Sensitive Detection and Quantification of the JAK2V617F Allele by Real-Time PCR

Abstract

A single G-to-T missense mutation in the gene for the JAK2 tyrosine kinase, leading to a V617F amino acid substitution, is commonly found in several myeloproliferative neoplasms. Reliable quantification of this mutant allele is of increasing clinical and therapeutic interest in predicting and diagnosing this group of neoplasms. Because JAK2V617F is somatically acquired and may be followed by loss of heterozygosity, the percentage of mutant versus wild-type DNA in blood can vary between 0% and almost 100%. Therefore, we developed a real-time PCR assay for detection and quantification of the low-to-high range of the JAK2V617F allele burden. To allow the assay to meet these criteria, amplification of the wild-type JAK2 was blocked with a peptide nucleic acid oligonucleotide. JAK2V617F patient DNA diluted in JAK2 wild-type DNA could be amplified linearly from 0.05% to 100%, with acceptable reproducibility of quantification. The sensitivity of the assay was 0.05% (n = 3 of 3). In 9 of 100 healthy blood donors, a weak positive/background signal was observed in DNA isolated from blood, corresponding to approximately 0.01% JAK2V617F allele. In one healthy individual, we observed this signal in duplicate. The clinical relevance of this finding is not clear. By inhibiting amplification of the wild-type allele, we developed a sensitive and linear real-time PCR assay to detect and quantify JAK2V617F.

In 2005, the involvement of the JAK2V617F somatic point mutation in myeloproliferative neoplasms (MPNs) became clear. JAK2V617F is caused by a G-to-T transversion in the Janus kinase 2 (JAK2) gene resulting in a valine-to-phenylalanine amino acid substitution at codon 617.^1–4 JAK2 is a nonreceptor tyrosine kinase involved in the JAK-STAT signaling pathway.⁵ The autoinhibitory pseudokinase JH2 domain is thought to be by the mutation altered in such a way that the JAK-STAT signaling pathway is constitutively activated, resulting in growth factor–independent cell proliferation.⁶

Since the discovery of JAK2V617F, other mutations in JAK2 and other genes, such as Exon 12, MPL, and TET2, have been described. These mutations occur in a relatively small percentage of patients with MPN.^7–10 JAK2V617F, in contrast, is found in many patients: 65% to 97% of polycythemia vera (PV) cases, 23% to 57% of essential thrombocythemia cases, and 35% to 57% of chronic idiopathic myelofibrosis cases.^11,12 The diagnostic value of JAK2 mutational analysis in MPN is now well established and endorsed in the classification of hematologic malignancies by the World Health Organization.¹³

Patients with PV often carry a JAK2V617F homozygous burden, in contrast to those with essential thrombocythemia, in whom homozygosity is rare.¹⁴ This finding was also recapitulated in a large multicenter study: PV (n = 323: 67.8% heterozygous and 32.2% homozygous) and essential thrombocythemia (n = 639: 40.2% wild-type, 57.6% heterozygous, and 2.2% homozygous).¹⁵ It is, therefore, more common for patients with PV to have a mutant allele burden >50%. Although the clinical value of the JAK2V617F allele burden is not yet fully understood, a correlation between disease phenotype and the proportion of mutant alleles has been postulated.^15,16 Splenomegaly, vascular events and pruritus in PV and splenomegaly, arterial thrombosis, and microvessel disease in essential thrombocythemia have been clinically correlated to mutant allele burdens.^17,18 Patients in the lowest quartile of mutant allele burden in idiopathic myelofibrosis had, overall, significantly shortened overall and leukemia-free survival compared with those with higher allele burdens and those negative for JAK2V617F.¹⁹ In addition, the amount of mutant allele burden in correlation to therapy may be of increasing interest in future diagnostic and predictive tests.²⁰

With the discovery of JAK2V617F, the detection of this mutation became routine procedure in many laboratories. Since 2005, many assays to detect, and sometimes quantify, JAK2V617F have been described, evaluated, and compared.^4,6,21–51 Today, numerous techniques are used: PCR–restriction fragment length polymorphism (RFLP), direct sequencing, pyrosequencing, allele-specific PCR/amplification refractory mutation system, allelic discrimination, real-time PCR, high-resolution melting curve analysis, PCR, denaturing high-pressure liquid chromatography, PCR–matrix-assisted laser desorption/ionization–time of flight (PCR-MALDI-TOF) mass spectrometry, and sequence-specific primer–single molecule fluorescence detection. The techniques differ in a variety of characteristics, such as sensitivity (ranging from 0.01% to 20%, mutant allele diluted in wild-type allele), the potential for high-throughput diagnostics, and equipment required. An overview of currently available assays is presented in Table 1.

