Clinical Performance of JAK2 V617F Mutation Detection Assays in a Molecular Diagnostics Laboratory Evaluation of Screening and Quantitation Methods

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1 Hematopathology / Method Comparison for JAK2 V617F Detection Clinical Performance of JAK2 V617F Mutation Detection Assays in a Molecular Diagnostics Laboratory Evaluation of Screening and Quantitation Methods Milena Cankovic, PhD, Lisa Whiteley, Robert C. Hawley, MD, Richard J. Zarbo, MD, DMD, and Dhananjay Chitale, MD Key Words: Myeloproliferative; JAK2 V617F; Mutation; Wild type; Screening; Quantitation DOI: 1.139/AJCPFHUQZ9AGUEKA Abstract The presence of the JAK2 V617F mutation is now part of clinical diagnostic algorithms, and JAK2 status is routinely assessed when BCR/ABL chronic myeloproliferative neoplasms (MPNs) are suspected. The aim of this study was to evaluate performance of 3 screening and 1 quantitative method for JAK2 V617F detection. For the study, 43 samples (27 bone marrow aspirates and 16 peripheral blood samples) were selected. The screening assays were the JAK2 Activating Mutation Assay (InVivoScribe, San Diego, CA), JAK2 MutaScreen kit (Ipsogen, Luminy Biotech, Marseille, France), and a home-brew melting curve analysis method. Ipsogen s JAK2 MutaQuant assay was used for quantification of mutant and wild-type alleles. The limit of detection was 1% for the kit-based screening methods and 1% for the melting curve method. The JAK2 MutaQuant assay demonstrated analytic sensitivity of.1%. All 4 methods detected cases of BCR/ABL MPNs and gave negative results with BCR/ABL+ chronic myelogenous leukemia, multiple myeloma, myelodysplastic syndrome, and normal cases. Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell malignancies characterized by hypersensitivity of hematopoietic progenitors to numerous cytokines and by clonal proliferation of one or several myeloid lineages. 1,2 In addition to thrombotic and hemorrhagic complications, leukemic transformation can occur. 3 Typically, the classic MPNs encompass 4 related entities: chronic myelogenous leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). A point mutation in the Janus kinase 2 gene (JAK2) was identified in several MPNs, 4-7 most frequently in PV (65%-97%), ET (23%-57%), and PMF (35%-57%). It is also infrequently present (3%-5%) in myelodysplastic syndrome, chronic myelomonocytic leukemia, and other atypical chronic myeloid disorders. 8 The common occurrence of this mutation in BCR/ABL MPNs can be used as a unique molecular marker for distinguishing PV, ET, and PMF from reactive hematopoietic disorders. 9 Also, JAK2 mutation has not been detected in Philadelphia chromosome+ CML, and these mutations are currently considered exclusive. JAK2 is a cytoplasmic tyrosine kinase that mediates growth factor receptor signaling. The V617F mutation is a G>T transversion at nucleotide 2343, resulting in substitution of phenylalanine for valine (V617F) in the JAK2 protein. This point mutation at amino acid residue 617 appears to cause constitutive activation of the JAK2 tyrosine kinase owing to loss of autoinhibitory control. 1 The mutant has enhanced kinase activity, and when overexpressed together with the erythropoietin receptor in cells, it causes hyperactivation of erythropoietin-induced cell signaling. This gain-of-function mutation of JAK2 may explain the hypersensitivity of PV progenitor cells to growth factors and cytokines. The annual Am J Clin Pathol 29;132: DOI: 1.139/AJCPFHUQZ9AGUEKA 713

2 Cankovic et al / Method Comparison for JAK2 V617F Detection incidence of MPNs is.5 to 6.5 per 1, people, 11 compared with about 1% of healthy subjects with no clinical and hematologic abnormalities harboring the JAK2 V617F mutation. Recent observations suggest that this mutation occurs at the level of the common myeloid progenitor or upstream at the level of a lymphomyeloid multipotent progenitor cell. 4,12,13 Identification of a JAK2 V617F mutation establishes the presence of a clonal disorder 14 and is an important diagnostic marker for these disorders. 9 Since the discovery of the JAK2 V617F mutation in 25, a number of different JAK2 mutation detection protocols became available. 