samples, and 40 healthy control samples. Differences between detect limitations of the two assays and their influence on the mutation detection rate were analyzed. The results showed that in some samples, the JAK2V617F burden was as low as nearly 1%, and thus more JAK2V617F-positive samples were detected by RQ-PCR than by SB-ASA assay due to the former higher detect limitation. Mutation allele ratios in PV and ET samples and their relevance to biological characteristics were also analyzed. The results showed that the mutation allele ratio was 0.436 ± 0.261 in PV, higher than the 0.216 ± 0.207 in ET; percentage of certainly homozygous mutation carriers in PV was 40.54%, higher than the 10% in ET. However, statistical analysis showed no relevance between mutation allele burden and sex or age. Our result shows that the pathogenesis of PV and ET may be related to the mutation allele burden of JAK2V617F.

Validity test study of JAK2 V617F and allele burden quantification in the diagnosis of myeloproliferative diseases

Several sensitive methods for the detection of JAK2 V617F mutation have been published recently, most of them based on Real Time polymerase chain reaction (PCR). However, only some of them have performed studies of diagnostic validity. This study compares three methods based on Real Time PCR to detect JAK2 V617F mutation: two based on hybridization probes (HP) and peptide nucleic acid probe (PNA) and a third employing allele specific oligonucleotide primers for JAK2 V617F quantification. One hundred forty-nine healthy subjects, 61 essential thrombocythemia (ET), 32 polycythemia vera (PV), 38 secondary thrombocytoses, and 35 secondary erythrocytoses were included. Validity test study for JAK2 617 HP PCR in PV Sensitivity (Se) was 88% and in Specificity (Sp), 100%. In ET, Se was 57% and Sp, 100%. For JAK2 617 PNA PCR in PV, Se was 94% and Sp, 97.8%. In ET, Se was 70% and Sp, 95.7%. In JAK2 V671F allelo-specific-oligonucleotide (ASO) quantitative PCR (qPCR), cutoff point of 1% was established by receiving operating characteristic (ROC) curves. In PV, Se was 93.8% and Sp, 98.5%. In ET, Se was 80% and Sp, 95.9%. Two percent of the healthy subjects were positive by JAK2 617 PNA PCR and 2% by JAK2 617 ASO qPCR. JAK2 V617F mutation was detected in healthy subjects by cloning and sequencing. JAK2 617 HP is an adequate test in differential diagnosis for both erythrocytosis and thrombocytosis. When JAK2 V617F allele burden is low, JAK2 617 ASO qPCR should be performed. Simultaneous determination of JAK2 V617F and PRV-1 overexpression does not improve the diagnostic value of JAK2 V617F tests in MPD.

Quantitation of the JAK2 V617F Mutation in Microdissected Bone Marrow Trephines: Equal Mutational Load in Myeloid Lineages and Rare Involvement of Lymphoid Cells

The JAK2V617F mutation is an essential oncogenic event in Philadelphia negative chronic myeloproliferative disorders (Ph-cMPD). It is still unclear how a unique tyrosine kinase mutation can give rise to the broad clinical and morphologic spectrum of Ph-cMPD. One possible explanation could be differences in the JAK2V617F gene dosage, or different maturation stages on which myeloid lineages are affected by the mutation. The extent of lymphoid lineage involvement in JAK2V617F-positive cMPD is still controversial. We comparatively studied the zygosity status of microdissected megakaryocytes, nonmegakaryocytic hematopoietic cells, and reactive as well as neoplastic lymphoid nodules from bone marrow trephines of 61 patients with Ph-cMPD. The presence of the mutation and mutant gene dosage were determined by allele-specific polymerase chain reaction and TaqMan analysis, respectively. The mutation was detected in 22/32 (68%) cases of essential thrombocythemia, all cases of polycythemia vera, and 4/8 (50%) idiopathic myelofibrosis. Comparison of whole bone marrow sections and the different myeloid lineages showed similar percentages of the mutated allele. Restriction to a particular lineage or major differences in allele dosage were not observed, except for 2 cases in which megakaryocytes revealed a higher frequency of the mutated allele. A heterozygous JAK2V617F mutation was detected in 3/8 "reactive" lymphoid nodule in patients with Ph-cMPD, whereas all concomitant non-Hodgkin lymphoma of B-cell type were negative. These results demonstrate that different myeloid lineages usually show similar frequencies of the JAK2V617F allele. The occasional detection of JAK2V617F in benign lymphocytes points to involvement of the lympho-myeloid stem cell.

