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From the Centre for Clinical Immunology and Biomedical Statistics,
* Institute for Clinical Research; Cellular and Tissue Therapies,
Royal Perth Hospital, Perth; and the Department of Haematology,
Pathwest, Royal Perth Hospital, Perth, Western Australia, Australia
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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Granulocyte Preparation, DNA and RNA Extraction, and cDNA Synthesis
Blood sampling was performed after informed consent either at diagnosis or during the follow-up. Granulocytes were separated by differential centrifugation using a density gradient (Polymorphprep; Axis Shield, Oslo, Norway), starting from 30 ml of peripheral blood collected in ethylenediaminetetraacetic acid within 24 hours from sampling. When necessary, contaminating red cells were removed by hypotonic lysis. Cell pellets, which contained >98% of granulocytes by visual inspection of cytospins, were extracted for DNA or RNA using the QIAmp DNA Blood Mini kit (Qiagen, Valencia, CA) and RNeasy Mini kit (Qiagen), respectively, both according to the manufacturer’s protocol. Concentrations were determined using NanoDrop technology (Biolab, Auckland, New Zealand). cDNA was reverse transcribed starting from 100 ng of RNA with oligo dT primers using Omniscript reverse transcriptase (Qiagen) according to the recommended protocol. For three patients with high granulocytic mutational load, we also measured mutational load from CD14+ or CD3+ cells using immunomagnetic beads (Invitrogen, Perth, Australia).
Quantitative Determination of JAK2 V617F by TaqMan
A polymerase chain reaction (PCR) assay using TaqMan (fluorescence-based, real-time PCR) and minor groove binding probes was designed for the specific and quantitative determination of DNA copy number of JAK2 V617F in clinical samples. The 7700 Sequence Detection System (ABI, Melbourne, Australia) amplifies and monitors an increase in fluorescence generated by binding of the allele-specific probe during the PCR cycle. In the intact probe, the quencher absorbs fluorescence emitted by the reporter. The 5' nuclease activity of the polymerase degrades the hybridization probe during replication, thereby releasing the reporter and producing an increase in fluorescent emission. The point at which the fluorescent signal rises above the background, or the crossing threshold (Ct), is directly proportional to the input DNA and is thus a measure of the starting template. The test uses a forward primer that has been designed with deliberate mismatches to specifically amplify the mutant sequence.
Twenty nanograms of DNA was added to MicroAmp Reaction Plate (ABI) tubes containing 20 µl of reaction mix. The working master mix contained 5 pmol of mutant-specific forward primer 5'-AGCATTTGGTTTTAAATTATGGAGTATaTT-3' containing a deliberate mismatch and 5 pmol of non-allele-specific reverse primer 5'-CAAAAACAGATGCTCTGAGAAAGG-3', 2x Taqman Universal Master mix [containing heat-activated AmpliTaq Gold DNA polymerase, AmpErase uracil-N-glycosylase for carryover prevention, dNTPs with dUTP, Passive Reference dye (Rox), and buffer components; ABI], and 5 pmol of a 5-carboxyfluorescein (FAM) fluorescently labeled probe specific for the reverse strand of the mutant allele, 5'-fam-ctccacagaaacatactc-3'.
JAK2 V617F amplifications were performed using the following cycling conditions. One cycle at 50°C for 2 minutes of incubation was performed for activation of AmpErase uracil-N-glycosylase, followed by a 10-minute incubation at 95°C for amplification of AmpliTaq Gold, then 40 cycles of denaturation at 95°C (15 seconds), and an annealing/extension step at 60°C for 1 minute. Reactions were kept on hold at 4°C. All samples and standards were run in triplicate.
Copy number for JAK2 V617F was calculated in absolute units by comparing the signal generated by the test samples to that generated by a set of external plasmid standards containing the sequence of interest. The stock plasmid standard was created by ligating a PCR product containing the JAK2 V617F sequence into a plasmid vector system (Zero Blunt Topo; Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The amount of plasmid DNA was determined by spectrophotometric analyses of the insert-containing plasmid DNA at A260 (1 optical density = 50 µg/ml plasmid DNA), and the copy number per milliliter was determined based on molecular weight. Dilutions of the plasmid ranged from 10 to 300,000 copies per reaction, and quantitative determination for clinical samples was determined by reading from this standard curve.
