Duplex quantitative PCR for molecular monitoring of <i>BCR‐ABL1</i>‐associated hematological malignancies
Authors: Gareth Gerrard, Katherine Mudge, Rebecca Stewart, Pierre Foskett, David Stevens, Jamshid S. Khorashad, Richard Szydlo, Katy Rezvani, David Marin, Alistair Reid, Jane Apperley, John Goldman, Letizia Foroni
Journal: American Journal of Hematology (2010)

Molecular monitoring of minimal residual disease (MRD) using quantitative real-time PCR (qPCR) to measure BCR-ABL1 transcript levels has become an essential tool in management of patients with chronic myeloid leukaemia (CML). The assay shows superior specificity and sensitivity compared with conventional morphology, cytogenetic, and fluorescent in situ hybridization. In an attempt to increase sample throughput and assay accuracy, we have developed a duplex assay that measures both BCR-ABL1 target and endogenous control transcripts in the same reaction with no loss of sensitivity compared with the conventional simplex test. The BCR-ABL1 fusion gene is a consequence of the t(9;22)(q34;q11) chromosomal translocation (resulting in the Philadelphia chromosome, Ph), a primary leukemogenic event in CML. Transcriptional control is dysregulated and the subsequent chimeric protein contains a constitutively activated tyrosine kinase with a multitude of downstream targets involved in cellular proliferation, avoidance of apoptosis and genomic instability [1]. The BCR-ABL1 fusion gene is also found in some cases of acute lymphoblastic leukemia (Ph+ALL) and in rare instances of acute myeloid leukemia. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become the standard tool for molecular assessment of residual disease in several hematological malignancies. As a result of the Europe against Cancer (EAC) initiative, optimized dual-labeled hydrolysis (TaqMan) probe and primer sets were developed for a wide range of leukemia-associated fusion genes, including BCR-ABL1 [2]. Endogenous control genes, used to normalize the target gene expression, were evaluated, and ABL1 was judged to be the most appropriate [3]. Particular effort has been expended recently to optimize and standardize the quantification of BCR-ABL1 transcripts especially following to the worldwide use of first- and second-generation tyrosine kinase inhibitor-based therapies [1–6]. Because of a high throughput of MRD samples, both in-house and referral, a routine assay was initially developed for use in our laboratory that quantitated BCR-ABL1 and ABL1 transcripts in separate qPCR runs (simplex tests). This assay was based on the EAC protocol but utilized different ABL1 primers and probes, hence referred to as the “Hammersmith” set, because of the presence of a G6PD sequence insert (part of an earlier competitive PCR assay) in the ABL1 exon 3 [7]. With the advent of a range of optimised, multiplex-capable reagent master mixes, the opportunity arose to streamline the simplex assay (Sx), which historically tested ABL1 and BCR-ABL1 in separate plate, into a single plate where both ABL1 and BCR-ABL1 reactions were carried out in a single well (hence referred to as duplex assay; Dx). This approach offers several advantages, including lower cost, time saving while providing higher throughput and increased precision due to the removal of inter-run variations. We took considerable care to ensure that gained benefits were not at the expense of assay sensitivity, especially because future CML trials may use the achievement of complete molecular response as a primary endpoint. Therefore, safe-guarding accuracy at the low end of the measurement scale was an essential consideration in the development stages of the duplex assay. This article describes the design, testing, and validation of a duplex assay, which has replaced standard simplex qPCR in our high throughput laboratory (average 500–600 tests per month). The rationale for the duplex assay design was to introduce as few modifications as possible to ensure minimal derivation from standard procedure. The TaqMan probes and primers were retained, with a modification to the reporter fluorophore of the ABL1 probe: this was changed from 6-carboxyfluorescein (FAM) to VIC amidite (VIC) to enable duplexing on the ABI 7900HT PCR platform (Applied Biosystems, USA); tetramethylrhodamine (TAMRA) remained the quencher fluorophore for all the probes. In addition, the multiplex capable Gene-Expression master mix replaced the standard Universal master mix (both from Applied Biosystem, ABI) and the standard 96-well reaction plate was changed to a lighter fast-mode plate (Table I). The latter modification was both economically advantageous and yielded a small decrease in run time. The duplex assay was optimized following