The impact of JAK2 and MPL mutations on diagnosis and prognosis of splanchnic vein thrombosis: a report on 241 cases

AbstractMyeloproliferative diseases (MPDs) represent the commonest cause of splanchnic vein thrombosis (SVT), including Budd-Chiari syndrome (BCS) and portal vein thrombosis (PVT), but their diagnosis is hampered by changes secondary to portal hypertension, while their influence in the outcome of SVT remains unclear. We assessed the diagnostic and prognostic value of JAK2 and MPL515 mutations in 241 SVT patients (104 BCS, 137 PVT). JAK2V617F was found in 45% of BCS and 34% of PVT, while JAK2 exon 12 and MPL515 mutations were not detected. JAK2V617F was found in 96.5% of patients with bone marrow (BM) changes specific for MPD and endogenous erythoid colonies, but also in 58% of those with only one feature and in 7% of those with neither feature. Stratifying MPD diagnosis first on JAK2V617F detection would have avoided BM investigations in 40% of the patients. In BCS, presence of MPD carried significantly poorer baseline prognostic features, required hepatic decompression procedures earlier, but had no impact on 5-year survival. Our results suggest that JAK2V617F testing should replace BM investigations as initial test for MPD in patients with SVT. Underlying MPD is associated with severe forms of BCS, but current therapy appears to offset deleterious effects of MPD on the medium-term outcome.

High prevalence of arterial thrombosis in JAK2 mutated essential thrombocythaemia: independence of the V617F allele burden

Approximately half of the patients with essential thrombocythaemia (ET) harbor the JAK2 V617F mutation. Despite a phenotypic mimicry of JAK2 V617F positive ET and polycythaemia vera (PV), the data on thromboembolic risk and correlation to JAK2 mutation status are ambiguous. On a strictly WHO defined ET cohort we evaluated possible clinical correlations to the JAK2 mutation status including a history of previous thrombosis. We used a highly sensitive quantitative real-time PCR (qPCR) assay for JAK2 V617F detection and allele burden quantification in a single institution study of 55 patients. A significantly increased prevalence of arterial thrombosis was recorded in JAK2 positive ET (p=0·001). There was no association between the mutational load and thrombosis. As compared to their JAK2 V617F negative counterparts, the JAK2 V617F positive patients had PV-like biochemical characteristics such as higher haemoglobin levels (p=0·02), lower platelet counts (p=0·002) and lower plasma EPO levels (p=0·04). The JAK2 V617F mutation per se but not the mutational load in patients with ET is associated with a PV-like phenotype and a higher prevalence of previous arterial thrombosis. This study adds further support to the contention of the JAK2 V617F mutation as a marker of increased risk of thrombosis.