4,15,16 Not all clonal cells express the JAK2 V617F point mutation, and granulocyte enrichment has been suggested for mutation detection assays. Because granulocyte separation is expensive and labor-intensive, with added loss of cells during the processing, testing of unfractionated whole blood and bone marrow samples is a more practical approach for most clinical molecular laboratories. For that reason, a sensitive system must be used for mutation detection, estimation of allele burden, and minimal residual disease (MRD) monitoring. The variety of available techniques provides flexibility of method design, which prompts a need for comparative analysis of these methods. 17,18 The goal of this study was to evaluate several currently available testing methods and to select a simple, preferably kit-based assay for use in our clinical molecular diagnostics laboratory. We report our experience with 4 different fluorescent polymerase chain reaction (PCR)-based assays (3 qualitative and 1 quantitative) for the detection of the JAK2 V617F point mutation. Materials and Methods Case Selection The samples for this study were received from patients who were referred for investigation of one or more of the following: high hemoglobin level, high platelet count, neutrophilia, or hematopathologic findings suggestive of MPN. A total of 33 cases were evaluated Table 1 : 27 bone marrow aspirate Table 1 Clinical Features of Patients Whose Samples Were Included in the Study * Case No./Sex/Age (y) Sample Type Hemoglobin (g/dl) Platelet Count ( 1 3 /μl) WBC Count (/μl) BM Diagnosis Cytogenetics 1/F/49 BM ,1 ET Normal 2/F/59 BM ,19 12,5 PV Normal 3/F/52 BM ,1 2E Normal 4/M/8 Blood ,3 MM dely (2%) 5/M/74 BM ,4 MDS del12q 6/F/7 BM ,3 2E Normal 7/M/66 BM ,2 ET Normal 8/F/33 BM , PV Normal 9/M/46 BM ,2 2G Normal 1/M/6 BM ,1 ET dely 11/M/85 BM ,4 PV Normal 12/F/73 BM ,3 ET Normal 13/M/47 BM ,6 ET Normal 14/M/57 Blood , PV Normal 15/M/54 BM ,976 14, ET Normal 16/F/36 BM ,6 2T Normal 17/M/28 Blood ,6 2E ND 18/F/48 BM ,6 ET Normal 19/M/82 BM ,1 ET Normal 2/M/59 BM ,3 PV Normal 21/M/82 BM 9.3 2,841 2,3 PMF Normal 22/F/73 BM ,2 ET Normal 23/F/19 BM 7. 1, T Normal 24/M/53 Blood ,4 2E Normal 25/M/53 BM ,2 PV Normal 26/M/53 BM ,7 2E ND 27/M/64 BM ,7 CMML Normal 28/M/72 BM ,2 2E + B CLL Normal 29/F/79 BM ,785 14, ET Normal 3/M/37 Blood ,6 A Normal 31/M/55 BM ,3 CML bcr/abl 32/M/51 Blood ,3 CML bcr/abl 33/M/28 BM ,6 CML bcr/abl 2E, secondary erythrocytosis; 2G, reactive granulocytosis; 2T, secondary thrombocytosis; A, iron deficiency anemia; B CLL, B chronic lymphocytic leukemia; BM, bone marrow; CML, bcr/abl+ chronic myelogenous leukemia; CMML, chronic myelomonocytic leukemia; ET, essential thrombocythemia; MDS, myelodysplastic syndrome; MM, multiple myeloma; ND, not done; PMF, primary myelofibrosis; PV, polycythemia vera. * Laboratory values are given in conventional units; conversions to Système International units are as follows: hemoglobin (g/l), multiply by 1.; platelet count ( 1 9 /L), multiply by 1.; and WBC count ( 1 9 /L), multiply by Am J Clin Pathol 29;132: DOI: 1.139/AJCPFHUQZ9AGUEKA

3 Hematopathology / Original Article specimens and 6 peripheral blood specimens. Cases were classified by the World Health Organization criteria as one of the following: PV (6 cases), ET (1 cases), PMF (1 case), BCR/ ABL+ CML (3 cases), multiple myeloma (1 case), secondary erythrocytosis (5 cases), secondary thrombocytosis (2 cases), reactive granulocytosis (1 case), myelodysplastic syndrome (1 case), chronic myelomonocytic leukemia (1 case), B chronic lymphocytic leukemia + secondary erythrocytosis (1 case), and iron deficiency anemia (1 case). In addition, 1 samples from healthy subjects were used as negative controls (results not shown). The samples in the myeloproliferative group were preselected based on hematologic diagnosis and were expected to have a higher incidence of JAK2 mutation compared with a randomly selected group. DNA Isolation and Quantification Genomic DNA was extracted from bone marrow aspirates and unfractionated peripheral blood samples using the QIAamp DNA mini kit (Qiagen, Valencia, CA). DNA concentration and sample absorbance at 26 and 28 nm were measured using SmartSpec Plus spectrophotometer (Bio- Rad, Hercules, CA). In addition, granulocytes were isolated from a subset of 1 specimens using density centrifugation with Ficoll-Paque Plus (GE Healthcare Biosciences, Upsala, Sweden). These