Quantification of residual leukemic cells at early time points during therapy can reliably predict the outcome in children with acute lymphoblastic leukemia (ALL). Recently, semiquantitative minimal residual disease (MRD) detection assays such as dot-blot hybridization have been replaced by real-time quantitative PCR. We tested the flexibility of the two most used real-time PCR machines: the SDS 7700 or ‘TaqMan’ (TM) (Applied Biosystems) and the LightCycler (LC) (Roche) instruments. Clonal T-cell receptor and immunglobulin gene rearrangements were used for MRD detection with germline hydrolyzation probes and clone-specific primers. Sensitivity tests for 65 clonal gene rearrangements and MRD quantification in 90 bone marrow samples during therapy of 49 children with ALL at diagnosis or relapse were performed with both machines. Both real-time PCR systems provided specific results for MRD quantification in all follow-up samples. In conclusion, we were able to demonstrate that TM and LC real-time PCR technologies produce similar MRD quantification results and that the quantification assays can be easily transferred from one detection system to the other. Using the same detection format, both techniques can be applied in combination in multicenter MRD studies.
Quantification of residual leukemic cells at early remission time points (TPs) enables a reliable and accurate risk assignment for children with acute lymphoblastic leukemia (ALL).1,2,3 Up to now, most PCR-based minimal residual disease (MRD) studies have used semiquantitative methods for the detection of clone-specific T-cell receptor (TcR) and immunoglobulin (Ig) gene rearrangements, which are present in more than 90% of childhood and adult ALL cases.4,5,6 More recently, semiquantitative techniques such as dot-blot hybridization (dbh), competitive PCR or limiting dilution assays7,8,9,10,11 have been replaced by real-time quantitative PCR (RQ-PCR).12,13,14,15 A major advantage of using RQ-PCR is that the position of the exponential phase is easily identified, thus allowing for an accurate quantification.11 Moreover, RQ-PCR permits easier and faster analyses in a closed system, thus minimizing the possible risk of PCR contamination. In addition, the amplification of an independent gene makes it possible to monitor the actual amount of input DNA and/or the presence of unwanted inhibitory effects.12,16
RQ-PCR technology on ABI-PRISM 7700 SDS (TaqMan, TM) for MRD quantification was first applied using clone-specific hydrolyzation probes.12 More recently, an equally efficient but more cost-effective approach has been described, consisting of the design of TM probes on the conserved germline region of Ig or TcR gene rearrangements.12,13,14,15
LightCycler (LC) technology represents an alternative for quantification of MRD in childhood ALL, using a clone-specific primer combined with a general detection method, consisting of either hybridization or hydrolyzation probes17 or the SYBR green dye.18
Up to now, no comparative data between TM and LC technologies have been reported using the same detection format (ie clone-specific amplification with germline probes).
Here, we report that by using hydrolyzation (TM) probes for consensus regions of Ig and TcR genes, comparable results can be obtained both in dilution experiments as well as during follow-up MRD monitoring by TM and LC technologies using the PCR mastermixes offered by the manufacturers or the same mastermixes in both machines.