JAK2 Mutations are present in all cases of polycythemia vera

The JAK2V617F point mutation has been described in 65 to 97% of patients with polycythemia vera (PV). Previously, 17 phenotypical cases of PV from a large group of patients from three institutions were initially reported as JAK2V617F negative. These cases were re-analyzed in detail to determine the cause, if any, of the JAK2V617F negativity. Reasons for this initial negativity included inaccurate clinical diagnosis, relatively insensitive laboratory techniques, insufficient material for re-testing, and possible treatment effect. It was predicted that when tested appropriately, a JAK2V617F mutation would be found in all new cases of PV. We developed a highly sensitive amplification refractory mutation system assay (ARMS) for the detection of JAK2V617F. The analytic sensitivity of the assay is 0.05-0.1% as defined by mixing JAK2V617F-containing HEL cells with normal peripheral blood mononuclear cells. At this level of sensitivity, the assay has a false positive rate of less than 1% (we found 1 positive case in 300 individuals without myeloid disorders). Pyrosequencing involves synthetic nucleotide extension by DNA polymerase. Unlike ARMS, the PCR pyrosequencing method reliably detects JAK2V617F only when the mutant allele is more than 5%. The sensitivity of the ARMS assay is therefore approximately 50 times greater than the pyrosequencing method. Of 105 PV cases diagnosed by Polycythemia Vera Study Group criteria, pyrosequencing detected the mutation in 96 cases. The ARMS technique detected JAK2V617F alleles in 8 of the remaining 9 cases. Of the 8, 2 had been treated by phlebotomy only, 4 had been on interferon for a median of 13 years, and 2 on imatinib for 3 years. In the last patient, who was negative by both assays, the JAK2 exon 12 deletion (E543-D544) was detected. This patient had been on imatinib for 3 years and remains in complete remission. We conclude that a sensitive assay is required to detect the JAK2 mutations in patients with polycythemia vera, especially in patients whose JAK2 mutation load might be affected by treatment.

Importance of JAK2 V617F Allele Burden in the Diagnosis of Myeloproliferative Diseases and Its Association to Age.

The aim of this study is to compare the diagnostic value of several methods, with different sensitivities, to detect JAK2 V617F mutation. Three new methods based on Real Time PCR to detect JAK2 V617F mutation were employed. The first two, based on hybridization probes and Peptide Nucleic Acid Probe (PNA) specific for wild allele; and the third employing Allele Specific Oligonucleotide (ASO) primers for JAK2 V617F mutant allele and MGB TaqMan probe. Healthy subjects (n 149) and patients with Essential Thrombocythemia (ET) (n 63), Polycythemia Vera (PV) (n 31), Secondary Thrombocythosis (ST) (n 38), Secondary Erytrocythosis (SE) (n 35) and other haematological malignancies (n 72) were included in the study. Results. Validity test study for JAK2 617 using HP PCR showed: A-. For PV patients: Sensitivity (Se) was 87.1%, Specificity (Sp) 100%, Positive Predictive value (PPV) 100%, and Negative Predictive Value (NPV) 97.9%. B-. For ET patients: Se was 61.3%, Sp 100%, PPV 100%, and NPV 93.9%. With JAK2 617 PNA PCR: A.- For PV patients: Se was 93.6%, Sp 97.8%, PPV 87.9%, and NPV 98.9%. B. - For ET patients: Se was 71%, Sp 95.7%, PPV 73.3%, and NPV 95.2%. With JAK2 V671F ASO quantitative PCR (JAK2 617 ASO qPCR): A.- For PV patients: Se was 93.5%, Sp 98.5%, PPV 90.6%, and NPV 98.9%. B.-For ET patients: Se was 80.6%, Sp 95.9%, PPV 75.8%, and NPV 96.7%. Cutoff point of 1% was established by ROC curves as mutation burden to distinguish PV or ET versus secondary causes. Three healthy subjects were positive (2%, 3/149) by JAK2 617 PNA PCR method. With JAK2 617 ASO qPCR, 16 healthy subjects would be positive (10%) if cutoff burden established in 0.1%. Significant differences were obtained when comparing Myeloproliferative Disease (MPD) patients over 40 years (n 64) to patients under 40 (n 15). JAK2 V617F median allele burden was 18.41% and 7.26% respectivily (p=0.016).Conclusions: JAK2 617 HP and ASO qPCR are the best tests in differential diagnosis of Polyglobulias and Thrombocytosis. When JAK2 V617F allele burden is low, the best test for MPD diagnosis is JAK2 617 ASO qPCR. The number of JAK2 V617F mutated cells is higher in patients over 40, which leads to think that age may influence in tumour burden.