samples were selected randomly, with the only criterion being that the samples be less than 24 hours old. The granulocyte layer was collected, and the residual RBCs lysed with RBC Lysing solution (Catalog No. V5, Gentra Systems, Minneapolis, MN). DNA from the granulocyte layer was isolated and processed in the same manner as DNA from the unfractionated populations. All samples were coded and assayed blindly in duplicate for the JAK2 V617F mutation. JAK2 Activating Mutation Assay The JAK2 Activating Mutation Assay (InVivoScribe, San Diego, CA), which identifies the presence of the JAK2 V617F activating mutation by restriction digestion of PCR products and capillary electrophoresis (RFLP-CE) was used per manufacturer s protocol. PCR was first performed to amplify the portion of the JAK2 region that acquires this mutation. The amplicon products were then digested with the BsaXI restriction enzyme. Results were interpreted as follows: human genomic DNA from any source should generate the initial 267-base-pair (bp) amplicon products before restriction digestion. Following BsaXI digestion, 3-, 97-, and 14-bp products are generated from normal samples. Samples harboring a homozygous JAK2 V617F activating mutation no longer have the BsaXI restriction site, which leaves the parent 267-bp amplicon intact. Digested PCR products were analyzed on an ABI 31 Genetic Analyzer (Applied Biosystems, Foster City, CA). Zygosity determination was based on restriction digest patterns Figure 1A. To standardize result interpretation, the peak height of the small 267-bp peak seen in low-positive samples was always compared with the small remnant 267-bp peak in the negative control sample. Only samples in which the height of the 267-bp fragment was at least 3 times as high as the remnant of the 267-bp peak observed in the negative control sample were interpreted as positive. JAK2 MutaScreen Assay (Allele-Specific PCR) The JAK2 MutaScreen assay (Ipsogen, Luminy Biotech, Marseille, France), which detects JAK2 wild-type and V617F alleles in genomic DNA using TaqMan allelic discrimination, was used according to the manufacturer s protocol. The assay is based on the simultaneous use of 2 specific TaqMan probes (Applied Biosystems) and the measurement of the respective fluorescence of the 2 alleles (FAM for V617F and VIC for wild-type) to differentiate the amplification of each allele. Briefly, after DNA extraction, the short fragment spanning the JAK2 mutation site was amplified using real-time PCR and a primer and probe mix. Reactions were performed using the following PCR conditions: 5 C for 2 minutes for uracil N-glycosylase activation, 95 C for 1 minutes for uracil N-glycosylase inactivation, followed by 5 cycles of 95 C for 15 seconds and 6 C for 1 minute. Quantitation of mutant and wild-type alleles was accomplished by using a Rotor-Gene 3 real-time PCR instrument (Corbett Research, Sydney, Australia). By using allelic discrimination analysis in the Rotor-Gene software, the results from both channels were displayed together on one screen. The software also automatically calculated the genotypes shown. The Excel (Microsoft, Redmond, WA) analyzed data export feature further allowed concentration analysis of mutant and wild-type alleles. The mean ratio of mutant to wild-type allele was calculated for each sample. The mean ratio values obtained for patient samples and for positive and negative control samples were compared with the mean ratio of the reference sample in each assay. When the mean ratio of the test samples was equal to or greater than the mean ratio of the reference sample, the results were interpreted as positive for the presence of JAK2 V617F mutant alleles. When the mean ratio of the test samples was less than the mean ratio of the reference sample, the results were interpreted as negative, meaning that only wild-type alleles were detected. Melting Curve Analysis Assay The genotyping assay using fluorescence resonance energy transfer probes and melting curve analysis was carried out using the primers and probes as described previously. 19 An amplicon of 177 bp in length was generated using a PCR forward primer, 5 -TTCCTTAGTCTTTCTTTGAAGCA-3 and a reverse primer, 5 -GTGATCCTGAAACTGAATTTTCT-3 (Applied Am J Clin Pathol 29;132: DOI: 1.139/AJCPFHUQZ9AGUEKA 715