Quantitative Determination of Cell Number by TaqMan
The number of cells per reaction was determined in a separate real-time PCR reaction using a previously validated assay for measuring the number of copies of a single-copy region of the human growth hormone gene (HGH).5, 6
Quantitative amplification of this highly conserved region of the nuclear-encoded human growth hormone gene used primer sets and probes designed using Primer Express software (ABI) according to primer design guidelines. The reaction used forward primer 5'-TATCCCAAAGGACAGAAGTATTCATT-3' and reverse primer 5'-TTGTGTTTCCCTCCCTGTTGGA-3' to amplify a 141-nucleotide product. The fluorophore-labeled probe used was VIC-ACCTCCCTCTGTTTCTCAGAGTCTATTCCGACA-quencher TAMRA-3'.
Amplification of the HGH product was performed separately to the JAK2 V617F assay using another aliquot of the same DNA and using cycling conditions identical to that of the JAK2 V617F assay. Each 20-µl reaction contained 20 ng of DNA, and the final concentration of each component was as follows: 2.5 pmol of probe, 40 pmol of forward primer, 40 pmol of reverse primer, and 2x Taqman Universal Master mix (ABI). All samples and standards were run in duplicate. Determination of the HGH copy number was similar to that of the JAK2 V617F assay, in that known dilutions of a plasmid containing the HGH sequence of interest were measured to generate a standard curve, from which quantitative determination of HGH copy number in clinical samples could be made. The number of cells per reaction was calculated by dividing HGH copy number by 2, based on every nucleated cell carrying two copies of the HGH sequence.
Quantitative Determination of JAK2 V617F per Cell
Determination of JAK2 V617F per cell was achieved by normalizing JAK2 V617F per PCR reaction (20 ng of input DNA) to cell number per PCR reaction (20 ng of input DNA). Because the measured fluorescence in the quantitative PCR assay is directly proportional to the (base 10) logarithm of the initial DNA content in the sample, results were expressed and analyzed in logarithmic form. Hence, calculations of JAK2 V617F/cell from the JAK2 V617F and nuclear quantitative PCR assay results used the formula log10JAK2 V617F DNA/cell = log10JAK2 V617F DNA – log10nDNA – log100.5. Final results were reported in logarithmic form as copies of JAK2 V617F per cell (or per 20 ng of input DNA).
Detection of JAK2 V617F by Sequence Analysis
To confirm the specificity of the real-time quantitative PCR for the G to T mutation, paired samples (n = 17) of granulocyte and lymphocyte populations were analyzed by sequencing reaction. Amplifications for 40 cycles used Platinum Taq polymerase (Invitrogen), 2 mmol/L MgSO4, and 10 pmol each of sense and antisense primers (forward primer 5'-TGTAAAACGACGGCCAGTATCTATGTCATGCTGAAAGTAGGAGAAAG-3' and reverse primer 5'-CAGGAAACAGCTATGACCCTGAATAGTCCTACAGTGTTTTCAGTTTCA-3'). PCR sequencing reactions used Big Dye terminator chemistry and M13 reverse primer and forward primer 5'-ttccttagtctttctttgaagc-3'. Products were resolved on an ABI Prism 3730 48 capillary sequencer. Typing for the mutation in JAK2 exon 12 was made with alignment of test sequence to a reference sequence in GenBank (accession NM_00497215) using Conexio software (Perth, Australia).
Dilution Method for Determining Sensitivity of JAK2 V617F Assay
DNA from an individual with polycythemia vera carrying 1.8 copies of the JAK2 mutation per cell as determined by our assay was diluted with DNA from a healthy control to provide mixtures of 100, 95, 80, 50, 20, 5, 1, 0.5, and 0.05%. Taqman JAK2 V617F measurement of duplicate dilutions was performed and used to assess the sensitivity of the assay to detect dilutions of the mutation.
Dilution Method for Determining Specificity of JAK2 V617F Assay
DNA (sample A) from a healthy control carrying 0.007 copies of the JAK2 mutation per cell as determined by our assay was diluted with DNA (sample B) from another healthy control carrying 0.006 copies of the JAK2 mutation per cell to provide mixtures of 100, 75, 25, and 0%. Similar dilutions of sample A were generated with distilled water instead of DNA. TaqMan JAK2 V617F measurement of triplicate dilutions was performed and used to assess the specificity of the assay.