Study on Clinical and Laboratory Characteristics of Chronic Eosinophilic Leukemia CEL/Hypereosinophilic Syndrome (CEL/HES).

Hypereosinophilic syndrome (HES) was a group of diseases associated with persistent eosinophilia without unknown causes, companied with tissue and organ impairments. In 2001, WHO classification of hematopoietic and lymphoid neoplasms classified chronic eosinophilic leukemia (CEL) / HES into the category of chronic myeloproliferative disease, and proposed that the principal basis for the identification of CEL and HES was whether to have the evidence of eosinophils clonal proliferation: CEL had the evidence of eosinophils clonal proliferation, while HES was lack of the evidence of eosinophils clonal proliferation. In this study, 20 cases of CEL/ HES patients were analyzed retrospectively, and nested RT-PCR was used to detect FIP1L1-PDGFRA (F/P) fusion gene; Allele-specific PCR (ASP) conjoint sequencing analysis was used to detect JAK2 V617F, and PCR-RFLP was adopted to detect the mutation status of JAK2 V617F; and PCR is applied to detect TCRγ rearrangement. The clinical and laboratory characteristics of CEL and HES were compared. The ratio of male and female in the 20 cases of patients was 19:1, and the median age was 33 (20–57). F/P detection was positive for 12 cases, and the sequencing confirmed that FIP1L1 break point was at intron 10–12, while PDGFRA break point was at exon 12. There was 1 case of patient found that had JAK2 V617F, and the mutation status analysis showed that it was the mutation on heterozygote. 6 cases were detected having TCRγ gene rearrangement, of which 4 cases were CEL patients, and other 2 cases were HES patients. Most of CEL patients had respiratory symptoms in the early stage, easily companied with circulatory systematic impairment and nervous systematic symptoms. The incidence of splenomegaly of CEL patients was obviously higher than that of HES patients (92.5% vs 42.5%, p=0.031), so did the incidence of anemia and myelofibrosis. There was no difference in EO, WBC, PLT and EO% in peripheral blood as well as bone-marrow eosinophils percentage and bone-marrow primitive cells' percentage between two groups. Abnormal morphology of eosinophils was often found in CEL patients, with the main manifestation of eosinopenia, basopenia and plasma vacuoles. Our data showed that1.Eosinophilia is often caught by male, mainly by the young;2.CEL patients have the main manifestation of the circulatory systematic, respiratory and gastrointestinal symptoms, and have a high incidence of anemia and thrombocytopenia. The routine examination has a little significance for the identification o CEL and HES, while the bone marrow smears morphological examination has a certain help for the diagnosis of CEL;3.Some HES patients have JAK2 V617F mutation, and further studies on the effect of JAK2 V617F mutation on the pathogenesis of HES can contribute to the diagnosis of such patients in the future and the development of the new targeted drug therapy;4.Some CEL patients have TCRγ rearrangement, while the relationship of CEL and TCRγ needs a further study.

JAK2 Mutations Are Present in All Cases of Polycythemia Vera.

