A Simple, Rapid, and Sensitive Method for the Detection

Hematopathology / S IMPLE AND S ENSITIVE PCR FOR JAK2 V617F A Simple,Rapid,and Sensitive Method for the Detection of the JAK2 V617F Mutation

Angela Y.C. Tan, PhD,1*David A. Westerman, MBBS, FRACP, FRCPA,1,2

and Alexander Dobrovic, PhD1,2

Key Words: JAK2; Mutation detection; V617F; Polymerase chain reaction; PCR; Method; Chronic myeloproliferative disorders

DOI: 10.1309/1U61JVXTLPPQ7YP1

A b s t r a c t

The point mutation 1849 (G→T) V617F in the JAK2 gene occurs at high frequency in several chronic myeloproliferative diseases. Although a number of

V617F mutation detection methods have been described, few are readily implemented in a diagnostic setting. We developed a simple and sensitive allele-specific competitive blocker polymerase chain reaction (ACB-PCR) assay to detect the V617F mutation. DNA was extracted from peripheral whole blood samples of 26 patients with chronic myeloproliferative disease. The ACB-PCR limit of detection was 1%. All positive samples detected by sequencing were detected by ACB-PCR. In 3 patients with essential thrombocythemia, the V617F mutation was readily detected by ACB-PCR but was near the detection limit of sequencing, confirming that ACB-PCR is more effective at detecting V617F when the mutant cell population is low. Detection of the monomorphic JAK2 V617F mutation using the ACB-PCR assay is easy to perform, rapid, sensitive, and cost-effective, which are key features of an ideal diagnostic method.

Janus kinase 2 (JAK2) is a member of a group of cyto-plasmic tyrosine kinases are that involved in the transduction of signals from growth factor receptors.1JAK2 is a key play-er in the JAK–signal transducer and activator of transcription (STA T) pathway. On autophosphorylation following activa-tion via ligand binding, JAK2 recruits STA T molecules, which are then phosphorylated and translocate to the nucleus to act as transcription factors.1

It has been recently reported that a somatic point muta-tion, 1849 (G→T), in the JAK2gene causing phenylalanine to be substituted for valine in codon 617 (V617F) is present in a number of myeloproliferative disorders such as polycythemia vera (PRV [65%-93%]), essential thrombocythemia (ET [23%-57%]), idiopathic myelofibrosis (IMF [43%-53%]), and, to a smaller extent (~5%), chronic myelomonocytic leukemia, myelodysplastic syndrome, systemic mastocytosis, and chronic neutrophilic leukemia.2-8This mutation disrupts the autoinhibitory J

2 domain of JAK2,9which leads to the constitutive activation of J AK2 and, subsequently, uncon-trolled proliferation.2No other mutations in JAK2have been detected, a consistent finding across the 4 independent groups who initially described the V617F mutation.2-5

The incidence of the V617F mutation in patients with bcr-abl– myeloproliferative disorders varies, but defining the presence or absence of this mutation is now part of clinical diagnostic algorithms,10and, in the future, will be part of the assessment for treatment with the potential introduction of JAK2 inhibitors.

Several groups have developed assays to enable detec-tion of the V617F mutation (recently reviewed by Greiner11). Some of these methods have been inappropriate for a rou-tine diagnostic laboratory because they are comparatively

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Tan et al / S IMPLE AND S ENSITIVE PCR FOR JAK2 V617F

labor-intensive methods with low detection sensitivities, eg, restriction enzyme (Bsa XI) digestion following polymerase chain reaction (PCR)12,13or sequencing.2-5Other methods are relatively complex, such as those approaches with a pyro-sequencer,7,8fluorescence probes,14-16or denaturing high-per-formance liquid chromatography,17considering the monomor-phic nature of the V617F mutation.