Quantitative Determination of PRV-1 Overexpression by TaqMan
Neutrophil PRV-1 expression levels were quantified similarly to the method described by Klippel et al7
using TaqMan and glyceraldehyde-3-phosphate dehydrogenase as the housekeeping gene. PRV-1 was amplified from cDNA using 20 pmol of forward primer 5'-CTGCGTGGCCCAACCTT-3' and reverse primer 5'-GCAGAGAAGATCCCGATTTGTCT-3' and detected using 3.5 pmol of a FAM-labeled probe 5'-CCAGCTTCTTGTTGAAC-3' in a 20-µl reaction with 2x Universal PCR Master Mix (ABI) using real-time quantitative PCR on the ABI 7700 SDS. Amplification was performed in triplicate samples in a two-step cycle (denaturation, 95°C for 15 seconds, annealing/extension at 62°C for 60 seconds) for 40 cycles. Data collected during cycling were analyzed with the FAM threshold set at 0.05.
Each glyceraldehyde-3-phosphate dehydrogenase reaction used another aliquot of the same cDNA and consisted of 20-µl reactions with 2x Universal MasterMix (ABI) and 20x glyceraldehyde-3-phosphate dehydrogenase premix control reagent (ABI). Reactions were performed in triplicate, as for the PRV-1 assay. Data collected during cycling were analyzed with the VIC threshold set at 0.05. A mean Ct value for each triplicate measurement was calculated. The PRV-1 Ct ratio for each sample was calculated from the mean value of triplicate PRV-1 Ct determinations divided by the mean glyceraldehyde-3-phosphate dehydrogenase Cts.
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Results |
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. Fluorescent detection of 20 ng of undiluted input template DNA from controls (n = 18) was detectable only after 35 cycles of amplification. To test whether fluorescence occurring after 35 cycles was an artifact of the assay arising from nonspecific priming, we diluted DNA from a healthy control (sample A) in water and also in DNA from another control (sample B). As shown in Figure 1b
, triplicate measures of different dilutions showed a poor relationship with mutational load, suggesting that nonspecific amplification occurs after 36 cycles of PCR.
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Samples of DNA from paired granulocyte (n = 17) and lymphocyte (n = 17) preparations were sequenced for semiquantitative detection of the mutation. All lymphocyte DNA failed to demonstrate the mutation by sequencing. The assay showed 100% concordance with sequence-based typing for detecting the presence of the mutation in granulocyte DNA samples with at least 0.08 copies per cell (n = 14). Three samples of granulocyte DNA with low mutational load showed no mutation by sequence-based typing, presumably because of limitations in assay sensitivity.
Quantitative determination of JAK2 V617F in monocytes and granulocyte preparations were similar for three individuals with high levels of the mutation. Measurements in lymphocyte fractions were just above those observed in healthy controls, presumably because of contamination by the myeloid lineage (data not shown). Amplifications in the granulocyte fraction occurred 11 to 12 cycles before detectable amplification in the lymphocyte fraction, indicating good discrimination between amplification of mutant wild-type alleles.
Variation in JAK2 V617F copies per cell among individuals referred for mutational typing and among 18 controls is given in Figure 2
. We observed a strongly bimodal distribution of the data for cases that grouped lower levels of mutation in the healthy controls and a number of the cases separately from logarithmically higher levels of mutation in a subset of the cases. Restricting to values that fell in the higher half of this bimodal distribution, mutational load was significantly increased among cases of PV compared with ET (P < 0.001). A small number of cases of IMF (n = 7) showed increased mutation load compared with cases of ET (P < 0.001). The frequency of the mutation, based on assigning the occurrence of the mutation to cases with mutation load above the threshold defining the bimodal distribution is given in Figure 2
and was within previously reported ranges.
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Clinical associations with occurrence of JAK2 V617F (based on assigning occurrence of mutation to levels of mutation load in the higher bimodal distribution) among myeloproliferative disease (MPD) cases (n = 93) of PV, ET, or IMF that we detected were restricted to increases in white cell and neutrophil counts at diagnosis and on treatment (Table 1)
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The precision and efficiency of the PCR is similar to that attained for real-time PCR quantitation of the Philadelphia chromosome (BCR-ABL) transcript,16 and the method lends itself to high throughput and cheap and readily available set-up. The sensitivity is favorably comparable with pyrosequence and amplification refractory mutation system-based methods.17, 18, 19 The performance characteristics of the assay presented here seem very similar to those reported recently15 in which locked nucleic acids are used with molecular beacons to quantify the mutant allele in a real-time PCR assay, with the notable exception that in our study, we have shown low-level detection of the mutation in control samples to be an artifact of the system.