The JAK2V617Fpoint mutation has been described in 65 to 97% of patients with polycythemia vera (PV). Previously, 17 phenotypical cases of PV from a large group of patients from three institutions were initially reported as JAK2V617Fnegative. These cases were re-analyzed in detail to determine the cause, if any, of the JAK2V617Fnegativity. Reasons for this initial negativity included inaccurate clinical diagnosis, relatively insensitive laboratory techniques, insufficient material for re-testing, and possible treatment effect. It was predicted that when tested appropriately, a JAK2V617Fmutation would be found in all new cases of PV. We developed a highly sensitive amplification refractory mutation system assay (ARMS) for the detection of JAK2V617F. The analytic sensitivity of the assay is 0.05-0.1% as defined by mixing JAK2V617F-containing HEL cells with normal peripheral blood mononuclear cells. At this level of sensitivity, the assay has a false positive rate of less than 1% (we found 1 positive case in 300 individuals without myeloid disorders). Pyrosequencing involves synthetic nucleotide extension by DNA polymerase. Unlike ARMS, the PCR pyrosequencing method reliably detects JAK2V617Fonly when the mutant allele is more than 5%. The sensitivity of the ARMS assay is therefore approximately 50 times greater than the pyrosequencing method. Of 105 PV cases diagnosed by Polycythemia Vera Study Group criteria, pyrosequencing detected the mutation in 96 cases. The ARMS technique detected JAK2V617Falleles in 8 of the remaining 9 cases. Of the 8, 2 had been treated by phlebotomy only, 4 had been on interferon for a median of 13 years, and 2 on imatinib for 3 years. In the last patient, who was negative by both assays, the JAK2 exon 12 deletion (E543-D544) was detected. This patient had been on imatinib for 3 years and remains in complete remission. We conclude that a sensitive assay is required to detect the JAK2 mutations in patients with polycythemia vera, especially in patients whose JAK2 mutation load might be affected by treatment.

Comparative Evaluation of ThreeJAK2V617FMutation Detection Methods

The correlation of JAK2V617F with a proportion of chronic myeloproliferative disorders has generated numerous studies focused on the development of molecular-based assays for JAK2V617F detection. The current parallel study comparatively evaluated 3 JAK2V617F molecular detection methods. Genomic DNA from blood or bone marrow was assayed by 3 laboratories using allele-specific polymerase chain reaction (AS-PCR) or kit-based restriction fragment length polymorphism methods, which used polyacrylamide gel or capillary electrophoresis analysis. In addition, samples were sequenced in 2 of the laboratories. Results found 100% concordance among the 3 methods, with analytic sensitivities of 5% for both kit methods and 0.01% for AS-PCR. The kitbased assays detect JAK2V617F with equal sensitivity regardless of analysis method, and, despite greater sensitivity of AS-PCR, all 3 methods yielded 100% concordant results for this 36-sample set. Consistent with other reports, direct sequencing was insufficiently sensitive to serve as an initial diagnostic tool for JAK2V617F detection.

Most pediatric patients with essential thrombocythemia show hypersensitivity to erythropoietin in vitro, with rare JAK2 V617F-positive erythroid colonies

Essential thrombocythemia (ET), a Philadelphia (Ph) chromosome negative chronic myeloproliferative disorder, is usually a disease of middle age and it is extremely rare in pediatric patients. In this report we studied 12 children diagnosed with ET and one child with thrombocytosis and family history of ET. We failed to detect JAK2 V617F mutation either in peripheral blood leukocytes or in separated platelets and granulocytes. Monoclonal hematopoiesis was noted in only one female patient. Erythroid progenitors of most of the patients displayed hypersensitivity to erythropoietin (Epo) in vitro; Epo-independent erythroid colonies (EECs) were detected in seven patients. Among EECs of three patients we observed rare colonies heterozygous or homozygous for the JAK2 V617F mutation. Our data suggest that childhood ET patients could bear minor JAK2 V617F-positive subclones.

JAK2V617F detection and dosage of serum erythropoietin: first steps of the diagnostic work-up for patients consulting for elevated hematocrit

The predictive values of common biological criteria for the diagnosis of polycythemia vera were studied in a cohort of patients with high hematocrit. We found JAK2V617F and erythropoietin assays were the most relevant first tests. Classification of patients according to their JAK2V617F status and erythropoietin levels facilitated the choice of further diagnostic investigations.

Comparison of whole blood vs purified blood granulocytes for the detection and quantitation of JAK2V617F

Comparison of whole blood vs purified blood granulocytes for the detection and quantitation of JAK2 V617F

A Novel and Quantitative Assay To Detect JAK2 V617F Allele by Real-Time AS-PCR and Its Applicability to PV Initiating Mutation.