We developed a diagnostic allele-specific competitive blocker (ACB)-PCR assay to identify the V617F mutation. The sensitivity of the assay allows it to be performed on peripheral whole blood DNA without the need to separate the granulocytes. The ACB-PCR assay is quick, easy, and cost-effective. In addition, it is highly sensitive, permitting detec-tion of the mutation in patients with low mutant cell levels. Materials and Methods

Samples

The peripheral blood samples of 26 patients with myelo-proliferative disease (PRV, 8; ET, 15; IMF, 2; postpoly-cythemic myelofibrosis and myeloid metaplasia, 1) and 4 patients with lymphoproliferative disease were analyzed by the Pathology Department, the Peter MacCallum Cancer Centre, East Melbourne, Australia. The patients were under-going studies for hematologic disease. Normal peripheral blood samples were obtained from 16 healthy volunteers. All samples were collected into tubes containing EDTA and were obtained in accordance with the Peter MacCallum Cancer Centre Ethics of Human Research guidelines.

DNA Extraction

DNA was isolated from the peripheral blood samples using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) according to the manufacturer’s instructions. ACB Assay

Approximately 100 ng of DNA was amplified in a total volume of 25 μL containing 200 nmol/L of Normal_f primer 5'-GCATTTGGTTTTAAATTATGGAGTATGTG-phosphate (a phosphate group was added to the 3' end to prevent exten-sion from the normal primer, blocking amplification of the normal allele), 400 nmol/L each of Mutant_f primer 5'-GCATTTGGTTTTAAATTATGGAGTATGAT and reverse primer 5'-ACTGACACCTAGCTGTGA TCCTG, 2 mmol/L of magnesium chloride, 50 μmol/L of each deoxynucleotide triphosphate (dNTP), 0.5 U of HotStar Taq (Qiagen, Hilden, Germany), and 1× buffer (Qiagen). All primers were pur-chased from Sigma Aldrich (Castle Hill, Australia). The cycling conditions were 95°C (15 minutes) and 45 cycles of 94°C (30 seconds), 64°C (30 seconds), 72°C (30 seconds),and a final 72°C extension for 10 minutes. The 139-base-pair products were visualized on a 2% ethidium bromide–stained agarose gel. The presence of a band indicated that the sample was positive for the V617F mutation. All samples were first amplified using the control PCR reaction of JAK2(see the next section). Each sample was set up in triplicate in the ACB-PCR assay to ensure that no false-negative results were obtained, and at least 3 negative control samples were run alongside the test samples to exclude false-positive results. Control Reaction PCR of JAK2

A control PCR reaction that amplifies exon 14 of JAK2 was performed for all samples to exclude false-negative results in case the extracted DNA samples were not of ade-quate quality to amplify. The amplification was performed as described for the ACB-PCR assay except the primers Control_f 5'-TTCCTTAGTCTTTCTTTGAAGCAG and Control_r 5'-ACTGACACCTAGCTGTGA TCCTG were used with 200 μmol/L of dNTPs. The 191-base-pair products were visualized on a 2% ethidium bromide–stained agarose gel. Direct Sequencing

To validate the ACB-PCR assay, each sample was ana-lyzed by direct sequencing of the control reaction PCR prod-ucts (exon 12, JAK2) using a standard fluorescent dye method. Briefly, products were incubated with EXO-SAP-IT (USB, Cleveland, OH), following the manufacturer’s instructions. The enzymatically cleaned PCR products were used as a tem-plate for the cycle sequencing reaction. Forward and reverse sequencing were performed using Big Dye Terminator version 3.1 chemistry (Applied Biosystems, Foster City, CA) accord-ing to the manufacturer’s instructions. After cycle sequencing was completed, products were ethanol precipitated and, after addition of Hi-Di (Applied Biosystems), analyzed on an ABI 3100 (Applied Biosystems).

Results

Optimization

The important variables for the ACB-PCR assay to detect the V617F mutation were the annealing temperature, dNTP concentration, and primer concentration. The majority of the conditions were modeled from a previous ACB-PCR assay.18 Annealing temperatures of 60°C, 62°C, 64°C, and 68°C were initially used with a low concentration of 50 μmol/L of dNTPs to test the specificity of the primers. A low concentration of dNTPs encourages the correct base to be incorporated during the PCR reaction, increasing fidelity and accuracy at the criti-cal 3' end of the primers. In addition, it was previously found that the ideal primer concentration was when the mutant primer and normal primer (with the 3' phosphate blocking

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group) were used at a 2:1 ratio. The conditions were deemed optimal when repeated negative control DNA samples did not amplify while the positive patient samples were robustly amplified. These conditions are those described in the “Materials and Methods” section. If the ACB-PCR assay is performed under nonoptimal conditions, mispriming may occur from the normal primer and amplification would be observed in the negative control samples.