Table 1.

Overview and Some Characteristics of Currently Available JAK2V617F Detection (and Quantification) Techniques

Technique	Sensitivity (%)	Advantages	Disadvantages	References
PCR-RFLP	1–20	No special equipment required, inexpensive	Risk of false positives due to incomplete digestion, labor intensive, post-PCR processing required	4, 6, 21–26
Direct sequencing	5–20	Simultaneous detection of other mutations	Labor intensive, sequencing equipment required, post-PCR processing required	4, 6, 22, 23, 25, 27, 28
Pyrosequencing	2–10	Simultaneous (real-time) detection of other mutations	Pyrosequencing equipment required, expensive	6, 28, 29
AS-PCR/ARMS	0.01–5	Highly sensitive	Post-PCR processing or real-time cycler required	6, 21–25, 28, 30–33
Real-time PCR	0.01–5	Highly sensitive, high-throughput, fast	Real-time cycler required	6, 21, 24, 26, 27, 33–41
HRM	0.5–10	Detection of other mutations	Real-time cycler required	6, 21, 25, 27, 38, 42–45
Allelic discrimination	0.1	Variable amplicon detection techniques possible	Real-time cycler required	46, 47
dHPLC	1–2.5	High-throughput, fast	Post-PCR processing required, technically challenging	6, 23, 48
PCR-MALDI-TOF	0.01–1	Highly sensitive, high-throughput, fast	MALDI-TOF equipment required, post-PCR processing required	49, 50
SSP-SMFD	5	High-throughput	Automated fluorescence cell sorter required, expensive	50

AS-PCR/ARMS, allele-specific PCR/amplification refractory mutation system; dHPLC, denaturing high-pressure liquid chromatography; HRM, high-resolution melting curve analysis; PCR-MALDI-TOF, PCR -matrix-assisted laser desorption/ionization -time of flight; PCR-RFLP, PCR -restriction fragment length polymorphism; SSP-SMFD, sequence-specific primer–single molecule fluorescence detection.

To increase the sensitivity of the JAK2V617F diagnostics, several techniques have been used in our laboratory during the past years. We started with an RFLP assay and subsequently used a semiquantitative real-time PCR and allele-specific minor groove–binding probes.⁴¹ We then developed a quantitative real-time PCR using a locked nucleic acid (LNA) oligonucleotide to block amplification of the JAK2 wild-type allele. We report herein replacement of the LNA moiety by peptide nucleic acid (PNA) for better blocking.

Materials and Methods

JAK2V617F-Positive Blood Sample

JAK2V617F-positive blood containing 97% mutant allele, estimated by RFLP, the absence of a wild-type peak in the sequence chromatogram, the presence of minimal wild-type signal in real-time PCR, and a hematologic analysis showing 3% lymphoid cells and 97% myeloid cells together with a high white blood cell count (23.2 × 10⁹/L)⁴¹ was derived from a patient with PV and generated a mean ± SD C_T value of 24.13 ± 0.02. A 100-fold dilution from DNA from this sample in JAK2 wild-type DNA was used as a 1% JAK2V617F-containing positive control.

A second positive blood sample, used for serial dilutions in this study, was obtained from a 64-year-old man diagnosed as having possible PV, with a high number of erythrocytes [7.64 × 10¹²/L (reference range, 4.4 to 5.8 × 10¹²/L)] and iron deficiency. JAK2V617F real-time PCR using the PNA oligonucleotide on genomic DNA derived from this sample generated a mean ± SD C_T value of 23.88 ± 0.07. Thus, the sample used in this study was considered to contain >97% JAK2V617F.

Cohort Healthy Donors

EDTA blood samples from 100 healthy blood donors—rendered anonymous—were used as a negative cohort. All the individuals were <30 years of age.