testing of primer-probe concentration matrices, different master mixes (from ABI and Qiagen) and four real-time hardware platforms (ABI 7900HT, ABI 7500, ABI Step-One-Plus and Qiagen Rotor-Gene Q). The primer and probe concentrations were matched to yield as equivalent as possible reaction efficiencies and similar Ct values for both the BCR-ABL1 and ABL1 targets, which in turn enables the use of the ΔCt calculation as an internal validation tool [8]. The resultant Dx assay was then validated against the standard Sx assay by replicating in parallel a set of samples runs over 4 consecutive days and the results statistically analyzed both for individual runs and collectively as a meta-analysis. A further validation plate was also performed, assaying only very low level samples (<10 transcripts per reaction volume) to ensure neither loss of sensitivity nor gain in background signal through using the duplex assay. Over 4 consecutive days, the standard MRD assay (one ABL1 plate and two BCR-ABL1 plates, 92 patient samples in total) was replicated using a duplex plate, without sample preselection. The data from these plates were analyzed on a per-day basis for validation purposes and as a collated meta-analysis; it is the latter that is presented here (Fig. 1). An extra duplex plate (24 patient samples) provided comparative data for samples that had previously been shown to have very low BCR-ABL1 expression (<10 transcripts). The plasmid standard curve slopes, intercepts and R2 values did not vary significantly between runs (Kruskal–Wallis and Dunn's post tests, all P > 0.05) and were equivalent between Sx and Dx assays (Wilcoxon test, P > 0.05) (median slope ABL1: −3.37 Sx, −3.35 Dx; intercept: 39.59 Sx, 40.10 Dx; R2: 1.00 Sx, 0.998 Dx; median slope BCR-ABL1: −3.41 Sx, −3.47 Dx; intercept: 39.82 Sx, 40.29 Dx; R2: 0.999 Sx, 0.998 Dx). Statistical analysis of the validation exercise between the standard simplex (Sx) and the duplex (Dx) assays. Graphs (A)–(D) show before-and-after plots for ABL1 (A), BCR-ABL1 (B), BCR-ABL1/ABL1 ratio (C) for 4 days consecutive runs (n = 92) and a separate very low BCR-ABL1 transcript (<10 copies) plate (n = 24) (D); the P-value shows the result of a Wilcoxon paired test between the Dx and Sx data sets, where P < 0.05 denotes statistical significance. Graphs (E)–(H) show Bland–Altman difference plots for ABL1, BCR-ABL1, ratio and low transcripts, respectively. The three dashed lines denote the 95% limit of agreement (upper and lower) and bias (central). The results from the total of 92 patient samples run in parallel showed that the median BCR-ABL1 transcripts, ABL1 transcripts, and derived percentage ratio was 88 (range 0–159,097), 4.48 × 104 (3.35 × 104 – 2.22 × 105), 0.31% (0–138.6%) and 109 (0–235,277), 4.09 × 104 (3.26 × 103 – 2.39 × 105), 0.28% (0–129.9%) for the Sx and Dx assays, respectively. The graphs (A–D) in Fig. 1 illustrating this data with the results of the associated statistical tests show that there was no significant difference in the results from the simplex (Sx) and duplex (Dx) assays, with the exception of the BCR-ABL1 (B) where a median difference between the data sets of 21.3 transcripts was found to be significant (P = 0.0086). This was reflected in the corresponding Bland–Altman difference plot (F), which returned a systemic bias of 19.23% indicating an increase in assay sensitivity in favor of the Dx assay but only by the equivalent of 0.2 Cts. The very low BCR-ABL1 transcript plate (n = 24) did not show this bias (H); however, there was a greater spread in the data (coefficient of variation = 68.07 and 33.57% for Dx and Sx, respectively), suggesting that with an increase in sensitivity there was also a corresponding increase in assay noise. It is important to note that these values are below the quantitative range of the standard curve and are therefore subject to inherent uncertainty. The ABL1 data (E) also showed a very small bias (1.65%), and the derived ratio (G) showed a bias of 17.98%. To further ensure that the Dx strategy did not deleteriously impact on assay sensitivity through, for example, coamplification competition, a serial dilution of cDNA from the ABL1 null, BCR-ABL1 positive cell line BV-173 was made using ABL1 positive, BCR-ABL1 negative cell line HL-60 cDNA as the diluent. These were then run in parallel using the Sx and Dx protocols and when compared showed no difference in Ct or slope characteristics, even down to <10 transcripts BCR-ABL1. This indicates that the Dx assay retains at least the same level of sensitivity as the Sx assay, with no evidence of coamplification competition. As the overall derivation was small, statistically nonsignificant and with a bias toward greater