4 Cankovic et al / Method Comparison for JAK2 V617F Detection A 4, 2, % 1, % 1,8 1, % 1, % 1, % B df/dt Mutant peaks Wild-type peaks Bin A 1% Mutant Bin B 2% Mutant 5ºC 55ºC 6ºC 65ºC 7ºC 75ºC 1% Positive 2% Positive 1% Positive 5% Positive 2.5% Positive 1.25% Positive % Positive Figure 1 Analytic sensitivity of the JAK2 V617F screening assays. A, JAK2 Activating Mutation Assay (InVivoScribe, San Diego, CA). B, Melting curve analysis assay. Biosystems). A sensor probe, 5 -ATGGAGTATGTGT- CTGTGG-fluorescein-3, and an anchor probe, 5 -LCR64- ACGAGAGTAAGTAAAACTACAGGCT-phosphate-3, (Roche Applied Sciences, Indianapolis, IN) were used to perform melting curve analysis. The assay conditions for this study were as follows: PCR was performed in.2-ml tubes on the Rotor-Gene 3 in a total volume of 25 μl. The final reaction contained 2 μmol/l of deoxynucleoside triphosphates, 4 mmol/l of magnesium chloride,.1 μmol/l of forward primerç.5 μmol/l of reverse primer, and.2 μmol/l each of the sensor and anchor probes. The following PCR program was used: initial denaturation at 95 C for 2 minutes; 5 amplification cycles at 95 C for 5 seconds, 55 C for 15 seconds, and 72 C for 1 seconds. Melting curve analysis was performed as follows: denaturation at 95 C for 15 seconds, cooling to 4 C for 6 seconds, holding at 5 C for 6 seconds, followed by a melting rate of 1. C per second from 45 C to 75 C. Melting curve analysis was set up as shown in Table 2. JAK2 MutaQuant Assay (Allele-Specific PCR) The JAK2 MutaQuant assay (Ipsogen, Luminy Biotech) detects the JAK2 V617F allele in genomic DNA by real-time detection of fluorescent signals using double-dye hydrolysis oligonucleotide probes, the same as in the JAK2 MutaScreen assay. Calibration standards at 4 different concentrations (included in the kit) were included with each PCR run, and wild-type and mutated alleles were detected in 2 separate reactions for the same sample. All samples were tested in duplicate using the Rotor-Gene 3 and the same protocol 716 Am J Clin Pathol 29;132: DOI: 1.139/AJCPFHUQZ9AGUEKA

5 Hematopathology / Original Article C Fluorescence Positive 1 5% Positive 1 2% Positive 1 1% Positive 1 5% Positive 1 2.5% Positive % Positive 1 Negative 1 1% Mutant % Mutant Reference sample % Mutant Cycle C, JAK2 MutaScreen assay (Ipsogen, Luminy Biotech, Marseille, France). Control DNA positive for JAK2 V617F mutation was serially diluted into negative control DNA to achieve the following concentrations of JAK2 V617F: 1% mutant DNA, 2% mutant DNA, 1% mutant DNA, 5% mutant DNA, 2.5% mutant DNA, 1.25% mutant DNA, and % mutant DNA. as for the JAK2 MutaScreen assay. Use of a standard curve in result analysis allowed precise quantitation of mutant and wild-type alleles. The results were expressed as the percentage of mutant allele in the total reaction mixture. Comparison of Detection Methods For establishing assay sensitivity and the limit of detection for each method, positive (1% mutant) control DNA (InVivoScribe) was serially diluted into negative (1% wildtype) control DNA (InVivoScribe) in the following ratios (and percentages of the mutant clone): 1:1 (5%), 1:1 (1%), 1:2 (5%), 1:4 (2.5%), 1:8 (1.25%), 1:16 (.625%), 1:32 (.313%), 1:64 (.156%), 1:1,28 (.78%), 1:2,56 (.39%), 1:5,12 (.195%), and 1:1,24 (.98%). The serially diluted samples were then analyzed using each of the protocols to determine the limit of detection for each method. Once assay performance had been evaluated, the patient samples were analyzed by each protocol for the presence of the JAK2 V617F mutation. Four of the samples (samples 2 to 23) were not analyzed by the JAK2 MutaQuant assay because of insufficient quantity. Results Analytic Sensitivity The dilution study demonstrated that the 2 semiquantitative kit-based screening methods, the JAK2 Activating Table 2 Melting Curve Analysis Bin Value ( C) Name GG 61 Wild type GT 53/61 Heterozygous TT 53 Homozygous mutant Mutation Assay (Figure 1A) and the JAK2 MutaScreen assay Figure 1C, reliably detected JAK2 V617F mutation with the limit of detection of 1.25% when JAK2 V617F+ DNA was serially diluted into negative control DNA. The melting curve assay proved to be less sensitive, with the dilution of 1% of mutant alleles in the wild-type background Figure 1B showing a hint of positivity with no clear mutant peak and only 2% and 1% of mutant demonstrating clear mutant and wild-type peaks. The quantitative allele-specific PCR technology demonstrated greater sensitivity of detection, as expected. In the serial dilution experiment, the JAK2 MutaQuant assay had a limit of detection of.98% of the mutant allele in the background of wild-type DNA. JAK2 V617F Detection in Patient Samples Comparison of the results for patient samples is summarized in Table 3. Four of the samples were evaluated only by the screening methods because insufficient sample was Am J Clin Pathol 29;132: DOI: 1.139/AJCPFHUQZ9AGUEKA 717