Genomic DNA Isolation from Blood

Genomic DNA was isolated from 200 μL of blood that had been anticoagulated with EDTA using the Qiagen blood mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA was eluted in DNase- and RNase-free water (Fisher Emergo B.V., Landsmeer, The Netherlands), quantified by photo spectrometer (Eppendorf BioPhotometer; Eppendorf, Hamburg, Germany), and diluted to 10 ng/μL in DNase- and RNase-free TE (10 mmol/L Tris-HCl, pH 8; 0.1 mmol/L EDTA).

JAK2V617F Real-Time PCR Assay with PNA and LNA Blocking

Primers and probes were designed by Applied Biosystems (Foster City, CA) using TaqExpress software as previously described.⁴¹ The wild-type probe was altered to function as a blocking oligonucleotide with PNA or LNA. Oligonucleotide sequences are listed in Table 2. The PCR (25 μL) contained 20 mmol/L Tris-HCl (pH 8.4), 50 mmol/L KCl, 3 mmol/L MgCl₂ [prepared from 10× PCR buffer and 50 mmol/L MgCl₂ solution delivered with Platinum Taq polymerase (Invitrogen, Breda, The Netherlands)], 0.75 U of Platinum Taq polymerase, 4% glycerol (molecular biology grade; CalBiochem, VWR International, Amsterdam, The Netherlands), 200 μmol/L of each deoxyribonucleotide triphosphate (Invitrogen), 0.5 μL of ROX reference dye (Invitrogen), 900 nmol/L primers and 200 nmol/L probes (Applied Biosystems), 1 μmol/L PNA oligonucleotide (PanaGene, Daejeon, Korea; alternate source: Biosynthesis Inc., Lewisville, TX) or LNA oligonucleotide (Sigma-Proligo, The Woodlands, TX), and 50 ng of target DNA. The ABI Prism 7500 Fast sequence detection system (Applied Biosystems) was used for amplification and detection (10 minutes at 95°C, 45 cycles of 3 seconds at 95°C, and 30 seconds at 60°C and an infinite holding step at 25°C in Fast 7500 mode). The threshold was set at 0.008, and baseline was set from cycles 6 to 15 for both reporters. C_T values were calculated using SDS version 1.3.1 software. A real-time PCR targeting the human albumin gene was used to test whether DNA extraction and dilution to 10 ng/μL were successful and to verify that the PCR was not inhibited. Amplification was performed as described previously herein with 900 nmol/L both primers and 200 nmol/L probe. Oligonucleotide sequences were described earlier and are denoted in Table 2.⁵²

Table 2.

Sequences of Primers, Detection Probes, and PNA and LNA Blocking Oligonucleotides to Detect and Quantify JAK2V617F and to Detect Albumin by Real-Time PCR

JAK2 V617F: primers and mutant probe41	Sequences
Forward primer	5′-AAGCTTTCTCACAAGCATTTGGTTT-3′
Reverse primer	5′-AGAAAGGCATTAGAAAGCCTGTAGTT-3′
Mutant probe	5′-FAM-TCCACAGAAACATAC-MGB-BHQ-3′
LNA wild-type blocking probe	5′-ACAGACACATA-3′
PNA wild-type blocking probe	5′-Acetyl-CTCCACAGACACATAC-3′

Albumin: primers and probe [53]	Sequences
Forward primer	5′-TGAAACATACGTTCCCAAAGAGTTT-3′
Reverse primer	5′-CTCTCCTTCTCAGAAAGTGTGCATAT-3′
Probe	5′-FAM-TGCTGAAACATTCACCTTCCATGCAGA-TAMRA-3′

BHQ, black hole quencher; MGB, minor groove binder.

Sensitivity and Linearity Testing of PNA versus LNA Blocking

To compare PNA and LNA blocking oligonucleotides, a JAK2V617F real-time PCR was performed on twofold (v/v) serial dilutions of genomic DNA from the patient sample containing >97% JAK2V617F. JAK2V617F DNA (10 ng/μL) was diluted in genomic DNA (10 ng/μL) of a JAK2 wild-type individual. The lowest JAK2V617F concentration tested was 0.006% v/v. All the dilutions were tested in triplicate.