Mutational load among healthy controls was extremely low, and as previously stated, we assumed that this represented nonspecific amplification of the wild-type allele because of mispriming or the presence of 3'-shortened mutation-specific primers (because it is only the very 3'-end base that confers specificity and 3'-shortened primers will amplify from both wild-type and mutant DNA). Groups using real-time PCR assays have previously reported that nonspecific amplification of a negative allele occurs after a certain number of cycles,20 and our assay also does not maintain specificity at these low levels of detection. For the purpose of diagnosing myeloproliferative disease, we set a threshold for reporting the presence of the mutation based on the ability to discriminate healthy controls from individuals with disease and based on the ability of the assay to show a linear relationship with dilutions of the mutation. Using this approach, we report a mutational frequency that is consistent with previous publications,8 although lower than might be expected for cases of PV, given the high sensitivity of the assay. One possible explanation may relate to differences in the ascertainment of cases of PV for different studies.
We chose to measure JAK2 V617F levels in granulocytes in an effort to maximize sensitivity of detection. However, our data indicate that the assay would easily detect the presence of the mutation in whole buffy coats, bone marrow aspirates, or mononuclear populations.
Our study detected an average of more than two copies per cell in a number of individuals who were diagnosed MPD (Figure 2)
. The veracity of these high mutational loads requires confirmation by an alternative approach. However, currently, our method is uniquely designed to detect changes in copy number greater than two, essentially because it is not based on ratios of wild-type to mutant alleles. Although probably unlikely, a copy number of greater than two copies per cell could also be due to loss of an HGH allele, and confirmation using an unrelated marker at a different location should be pursued. Despite these limitations, the study provides supporting data for a hypothesis that disease evolution involves further gene duplication associated with genetic instability. Homozygosity in the JAK2 mutation has been reported to result from uniparental disomy. However, we require an alternative model to explain more than two copies per cell. One possibility is that in-frame internal tandem duplication occurs, similar to that reported for the mutation in FLT-3.21
Interpreting the clinical relevance of mutational load to disease evolution and phenotype variation within the spectrum of myeloproliferative disorders represents the next significant challenge. A continuum model22
would predict significant correlations between clinical phenotype and mutational load that extends across current classifications of essential thrombocythemia and polycythemia vera (Figure 3)
. Our investigation certainly shows that cross-sectional measures of mutational load are significantly different according to disease phenotypes observed in MPD. We also observe a significant positive relationship between mutational load with PRV-1 overexpression and with myeloproliferative markers, consistent with the findings of Vannucchi et al18
and Lippert et al.23
The Lippert et al group also reported significant correlations between JAK2 V617F levels and leukocyte counts. However, unlike our study, they did not find that mutational levels at diagnosis were linked with hemoglobin level, hematocrit level, or platelet counts. Further investigation that accounts for these differences across studies is therefore required. If, as our data indicate, mutational load does reflect myeloproliferative aspects of PV and ET phenotype, then its measurement would provide a useful tool for monitoring disease progression and for providing insights into the molecular process of disease. Potentially, this type of assay could be similar to the way that real-time PCR quantitation of BCR-ABL mRNA in the blood of patients with chronic myeloid leukemia has become the predominant molecular monitoring technique. Other studies have indicated that the mutation load is also correlated with increases in a number of activation markers of myeloid cells, although exactly how these cellular activations relate to thrombo-hemorrhagic and fibrotic complications is still unclear.18, 24
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In summary, the data indicate that the TaqMan quantitative JAK2 V617F test provides an attractive approach for monitoring disease progression, the efficacy of curative interventions, or early indications of emerging drug resistance. Further investigation is now needed to determine whether variation in this measurement can elucidate the pathogenic mechanism and contribute to clinical management decisions.
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Footnotes |
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Supported by the Ray and Bill Dobney Trust.
Accepted for publication November 8, 2006.
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References |
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