6 Cankovic et al / Method Comparison for JAK2 V617F Detection available for the quantitative assay. Of the 33 cases evaluated, 15 (45%) tested positive by at least one of the methods. Of these, 6 were diagnosed as PV, 8 as ET, and 1 as PMF. Two of the ET samples showed no evidence of the mutant allele. In general, good concordance among all 4 testing methods was observed at higher concentrations of the mutant allele, while the melting curve assay proved less reliable at lower levels. The kit-based screening methods proved superior, showing equal sensitivity in identifying all of the V617F+ cases, except for 1 specimen. Case 25 (a bone marrow aspirate sample) tested as very low-positive with the JAK2 Activating Mutation Assay and was negative with the JAK2 MutaScreen assay on repeated testing. The clinical picture for this patient was indicative of PV, thus JAK2 V617F results were expected to be positive. We interpreted the RFLP-CE result with caution because the inherent weakness of this method is difficulty in distinguishing a low-positive peak from the negative peak owing to incomplete digestion of the wild-type JAK2 sample. In addition, a previous blood specimen from the same patient tested negative by both screening methods. Quantitative testing demonstrated that the mutant clone Figure 2 represented less than.1% of the total population, and the final result was interpreted as positive. To evaluate analytic and clinical specificity, we included control samples that were not expected to carry the JAK2 V617F mutation. The 3 BCR/ABL+ CML cases tested negative by all 4 methods. The 1 normal control samples also tested negative, as did samples from patients diagnosed with conditions other than classic BCR/ABL MPNs. JAK2 V617F Detection in Purified Granulocyte Populations To determine whether enriching for the granulocyte subpopulation would increase assay sensitivity, we compared the analytic performance of the 3 screening methods on a small subset of 1 randomly selected cases. The results between unfractionated populations and the granulocyte populations were identical for all 3 protocols (data not shown), with no improvement in the quality of signals with the granulocyte population. Use of the granulocyte-enrichment protocol before DNA isolation increased hands-on time and complexity of the testing without increasing the sensitivity of the assay. Table 3 Summary of Parallel Results Case No./Sex/Age (y) Sample MutaScreen Assay JAK2 Assay Results JAK2 Melt Curve MutaQuant Assay Concordant 1/F/49 BM Yes 2/F/59 BM Yes 3/F/52 BM Yes 4/M/8 Blood. Yes 5/M/74 BM. Yes 6/F/7 BM. Yes 7/M/66 BM. Yes 8/F/33 BM Yes 9/M/46 BM. Yes 1/M/6 BM Yes 11/M/85 BM Yes 12/F/73 BM Yes 13/M/47 BM No 14/M/57 Blood Yes 15/M/54 BM Yes 16/F/36 BM. Yes 17/M/28 Blood. Yes 18/F/48 BM No 19/M/82 BM. Yes 2/M/59 BM ND Yes 21/M/82 BM ND Yes 22/F/73 BM ND Yes 23/F/19 BM ND Yes 24/M/53 Blood.1 Yes 25/M/53 BM +.1 No 26/M/53 BM. Yes 27/M/64 BM. Yes 28/M/72 BM. Yes 29/F/79 BM Yes 3/M/37 Blood. Yes 31/M/55 BM. Yes 32/M/51 Blood. Yes 33/M/28 BM. Yes BM, bone marrow; ND, not done. 718 Am J Clin Pathol 29;132: DOI: 1.139/AJCPFHUQZ9AGUEKA