Results

Sensitivity and Linearity Testing of PNA versus LNA Blocking

LNA nucleotides are generally used as blocking oligonucleotides.^26,39,53 Because our data using the LNA oligonucleotide showed suboptimal sensitivity of 0.2%, we introduced a PNA blocker approach. To determine the sensitivity and linearity of the real-time PCR test using the PNA oligonucleotide versus the LNA oligonucleotide, twofold (v/v) serial dilutions of genomic DNA from a patient with PV containing 0.006% to >97% JAK2V617F were tested. The assay combined with the PNA oligonucleotide linearly detected JAK2V617F with a for quantification acceptable (mean C_T value ± 1 C_T) reproducibility ranging to be approximately 97% to 0.05% (n = 3 of 3). This range was determined for the real-time PCR with the LNA oligonucleotide to be 97% to 0.2% (n = 3 of 3; Figure 1).

Figure 1.

Standard curves (using twofold dilution steps) generated by JAK2V617F real-time PCR using PNA blocking (squares) and LNA blocking (triangles). Data are given as mean Ct ± SD.

Cohort Healthy Donors

One hundred healthy EDTA blood donors were tested in duplicate using the real-time PCR and the PNA blocking oligonucleotide. All individuals tested positive for the albumin housekeeping gene (C_T: mean ± SD, 23.16 ± 0.19; range, 22.60 to 23.88). All DNA isolations were considered successful with this value, and none of the samples showed PCR inhibition. Ninety-one patients (91%) were JAK2V617F negative. In nine patients (9%), a background signal corresponding to ±0.01% mutant allele was observed in one or both duplicates (eight patients and one patient, respectively). Two representative amplification plots are depicted in Figure 2. The results are summarized in Table 3.

Figure 2.

Amplification plots of JAK2V617F real-time PCR of 25 (A) and 22 (B) healthy individuals in duplicate (JAK2V617F negative cohort) and one 1% JAK2V617F positive control. The amplification plots shown are representative of the entire cohort. Gray horizontal line (at Delta Rn 0.8e-002) indicates threshold; black vertical line, cutoff C_T value of 37.9.

Table 3.

JAK2V617F Test Results from a Negative Cohort of 100 Healthy Individuals

Result	n	Description
Total results	200	100 healthy individuals tested in duplicate
Negative results	190	Negative for JAK2V617F/positive for albumin
Background CT ≳ 37.9	10	Mean background signal ± 3 SD (99.7% CI); eight patients single background signal; one patient in duplicate background signal

The samples were amplified in duplicate (n = 2 × 100). Signals were considered background when C_T ≳ 37.9.

Discussion

We developed a highly sensitive JAK2V617F detection and quantification method by inhibiting amplification of the JAK2 wild-type allele by means of a PNA wild-type blocking oligonucleotide. All C_T values generated during sensitivity and linearity testing that were higher than the cutoff C_T value of 37.9 [determined with the cohort of healthy donors = mean C_T value ±3 SD (99.7% CI)] were excluded. Compared with LNA blocking, PNA blocking resulted in a more sensitive and linear assay using the dilution series of JAK2V617F DNA in JAK2 wild-type DNA: 0.2% to ±97% JAK2V617F DNA versus 0.05% to ±97% JAK2V617F DNA (Figure 1). More efficient/stable blocking by the PNA oligonucleotide probably causes the difference in sensitivity and linearity of the real-time PCR using the PNA blocker versus the LNA blocker. Unlike DNA or DNA analogues such as LNA, PNAs do not contain any phosphate groups, resulting in a noncharged backbone. As a result, PNA-DNA complexes lack charge repulsion and are, therefore, more stable than are DNA-DNA and DNA-LNA complexes.^54,55

Although the clinical significance of a low JAK2V617F allelic burden in newly diagnosed patients is not clear,^38,40,52 highly sensitive mutant allele detection is currently used for minimal residual disease detection after allogeneic bone marrow transplantation.⁵⁶ In addition, quantification of JAK2V617F will help in monitoring new therapeutic strategies. A prerequisite for reliable quantification is the linearity of the assays from high to low allele burdens.⁵⁷