Polycythemia vera (PV) arises due to a somatic mutation(s) of a single hematopoietic stem cell leading to clonal hematopoiesis. Greater than 80% of PV patients carry a somatic mutation in JAK2 (V617F). Growing evidence suggests that increased frequency of the JAK2V617F allele may have a prognostic impact on certain clinical aspects of PV, and, possibly, in other myeloproliferative disorders associated with this mutation. We have developed a novel approach to primer design for Real-Time quantitative allele-specific PCR. Allelic discrimination is enhanced by the combined synergistic effects of an artificial mismatch introduced in the −1 position, starting from the 3′ end of the primer, and the use of a locked nucleic acid (LNA) modified nucleoside placed at the −2 position. We provide evidence that the −2 LNA assists in stabilizing the 3′ end, while the −1 mismatch provides specificity but not stability. The difference in cycle number between the two allele-specific reactions is used to calculate the relative allele frequencies. We demonstrate the robustness, sensitivity and reproducibility of our design. The proportion of mutant JAK2 allele determined by pyrosequencing and kinetic allele-specific PCR was highly concordant with an average allele frequency deviation of 2.6%. Repeated determination of allelic ratios in multiple patient samples was highly reproducible with a standard deviation of 1.5%. We have also determined that the design and assay is highly sensitive; as little as 0.1% mutant allele in 40–50 ng of genomic DNA can be detected. We further tested the applicability of this technique to the analysis of individual BFU-E colonies in order to address the question whether the JAK2V617F is the disease initiating mutation. Less than 10% of a single isolated BFU-E colony, originating from a single progenitor, is sufficient for determination of allele frequency. The remainder of the colony may be used for other analyses. A proportion of 0 or 50 or 100 percent JAK2 mutant allele is expected from each individual BFU-E colony, which was indeed observed. However, when we tested granulocytes from PV females, wherein the granulocytes were found to be clonal by the X-chromosome transcriptionally based clonality assay, we found 3 females <50 (27.5 ±11) and 7 females >50 (75 ±10.5)percent mutant JAK2 allele frequencies. This result suggests the presence of a heterogeneous population of cells with differing genotypes regarding the JAK2 mutant allele, and is further supported by our genotyping results with individual BFU-E colonies as described above. Our PV data suggest that the JAK2V617F may not be the PV initiating mutation. This novel primer design is simple, does not require tedious optimization of reaction conditions, and can be applied to any kinetic PCR platform for reliable and sensitive determination of allele frequencies. Potential applications are varied, such as, quantitative determination of mosaicism, proportion of fetal cells in maternal circulation, detection of minimal residual disease associated with known somatic mutation (such as reduction of malignant cells by chemotherapy or reappearance of resistant clone), rapid monitoring of efficacy of new drugs in both “in vitro” systems as well as clinical trials, and many others that require quantitation of allele frequencies.

Mutation JAK2 V617F in Patients with Myeloproliferative Disorders and Technical Aspects of Its Detection.