Detection Sensitivity

The sensitivity of the ACB-PCR assay was assessed using serial dilutions of the HEL cell line DNA (which carries the V617F mutation) with DNA of normal control peripheral blood samples ?Image 1A ?. Amplification products were visi-ble to the level of 1%. This level of sensitivity is higher than that of sequencing (10%-40%),2-5pyrosequencing (5%-10%),7,8and melt curve analysis (1%-10%).14-16Furthermore,

peripheral whole blood extraction using a commercial kit fur-ther simplified the assay because extra steps to isolate granu-locytes for low-level disease detection were unnecessary owing to the high sensitivity of the ACB-PCR assay.Patient Samples

Samples from 26 patients with myeloproliferative disease were analyzed using the ACB-PCR assay ?Image 1B ?, and the results were correlated with the sequencing results ?Table 1?.All ACB-PCR results were concordant with the direct sequencing results. However, analysis of the sequencing chro-matograms of 3 patients with ET revealed that in 2 patients,the mutation could not be distinguished from the background and in 1 patient, the mutation was observed but at the limit of detection. No amplification occurred in the ACB-PCR assays of samples from the 16 healthy subjects and 4 patients with lymphoproliferative diseases.

Hematopathology / O RIGINAL A RTICLE

HEL

50%25%10%

0.5%NTC

N 1

N 2

N 3

N 4

N 5

N 6

NTC +

T 1

T 2

T 3

T 4

T 5

T 6

T 7

T 8

T 9

T 10

?Image 1?The allele-specific competitive blocker polymerase chain reaction (ACB-PCR) assay. A , Sensitivity of the assay. DNA from the HEL cell line (homozygous for the V617F mutation) was serially diluted with DNA from a normal individual (N) and amplified in the ACB-PCR assay. HEL, undiluted (100%); 50%, HEL diluted 1/2; 25%, HEL diluted 1/4; 10%, HEL diluted 1/10;5%, HEL diluted 1/20; 1%, HEL diluted 1/100; and 0.5%, HEL diluted 1/200. The marker (M) is pUC19/HpaII. NTC, no template control. B , Detection of the V617F JAK2 mutation in patients undergoing studies for myeloproliferative disease. DNA was

extracted from the peripheral blood samples of 16 healthy volunteers and 26 patients with myeloproliferative disease from the Peter MacCallum Cancer Centre, East Melbourne, Australia (in compliance with the institute’s ethics and human research

guidelines) and subjected to the ACB-PCR assay. T en test (T) samples are shown and compared with 6 normal control samples (N 1-N 6), an NTC and a V617F+ patient control sample (+). The M is pUC19/HpaII. Amplified samples T 3-T 6were positive by the ACB-PCR assay and were confirmed V617F+ by direct sequencing. Samples T 1, T 2, and T 7-T 10were negative by the ACB-PCR assay and also confirmed negative by direct sequencing.

Tan et al / S IMPLE AND S ENSITIVE PCR FOR JAK2 V617F

Discussion

The ACB-PCR (first described by Orou et al19) is based on the amplification refractory mutation system method20in which relevant alleles are targeted using specific primers. Allele-specific assays such as the amplification refractory mutation system are efficient techniques for the detection of monomorphic mutations because they are simple and easy to perform. However, standard allele-specific assays can be prone to mispriming.12,21,22Therefore, we chose to adopt an ACB-PCR assay to detect the V617F JAK2mutation. In an ACB-PCR assay, 3 primers are combined in a single tube, a forward primer with a modified 3' end, a second forward primer that targets the mutant allele, and a reverse primer. Under optimized conditions, only the mutant allele (the allele of interest) is amplified, and owing to the 3' modification of the normal primer, amplification of the normal allele is inhibited.

Theoretically, any blocking group can be incorporated at the 3' end of the normal primer to prevent amplification, ie, a dideoxynucleotide. In this study, a phosphate group was select-ed because it was an inexpensive modification that was widely available from commercial oligonucleotide companies.