7 Hematopathology / Original Article Normal Fluorescence Wild-type amplification 75,254 copies Normal Fluorescence Mutant amplification 6 copies Threshold Cycle Cycle Figure 2 Quantitative JAK2 V617F mutation detection in case 25. Discussion The JAK2 V617F assay is an important tool in the evaluation of MPNs, and commercial and home-brew methods are available. Different testing methods offer different levels of sensitivity and specificity, and these factors, in addition to cost associated with this testing, are important factors to consider when selecting the method best suited to a particular laboratory. In selecting the particular combination of cases for the study, we attempted to put together a group of samples with well-defined clinical and morphologic characteristics in which classic BCR/ABL MPN cases, BCR/ABL+ CML, and cases from conditions other than classic MPN would be represented. These samples were from patients referred for investigation of one or more of the following: high hemoglobin level, high platelet count, neutrophilia, or hematopathologic findings suggestive of MPNs at the time the study was initiated; hence, there are variable numbers of samples in each group. As part of our method validation study, we evaluated 3 different screening methods and 1 quantitative method. Two of the kit-based screening methods, the JAK2 Activating Mutation Assay and the JAK2 MutaScreen assay, demonstrated good performance in detecting the JAK2 V617F mutation, with the limit of detection of 1.25%. Both assays also showed reliable performance with patient samples. In terms of the assay complexity and hands-on time, the Ipsogen JAK2 MutaScreen assay has features that can be very useful in a clinical setting. It is based on the real-time PCR protocol, allowing for ease of use and minimal amount of sample manipulation. The result interpretation was also simple and straightforward because the cutoff point between negative and positive results was set up by the reference sample (provided with the kit). When the mean ratio of the test sample was greater than the mean ratio of the reference sample, the results were interpreted as positive, and when the mean ratio of the test sample was less than the mean ratio of the reference sample, the results were interpreted as negative. In subsequent patient testing (results not shown), we encountered several cases in which the mean ratios of the patient sample and the reference sample were identical. For those cases, subsequent use of the quantitative assay demonstrated low levels (1%-3%) of V617F. The RFLP-CE assay from InVivoScribe also allowed for sensitive and reliable detection of the JAK2 V617F mutant, but post-pcr manipulation required more hands-on time and longer time to obtain results. In this system, PCR is first performed to amplify the portion of the JAK2 region that acquires this mutation. The amplicon products are then digested with the BsaXI restriction digestion enzyme. Differential product detection and relative quantification are determined using capillary electrophoresis. The results are presented as electrophoretograms and are easy to interpret (Figure 1A). Normal samples yield 3-, 97-, and 14-bp products after BsaXI restriction digestion. Because the restriction site is lost in JAK2 mutants, only the original parent 267-bp amplicon products are seen with homozygous mutants. The samples harboring a heterozygous JAK2 V617F will have a mixture of digested and nondigested peaks. The greatest challenge with the RFLP-CE assay in our experience was in interpretation of results for very low-positive samples. Regardless of the mutation status, a small undigested peak was always seen following restriction digestion in all samples, including the negative control sample, which made it challenging to set the cutoff point between the positive and the negative signals with low-positive samples. The melting curve analysis assay demonstrated lower analytic sensitivity and inferior performance compared with Am J Clin Pathol 29;132: DOI: 1.139/AJCPFHUQZ9AGUEKA 719

8 Cankovic et al / Method Comparison for JAK2 V617F Detection the 2 kit-based assays. When the mutant allele was serially diluted into a background of the wild-type allele, this method failed to detect fewer than 1% of mutant copies. This is lower sensitivity than 5% of mutant copies in the wildtype background originally reported for the LightCycler system. 19 This method also gave variable performance with patient samples. For quantitative V617F detection, the JAK2 MutaQuant assay had an analytic sensitivity of.1%. It performed well in patient testing, especially with very low-positive samples. Opinions vary as to what is the optimal sensitivity of the JAK2 assay. Until recently, most of the literature suggested that a sensitivity of 1% to 5% was clinically appropriate and sufficient, but lately, there have been reports of the use of this assay for MRD monitoring. 