In the cohort of 100 healthy individuals (Table 3), 9% generated JAK2V617F background signals (Figure 2). A weak positive/background JAK2V617F signal was observed in duplicate in one healthy individual. First, these background signals could be due to a technical issue, such as incomplete blocking of wild-type amplification. Second, JAK2V617F could be present in these patients in an extremely small number of circulating cells. Although this phenomenon has been described for other JAK2V617F assays, its significance is not clear.^38,40,52 To prevent false positives in the clinical setting, a cutoff C_T value was determined. The mean C_T value (C_T values >40 are generally considered unreliable and were, therefore, excluded) ± 3 SD of the negative cohort was determined, and signals with a generated value of C_T ≥ 37.9 (CI = 99.7%) were considered to be background. One patient generated duplicate JAK2V617F background signals (Table 3). Although both C_T values were ≥37.9 and should be considered background, further analysis of this material may be of interest. Cloning and subsequent sequencing of these amplicons could be of value to determine whether the signals were nonspecific or that JAKV617F was, in fact, present. In addition, an assessment of the presence of erythropoietin-independent erythroid colonies might be useful in this and similar cases.⁵⁸ In addition, such a patient could undergo testing for the presence of the JAK2V617F predisposing haplotype.^59–61 In the routine diagnostic setting, a JAK2V617F allele burden of 0.1% to 1.0% is considered weak positive, and a remark stating that the clinical relevance of this finding is unclear is added to the test report. In addition, we have requested a follow-up sample after 6 months.

Although sequencing is the gold standard for mutation screening, it is less suitable for the detection of JAK2V617F because the mutation can be present at low levels relative to the wild-type sequence. PCR-RFLP, pyrosequencing, sequence-specific primer–single molecule fluorescence detection, high-resolution melting curve analysis, and denaturing high-pressure liquid chromatography are all less sensitive than is the real-time PCR described herein (Table 1), which has a sensitivity of 0.05% and, thereby, is comparable with techniques such as allele-specific PCR/amplification refractory mutation system, allelic discrimination, and PCR-MALDI-TOF (Table 1).^4,21–51 In addition, interpretation of PCR-RFLP results is complicated by the risk of incomplete digestion, resulting in JAK2V617F false positives (Table 1).

Besides the higher or equal sensitivity, the real-time PCR using the PNA oligonucleotide has some additional advantages compared with other assays: i) it is fast (<2 hours for DNA extraction and real-time PCR); ii) it eliminates the need of post-PCR processing, a key step in techniques such as allele-specific PCR/amplification refractory mutation system, sequencing, PCR-RFLP, PCR, and PCR-MALDI-TOF, reducing the risk of contamination; and iii) it has a reduced hands-on time of approximately 30 minutes.

In 2006, Sidon et al^39,40 described a similar real-time PCR as the assay described in this study using an LNA blocking oligonucleotide and claimed a sensitivity of their assay of 0.01% JAK2V617F diluted in wild-type allele. The sensitivity of this test, however, was determined using a HEL cell line, which is known to have more than two mutant alleles per cell, instead of patient DNA.²⁹

Prospective evaluation of the real-time PCR with PNA oligonucleotide in a diagnostic setting for 1 year now included the analysis of 293 patients suspected of having MPN. Fourteen patients (4.8%) showed a JAK2V617F allele burden >50%, 16 (5.5%) between 1% and 50%, and 2 (0.7%) between 0.1% and 1%. The results generated during the performance of routine JAK2 diagnostics indicate a robust and rapid test with excellent reproducibility: the overall CV with different batches of controls, reagents, and various real-time PCR cyclers was 1.5% for the 1% control (n = 27) and 1.4% for the 50% control (n = 24). Reproducibility is essential for a reliable quantitative assay⁵⁷; therefore, these observations further confirm that the developed assay is well suited for the application.

In summary, blocking of wild-type JAK2 with the PNA oligonucleotide resulted in a more sensitive real-time PCR assay to detect and quantify JAK2V617F with a longer range of linearity compared with the LNA oligonucleotide approach. Nine percent of healthy individuals in this negative cohort generated background JAK2V617F signals. Therefore, a cutoff C_T value was introduced to prevent false-positive test results in a clinical setting.