Similar to any PCR test, some optimization of the ACB-PCR assay was required but was minimal owing to the attach-ment of the 3' phosphate group on the normal primer. Under optimal routine conditions, the ACB-PCR assay was able to detect the V617F mutation at the level of 1% or approxi-mately 1 mutant cell per 100 normal cells. This high level of detection sensitivity was equivalent to that of previous allele-specific assays.18

The advantage of such a high detection level of the ACB-PCR assay was made apparent when studying a cohort of ET cases. In 3 ET cases, the V617F mutation would not have been detected by sequencing because the mutant peak was at the level of background on the chromatograms. Low levels of V617F in patients with ET may be due to heterogeneity in the granulocyte population, and the potential to miss the V617F mutation in some patients with ET using less sensitive tech-niques has been reported.23,24

We detected the JAK2V617F mutation in 6 of 8 patients with PRV, 10 of 15 with ET, and 1 of 2 with IMF. The pattern of incidence in each of the disease states is consistent with previous reports.2-5,7The mutation was not present in the patient with postpolycythemic myelofibrosis and myeloid metaplasia.

The ACB-PCR assay described in this article has subse-quently been implemented in the routine diagnostic laborato-ry of the Pathology Department, the Peter MacCallum Cancer Centre. Currently, the V617F mutation has been identified in 15 (83%) of 18 patients with PRV, 14 (56%) of 25 with ET,

?Table 1?

Clinical Details and Mutation Status of Samples From 26 Patients With Myeloproliferative Diseases

Case No./Sex/Age (y)Diagnosis V617F ACB-PCR Direct Sequencing

1/M/69PRV transformed to AML Positive Positive

2/F/73ET Positive Positive

3/M/41ET Negative Negative

4/F/86PRV Positive Positive

5/M/51IMF Negative Negative

6/F/46PRV Positive Positive

7/M/66IMF Positive Positive

8/M/37PRV Positive Positive

9/M/74PRV Positive Positive

10/F/49ET Negative Negative

11/F/54ET Negative Negative

12/M/70PRV Negative Negative

13/M/87ET Positive Positive

14/M/67PRV Negative Negative

15/F/46ET Positive Positive

16/F/25ET Positive Positive

17/F/45ET Negative Negative

18/F/70PPMF*Negative Negative

19/F/91ET Positive Positive

20/M/57PRV Positive Positive

21/M/85ET Negative Negative

22/F/55ET Positive Indeterminate?

23/F/79ET Positive Indeterminate?

24/M/72ET Positive Positive

25/F/43ET Positive Positive

26/M/50ET Positive Indeterminate?

ACB-PCR, allele-specific competitive blocker polymerase chain reaction; AML, acute myeloid leukemia; ET, essential thrombocythemia; IMF, idiopathic myelofibrosis; PPMF, postpolycythemic myelofibrosis and myeloid metaplasia; PRV, polycythemia vera.

*Reactive; initially thought to be a myeloproliferative disease but was later classified as a reactive condition.

?The mutation peaks were near the detection limit of sequencing.

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and 5 (71%) of 7 with IMF. The ACB-PCR assay is rapid and highly sensitive. It is a suitable technique for V617F mutation detection with wider appeal for routine diagnostic laboratories because it uses standard equipment, has few sample manipu-lation steps, and can be performed directly on DNA extracted from peripheral whole blood samples.

From the Departments of Pathology, 1Division of Haematology and Medical Oncology, Peter MacCallum Cancer Centre, East Melbourne; and 2the University of Melbourne, Parkville, Australia.

Address reprint requests to Dr Tan: Dept of Pathology, Peter MacCallum Cancer Centre, Locked Bag No. 1 A’Beckett Street, Melbourne, Victoria, 8006, Australia.

*Dr Tan’s position is funded by Novartis Pharmaceuticals, North Ryde, Australia.

Acknowledgments: We thank Serge Kovalenko and Michelle McBean, Molecular Pathology Diagnostic Haematology Laboratory, Peter MacCallum Cancer Centre, for making their results available to us.

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Hematopathology / O RIGINAL A RTICLE

Am J Clin Pathol2007;127:977-981 981

DOI: 10.1309/1U61JVXTLPPQ7YP1

? American Society for Clinical Pathology