2 Whether this level of sensitivity should be the goal for clinical testing depends on how the test results will be used in clinical practice. It has been shown that the V617F somatic mutation can be present in only a subset of cells, supporting that a highly sensitive method is needed to reveal the mutation, especially in early stages of disease. On the other hand, sensitivity of the JAK2 assay has to be evaluated in the context of other clinical data because low levels of V617F in healthy people have been reported in several studies. Sidon and colleagues 21 tested blood samples from 57 healthy donors for the JAK2 mutation using molecular beacon probes with a quantitative sensitivity of.1%. They detected a low level of the mutation, corresponding to less than 1 copies of the mutant allele, in 1% of the control samples. In 2 other studies, the JAK2 V617F mutant was found in.94% of 3, and in about.7% of normal control samples. Finally, in their study of allogeneic stem cell transplant recipients with myelofibrosis, Kröger and colleagues 2 detected the mutation in 4 (7%) of 6 healthy control subjects. The significance of these findings is not clear at present. The discovery of the JAK2 mutation is recent enough that long-term clinical follow-up of people who clinically and hematologically have no abnormalities but are JAK2+ is lacking. The 1 normal control samples in our study were all negative for the mutation, yet it is possible that this group was too small to be representative of a much larger population. Interpreting the clinical relevance of mutational load to disease evolution and phenotype variation within the spectrum of myeloproliferative disorders is another challenge. Studies suggest that gene dosage might have a role in determining whether a patient will develop PV, PMF, or ET. Approximately 25% of patients with PV have been shown to be homozygous for the JAK2 V617F allele, whereas most patients with ET are heterozygous or have the wild type. Homozygosity increases the expression of the mutated protein and eliminates the competition with wildtype protein. These data suggest that higher levels of JAK2 V617F expression support an erythroid phenotype and lower levels support a megakaryocytic phenotype. A recent report demonstrated a correlation between hematologic improvement and a reduction in the proportion of the JAK2 V617F mutant alleles. 23 Quantitative measurement of the V617F allele burden could thus become a valuable tool in predicting the disease course In terms of treatment, targeted drugs might become available for patient treatment in the near future, 27,28 and their use will necessitate evaluating the JAK2 V617F allele burden at diagnosis and for subsequent patient monitoring. As per 28 World Health Organization criteria and diagnostic algorithms, 29 peripheral blood JAK2 V617F screening is currently the preferred initial test for evaluating a patient with suspected PV. Because JAK2 V617F also occurs in approximately 5% of patients with ET or PMF, it is advisable to include JAK2 mutation screening in the diagnostic workup of thrombocytosis and bone marrow fibrosis. Our data indicate that all kit-based methods, the JAK2 Activating Mutation and the JAK2 MutaScreen assays and the JAK2 MutaQuant assay, allow for highly precise and sensitive qualitative and quantitative determination, respectively, of patient status at diagnosis and during MRD follow-up. Based on this study, the JAK2 MutaScreen assay, in particular, is a simple, fast, and reliable screening test in the differential diagnosis of PV, ET, and PMF for a majority of patients. For rare cases in which JAK2 results are negative with the screening method but a patient has clinical features strongly suggestive of MPN, the quantitative assay can be subsequently used for greater sensitivity and to possibly determine the precise JAK2 V617F allele burden. From the Department of Pathology and Laboratory Medicine, Henry Ford Hospital, Detroit, MI. Address reprint requests to Dr Cankovic: Dept of Pathology and Laboratory Medicine, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, MI References 1. Levine LR, Gilliland DG. Myeloproliferative disorders. Blood. 28;112: Spivak JL. The chronic myeloproliferative disorders: clonality and clinical heterogeneity. Semin Hematol. 24;41: Levine RL, Loriaux M, Huntly BJP, et al. The JAK2 V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia. Blood. 25;16: Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative diseases. Lancet. 25;365: James C, Ugo V, LeCouedic JP, et al. A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera. Nature. 25;434: Am J Clin Pathol 29;132: DOI: 1.139/AJCPFHUQZ9AGUEKA