Myeloproliferative disorders (MPD) are a heterogeneous group of diseases that involve chronic myeloid leukemia (CML), polycythemia vera (PV), essential trombocythemia (ET), chronic idiopatic myelofibrosis (IMF), hypereosinophilic syndrome and chronic neutrophilic leukemia. Recently, it has been found that in Ph-negative MPD, acquired somatic mutation in JAK2 gene can be identified, causing substitution of valine for phenylalanine at aminoacid position 617 (V617F). Experimental evidence indicates that JAK2 V617F might cause erythropoietin-independence of erythroid colonies and receptor hypersensitivity, thus be implicated in the pathogenesis of PV.The incidence of V617 in PV is high, reaching 96% of cases; in ET or IMF, the mutation is less frequent. Nevertheless, the percentage of JAK2 V617F-positive patients differs greatly among studies, depending on the detection technique used. Sequencing analysis, as one of the options, can underrate the number of positive cases, as its sensitivity in authentic samples with varying proportion of mutant granulocytes is unsatisfactory. The nucleotide context of the V617F site is complicated (direct repeat TGTG/TT), and thus prone to sequencing artifacts.For specific and sensitive detection of V617F mutation in JAK2 gene, we have developed a novel allelic discrimination assay, based on the usage of Real-Time PCR technology and LNA-modified fluorescently labeled probes (Locked Nucleic Acids). This technology is distinguished by 100% discrimination efficiency, and sensitivity detection rate reaching 10% of mutant granulocytes in an authentic sample. LNA-modified probes are extremely powerful tool for precise genotyping, since they are short (12-mers), and due to the interspersed LNA nucleotides, melting temperature (Tm) of the probe/target hybrid is high. Hence, the LNA-based genotyping technology combines the desired features of high sensitivity and absolute specificity.Herein, we present an analysis of JAK2 V617F detection in our group of 157 consecutive patients with the diagnosis of MPD or suspected MPD. Using our novel LNA-based Real-Time PCR genotyping technique, we were able to identify varying proportions of the mutant JAK2 allele in 87 cases (55.4%); in the remaining 70 patients the mutation was not detected.The LNA-based Real-Time PCR technique is extremely precise and efficient in identifying even subtle proportion of JAK2 V617F mutant granulocytes, which in cases of diagnostic uncertainties on the origin of the polycythemia might be of great importance.

Detection of the JAK2V617F Mutation in Myeloproliferative Disorders by Melting Curve Analysis Using the LightCycler System

Abstract Context.—A specific mutation, JAK2V617F, was recently recognized as having diagnostic value for myeloproliferative disorders. No practical assay is currently available for routine use in a clinical laboratory. Objective.—We report the development of a real-time polymerase chain reaction melting curve analysis assay that is appropriate for molecular diagnostics testing. Design.—Specific primers and fluorescence resonance energy transfer probes were designed, and patients with a previously diagnosed myeloproliferative disorder, de novo acute myeloid leukemia, or reactive condition were selected. The DNA was extracted from fresh and archived peripheral blood and bone marrow specimens, and real-time polymerase chain reaction melting curve analysis was performed on the LightCycler platform (Roche Applied Science, Indianapolis, Ind). Results.—The JAK2 region was successfully amplified, and wild-type amplicons were reproducibly discriminated from JAK2V617F amplicons. Titration studies using homozygous wild-type and mutant cell lines showed the relative areas under a melting curve were proportional to allele proportion, and the assay reliably detected one mutant in 20 total cells. JAK2V617F was identified in patients previously diagnosed with a myeloproliferative disorder or acute myeloid leukemia transformed from myeloproliferative disorder, whereas a wild-type genotype was identified in patients with reactive conditions or de novo acute myeloid leukemia. Conclusions.—These findings demonstrate the suitability of this assay for identifying JAK2V617F in a clinical laboratory setting. Furthermore, the semiquantitative detection of JAK2V617F in archived specimens provides a new tool for studying the prognostic significance of this mutation.