Materials and methods
Patients and cell samples
ALL patients at diagnosis (n=29) or at the time of relapse (n=20) were included in the study. Diagnostic bone marrow (BM) samples were collected prior to chemotherapy and malignant cells constituted 70–95% of the specimens. Diagnosis of ALL was made by BM morphological evaluation and immunophenotyping according to standard procedures.19,20
Mononuclear cells (MNCs) from samples taken at diagnosis, at relapse or during follow-up were isolated by Ficoll density gradient centrifugation. High molecular weight DNA was isolated using either the QIAamp Kit (QIAGEN, Hilden, Germany) or by standard phenol–chloroform extraction method.21
TPs 1 and 2 after diagnosis correspond to the end of the induction phase Ia (day +33) and before the maintenance phase (day +78) of the front-line therapy, according to a Berlin–Frankfurt–Munster (BFM) treatment-based protocol.22 Those TPs have been shown to be the most informative for patient risk of relapse.23
In relapsed ALL patients, TP1 and TP2 correspond to day 15 (after therapy course F1) and day 36 (after therapy course F2) of the ALL BFM-REZ treatment, respectively.24,25 We recently reported that TP2 (day 36) is highly prognostic in discriminating patients with different outcomes after relapse.26
Identification of clonal markers
TcR delta (TcR-δ), TcR gamma (TcR-γ) and Ig heavy (IgH)- or light Kappa (Igκ)-chain gene rearrangements were used as clone- and patient-specific targets for PCR-based MRD detection. Igκ and TcR clonal gene rearrangements were identified by single27 or by multiplex PCR.28 The screening for IgH clonal rearrangement was performed with framework 1 family-specific primers and a general downstream primer,29 or with VH- and JH-family-specific primers as described by Szczepanski et al.30 The clonality was confirmed by homo–heteroduplex gel electrophoresis analysis.31,32 Monoclonal PCR products were directly sequenced using Big Dye Terminator Cycle Sequencing on the ABI Prism 377 Automated Sequencer or on the ABI Prism 3100 (Applied Biosystems, Foster City, CT, USA). In the case of biclonality, bands were excised from the homo–heteroduplex gel, reamplified and then directly sequenced, or the PCR product was cloned into a plasmid vector (TOPO TA cloning kit, Invitrogen, Groningen, The Netherlands) and then sequenced. Oligoclonal samples were excluded from the study. In total, 65 clonal rearrangements were identified, and 132 samples were then analyzed with the two techniques.
Real-time PCR quantification
In both detection systems, we performed the same MRD assays, with germline TM fluorescent probes and clone-specific primers for all rearrangements. FAM (6-carboxy fluorescein) was chosen as reporter dye at the 5′-end of the probe and TAMRA (6-carboxytetramethyl rhodamine) as the quencher dye at the 3′-end. The germline probes/primers and the clone-specific forward primers were designed for each target using Primer Express (Applied Biosystems, Foster City, CT, USA), the OLIGO 6.0 (Molecular Biology Insights, Cascade, CO, USA) and/or the JAVA OLIGO program (TIB MOLBIOL). Different sets of consensus primers and hydrolyzation probes were used: probe set 1 and probe set 2 (Table 1). Each clone-specific primer was designed to include at least part of the junctional region at the 3′ extremity.
In detail, the PCR mixture 1 (PCR mix 1) was as follows: 5 mM (for TcR-δ, Igκ, IgH, β-globin genes) or 7.5 mM (for TcR-γ gene) MgCl2, 1 U platinum DNA Taq polymerase (Invitrogen, Karlsruhe, Germany) and 2 μl 10 × PCR supplied buffer, 200 μ M dNTPs, 5 μg BSA (Sigma, Deisenhofen, Germany) (only for LC), 1.5 μ M ROX dye (TIB MOLBIOL, Berlin, Germany) (only for TM), 500 nM primer, 100 nM hydrolyzation probe (TIB MOLBIOL or Applied Biosystems, Monza, Italy) and 500 ng DNA. The final PCR volume was 20 μl.
PCR mixture 2 (PCR mix 2) was as follows: TM buffer A (Applied Biosystems) (including ROX dye, 5 mM MgCl2, 200 μ M dNTPs and 1.25 U Ampli-TaqGold), 300 nM primers, 100 nM TM probe and 500 ng DNA. The final PCR volume was 25 μl test (A) and 20 μl test (B) (see below).
The amplification protocol was as follows: (i) for the TM: predenaturation 94°C/5 min (/10 min for TaqGold) followed by 50 cycles: 94°C/45 s and 60–69°C/1 min; (ii) for the LC: predenaturation 94°C/5 min (/10 min for TaqGold), followed by 50 cycles: 94°C/8 s and 60–69°C/23 s. For the TM, PCR was performed in 96-well microtiter plates (Applied Biosystems), whereas glass capillaries were used for the LC (Roche Diagnostics, Mannheim, Germany).
A standard curve was prepared as a test for the sensitivity of the assay and as a reference for the quantification. The standard curve was made from six points corresponding to 10-fold DNA dilution series of 105–1 leukemic cells in DNA derived from equivalent mixtures of blood MNCs from five to 10 healthy volunteers. Each PCR tube contained 500 ng DNA, which corresponds to about 105 cells (5–6 pg DNA/cell). DNA follow-up samples were measured in triplicate, standard dilution series in duplicate.