9 Hematopathology / Original Article 6. Kralovics R, Passamonti F, Buser AS, et al. A gain-offunction mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 25;352: Levine RL, Wadleigh M, Cools J, et al. Activating mutation of the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell. 25;7: Steensma DP, Dewald GW, Lasho TL, et al. The JAK2 V617F activating tyrosine kinase mutation is an infrequent event in both atypical myeloproliferative disorders and myelodysplastic syndromes. Blood. 25;16: Campbell PJ, Scott LM, Buck G, et al. Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study. Lancet. 25;366: Lindauer K, Loerting T, Liedl KR, et al. Prediction of the structure of human Janus kinase 2 (JAK2) comprising the two carboxy-terminal domains reveals a mechanism for autoregulation. Protein Eng. 21;14: Johansson P, Kutti J, Andréasson B, et al. Trends in the incidence of chronic Philadelphia chromosome negative (Ph ) myeloproliferative disorders in the city of Goteborg, Sweden, during J Intern Med. 24;256: Lasho TL, Mesa R, Gilliland DG, et al. Mutation studies in CD3+, CD19+ and CD34+ cell fractions in myeloproliferative disorders with homozygous JAK2 (V617F) in granulocytes. Br J Haematol. 25;13: Jamieson CH, Gotlib J, Durocher JA. The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation. Proc Natl Acad Sci U S A. 26;13: James C, Delhommeau F, Marzac C, et al. Detection of JAK2 V617F as a first diagnostic test for erythrocytosis. Leukemia. 26;2: Jones AV, Kreil S, Zoi K, et al. Widespread occurrence of the JAK2 V617 mutation in chronic myeloproliferative disorders. Blood. 25;16: Chen Q, Lu P, Jones AV, et al. Amplification refractory mutation system, a highly sensitive and simple polymerase chain reaction assay, for the detection of JAK2 V617F mutation in chronic myeloproliferative disorders. J Mol Diagn. 27;9: Frantz C, Sekora DM, Henley DC, et al. Comparative evaluation of three JAK2 V617F mutation detection methods. Am J Clin Pathol. 27;128: Greiner TC. Diagnostic assays for the JAK2 V617F mutation in chronic myeloproliferative disorders. Am J Clin Pathol. 26;125: Murugesan G, Aboudola S, Szpurka H, et al. Identification of the JAK2 V617F mutation in chronic myeloproliferative disorders using FRET probes and melting curve analysis. Am J Clin Pathol. 26;125: Kröger N, Badbaran A, Holler E, et al. Monitoring of the JAK2-V617F mutation by highly sensitive quantitative real-time PCR after allogeneic stem cell transplantation in patients with myelofibrosis. Blood. 27;19: Sidon P, El Housni H, Desars B, et al. The JAK2 V617F mutation is detectable at very low level in peripheral blood of healthy donors [letter]. Leukemia. 26;2: Xu X, Zhang Q, Luo J, et al. JAK2 V617F : prevalence in a large Chinese hospital population. Blood. 27;19: Jones AV, Silver RT, Waghorn K, et al. Minimal molecular response in polycythemia vera patients treated with imatinib or interferon alpha. Blood. 26;17: Campbell PJ, Baxter EJ, Beer PA, et al. Mutation of JAK2 in the myeloproliferative disorders: timing, clonality studies, cytogenetic associations, and role in leukemic transformation. Blood. 26;18: Vannuchi AM, Antonioli E, Guglielmelli P, et al. Prospective identification of high-risk polycythemia vera patients based on JAK2(V617F) allele burden. Leukemia. 27;21: Vannuchi AM, Antonioli E, Guglielmelli P, et al. Clinical profile of homozygous JAK2 V617F mutation in patients with polycythemia vera or essential thrombocythemia. Blood. 27;11: Walz C, Cross NPC, Van Etten RA, et al. Comparison of mutated ABL1 and JAK2 as oncogenes and drug targets in myeloproliferative disorders. Leukemia. 28;22: Morgan KJ, Gilliland DG. A role for JAK2 mutations in myeloproliferative diseases. Annu Rev Med. 28;59: Tefferi A, Vardiman JW. Classification and diagnosis of myeloproliferative neoplasms: the 28 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia. 28;22: Am J Clin Pathol 29;132: DOI: 1.139/AJCPFHUQZ9AGUEKA 721