Detection of JAK2 V617F as a first intention diagnostic test for erythrocytosis

The diagnosis of polycythemia vera (PV) is currently based on clinical and biological criteria defined either by the World Health Organization (WHO) or the Polycythemia Vera Study Group (PVSG).1, 2 Both of these classifications use clinical and biological markers organized in major and minor criteria, allowing to diagnose a PV when a define combination of major and minor criteria is present. Recently, a somatic point mutation of the tyrosine kinase JAK2 (JAK2 V617F) has been described in about 80% of PV patients as well as 30% of essential thrombocythemia (ET) and 50% of idiopathic myelofibrosis (IMF)3, 4, 5, 6 and several in vitro and in vivo experiments demonstrated that this mutation of JAK2 was the molecular event at the origin of PV.3 It was therefore tempting to use the detection of JAK2V617F as a diagnostic test for erythrocytosis. Such an attitude supposes the development of other techniques than sequencing, as this latter is time consuming and not always feasible in hematology laboratories. We thus compared sequencing with two techniques of real-time PCR-based mutation detection (one using the LightCycler® instrument, the other the Taqman® ABI Prism 7500), for the efficiency to detect the JAK2 V617F mutation in 119 samples from patients with suspicion of myeloproliferative disorder (MPD). The three techniques were equivalent in all samples but one, where sequencing failed to detect the mutation revealed by both LightCycler® and Taqman® technologies. To evaluate the sensitivity of these techniques, we tested serial dilutions of the homozygously mutated HEL cell line DNA in the nonmutated TF-1 cell line DNA, and serial dilutions of the genomic DNA from a patient homozygous for the JAK2 V617F mutation in normal DNA. Sequencing failed to detect the mutated allele under 5% of HEL cell line DNA diluted in TF-1 cell line DNA, and under 10% of the homozygously mutated patient's DNA diluted in normal DNA. The sensitivity of LightCycler® and Taqman® techniques were equivalent, slightly higher than sequencing, reaching 0.5–1% of HEL cell line DNA diluted in TF-1 cell line DNA (Figure 1), and 2–4% of a homozygously mutated patient's DNA diluted in normal DNA.

Detection of JAK2 V617F in the Diagnosis of Erythrocytosis: Feasibility and Diagnostic Value in Clinical Practice.

Polycythemia Vera (PV) is a myeloproliferative disorder (MPD), whose diagnosis is currently based on an association of clinical and biological criteria following the WHO or the PVSG classification. It is characterized by a primitive absolute erythrocytosis, and formation of endogenous erythroid colonies (EEC). Recently, we described a point mutation in JAK2 (JAK2 V617F) and showed that this activating mutation was the cause of the disease (James et al, Nature, 2005). This molecular abnormality was therefore likely to be a diagnostic marker of PV, as bcr-abl for chronic myeloid leukemia (CML). Nevertheless, JAK2 V617F is also found in other MPDs, sharing some common features with PV, as essential thrombocythemia, idiopathic myelofibrosis, and other rare MPDs. One major criterion for the diagnosis of PV requires the demonstration of increased red cell mass as measured by isotopic methods. We assessed the value of detection of JAK2 V617F as a first intention diagnostic test in 88 patients with hematocrit values above 51% (=erythrocytosis) and showed that the mutation correlated with the diagnosis of PV according to the WHO (R=0.879) and the PVSG (R=0.717) criteria, with a positive predictive value of 100% in the context of erythrocytosis. Besides, the presence of the mutation strongly correlated with EEC formation in 81/87 patients (R=0.824) and only weakly with the serum erythropoietin level (R=0,416). PCR-based genotyping techniques are less time-consuming, less expensive, than DNA sequencing and are easier to perform in hematology diagnostic laboratories. Therefore, we studied the feasibility of the detection of JAK2 V617F with widespread instruments commonly used in routine. We analyzed 119 samples from patients with a suspicion of myeloproliferative disease and showed that JAK2 V617F was efficiently detected by LightCycler® and TaqMan® genotyping technologies, these latter being a little more sensitive than sequencing. For 50 patients, peripheral blood and bone marrow samples were both available. In all cases (34 mutated, 16 non-mutated) the mutation was identically detected. Based on these results, we propose that the detection of JAK2 V617F in granulocytes has a first place in the diagnostic chart of an erythrocytosis, as bcr-abl in CML, avoiding, if positive, an isotopic red cell mass measurement and bone marrow EEC assays. The presence of JAK2 V617F in a patient with erythrocytosis would then lead to the diagnosis of MPD of PV type. Further prospective studies will be necessary to assess if all the MPDs patients bearing JAK2 V617F can be grouped in a new subset within the MPD entity, especially in term of thrombotic and neoplasic risk.