sensitivity (albeit with a corresponding increase in low-level noise), the duplex assay was accepted for validation and is now the standard MRD assay for e13a2 and e14a2 transcripts. This assay is also been in a validation stage for other transcript types, including e1a2, e19a2 and may be applicable to other targets such as PML-RARA and WT1. Further work may refine and improve the assay, such as the introduction of nonfluorescent quencher systems to reduce spectral interference and to free up the TAMRA channel for a third color triplex assay. The use of industry standard dual-labeled probe technology means that although this assay is currently optimized for use on ABI qPCR platforms, it should be easily portable to hardware from other manufactures. In conclusion, we have implemented a practical cost-effective and sensitive test, which saves time, reagents, and patient material while retaining the sensitivity and specificity of conventional simplex tests. In total, 92 patient samples were collected and processed according to standard procedure and in accordance with the EAC recommendations [2,6]. Briefly, all samples were peripheral blood in EDTA and processed within 72 hr of being drawn. Red cell lysis buffer (ammonium chloride, in-house) was used to isolate the leukocytes, which were then lysed in RNeasy RTL buffer (Qiagen, UK) and manually disrupted using 18-gage nonbevelled needles fitted to 5-mL syringes. RNA was then extracted into 60-μL aliquots using QIAcube automated platform (v2 protocol) with RNeasy spin-columns and reagents (Qiagen, UK). cDNA was then produced using first-strand synthesis with M-MLV reverse transcriptase (Invitrogen, UK) and random hexamer primers. The validated assay used was for the CML associated e13a2/e14a2 variants, with forward primer in BCR exon 13 and reverse primer and probe in ABL1 exon 2. ABL1 set targeted exon 3 and 4 of ABL1. Primers were as follows: BCR-ABL1 (EAC [2]) ENF501: 5′ TCC GCT GAC CAT CAA YAA GGA 3′; ENR561:5′ CAC TCA GAC CCT GAG GCT CAA 3′; BCR-ABL1 probe: ENP541: 5′ FAM-CCC TTC AGC GGC CAG TAG CAT CTG A-TAMRA 3′. The ABL1 (Hammersmith [7]) primers: ABL146F: 5′-GAT ACG AAG GGA GGG TGT ACC A-3′; ABL240R: 5′-CTC GGC CAG GGT GTT GAA-3′; ABL probes: ABL183T:5′ FAM-TGC TTC TGA TGG CAA GCT CTA CGT CTC CT-TAMRA 3′; ABL183VIC: 5′ VIC-TGC TTC TGA TGG CAA GCT CTA CGT CTC CT-TAMRA 3′. VIC is a proprietary fluorophore of ABI, so alternative reporters such as HEX or JOE (or equivalent) may be substituted if sourcing from another supplier, subject to individual validation and with the caveat that VIC is the dye of choice for use on ABI qPCR hardware due to its optimized signal characteristics. Primer-probe single-use aliquots were prepared in advance from stock solutions: BCR-ABL1 and ABL1 (FAM) as two separate aliquots for the simplex assays; and BCR-ABL1 and ABL1 (VIC) combined as a single aliquot for the duplex assay. The final concentrations were as described in Table I. For all assays, 3 μL of cDNA was used in a 25-μL final volume reaction. Simplex tests were performed testing ABL1 in duplicate and BCR-ABL1 in triplicates using the ABI universal master mix (cat: 4318157). In the duplex assay, all samples were tested in triplicates with the ABI Gene Expression master mix (cat: 4369542). An ABI 7900HT Fast PCR system was used for all runs, using standard mode conditions: denature at 95°C for 10 min; cycle at 95°C for 15 s/60°C for 60 s for 45 cycles; acquiring to FAM and VIC (duplex assay only) channels at the end of each cycle. The standard curves were generated from log10 dilutions of a BCR-ABL1-containing plasmid (in-house), calibrated against third party standards (Ipsogen, France) to cover the dynamic range of the assays (101–106 transcripts/3 μL). Two quality control samples were also included in each run, derived from BCR-ABL1 positive and negative cell lines and calibrated by the Wessex Genetics Reference Laboratory to yield percentage ratios of 10 and 0.1%. The 7900HT was running SDS v2.3 software. Ct threshold was set at 0.1 with auto-baseline, and transcript quantities were extrapolated by the SDS software from the relevant standard curve. The run data were exported to a spreadsheet for postprocessing and analyzed using the statistical package Prism v4.03 (Graphpad, USA). The data sets were found to be nonparametric using the D'Agostino & Pearson omnibus test and thus differences between paired sample data sets were assessed using the Wilcoxon matched pairs test. Interassay bias was tested for using Bland–Altman percentage-difference plots. The authors thank Prof Nick Cross and Dr Helen White of the Wessex Genetics Reference Laboratory for the preparation and donation of the quality control samples.