As the slope of the standard curve is an indirect measure of PCR efficiency, values between −3.7 and −3.0 were considered as indicators of an acceptable PCR system. Moreover, for a precise quantification, standard curves were accepted with a correlation coefficient of at least 0.95. Controls for nonspecific amplification were carried out in triplicate. The differences of Ct (cycle threshold) value between samples measured in duplicate or triplicate were only accepted when there were not higher than 1.5 Ct. The reproducible sensitivity was defined as the lowest dilution point with two positive wells and 3 Ct lower than the lowest Ct of the background signals of MNC. The maximum sensitivity of a PCR system was less reproducible and defined with two positive wells with the highest Ct at least 2 Ct lower than the nonspecific amplification in normal MNC.
Specificity and sensitivity had been optimized for each tested rearrangement, both by increasing the annealing temperature and/or by designing new primers.
Table 2 indicates all the tests performed to compare the two machines in different conditions. In both real-time PCR machines, the same detection format (germline primer and TM probe with clone-specific primer) was used.
(A) PCR mix 2/probe set 2 was used on both machines. The PCR mixes were prepared in duplicate, DNA was added in the double amount to each double PCR mix tube, carefully and gently mixed and finally divided in LC capillaries or in a 96-well plate for the TM. The total volume was always 20 μl each. Experiments were performed in one laboratory.
(B) The same procedure as described in (A) was performed; however, PCR mix 1/probe set 1 was used. Experiments were performed in one laboratory.
In tests A and B, possible variables of the RQ-PCR as PCR mix, primers and probes, the laboratory and the person preparing the PCR were identical for both machines. Test A evaluated the influence of the ROX dye on the LC machine, while test B evaluated the use of BSA on the TM.
(C) PCR mix 1/probe set 1 was used on both machines. The same DNA was sent from one laboratory to the other and the experiments were performed in two different laboratories, thus representing the only variable.
(D) PCR mix 1/probe set 1 on the LC, and PCR mix 2/probe set 2 on the TM were used. The experiments using TM and LC technologies in parallel were performed independently in two different laboratories. In this test, several variables were different between both machines: PCR mix, probe set, laboratory and person.
In tests C and D, the MNC DNA and the dilution series were separately prepared and used in each laboratory (this would mimic the case that the MNC DNA for the sensitivity test and MRD quantification during therapy does not have the same origin). The stability of the dilution series is very sensitive; therefore, the dilution series for the standard curve has been newly prepared for each new quantification and each new sensitivity test.
The main aim of this study was the comparison of two different RQ-PCR machines through the tests A–C. In addition, test D was introduced to mimic the influence of several variables, a situation that often occurs in multicenter trials.
Seven different Ig and TcR gene consensus hydrolyzation probes (as listed in Table 1) allowed the quantification of the most frequent Ig and TcR gene recombinations occurring in childhood ALL, using TM and LC technologies.
Figure 1 shows a representative experiment of PCR amplification of a VH3JH6 rearrangement performed by both techniques using the same PCR mix 1.
Comparison of real-time PCR and dot-blot hybridization
In 16 cases, dbh was performed in parallel with RQ-PCR, by using patient- and clone-specific radioactive probes complementary to the junctional region (N region), as previously described.23 In seven of 16 cases analyzed in dilution experiments, concordant results were obtained by dbh and RQ-PCR. In the remaining nine cases, the sensitivity by the dbh method led to at least one logarithm less than those obtained by TM and LC technologies (data not shown).
Sensitivity and quantification tests
Tests A and B were performed by preparing double DNA quantity in double amount of PCR mix, followed by splitting of the mix for analysis in LC or TM, always using 20 μl, in a single laboratory having both machines (Table 2). In test A, the PCR mix 2/probe set 2 was used; while in test B, the PCR mix 1/probe set 1 was used. These two tests assessed the crossed use of the ROX dye (contained in PCR mix 2) in LC and BSA (contained in PCR mix 1) in TM.
In total, 14 different rearrangements were tested (four TcR-δ, four TcR-γ, two IgH, four Igκ). The sensitivity was the same in 11 of 14 rearrangements. In three cases, the TM had better sensitivity and in one case the LC had better sensitivity (Table 3, panel a).
Three follow-up TPs of each patient were tested. All but one negative follow-up samples were consistently negative on both machines. Sample #1118, Vδ2–Dδ3 was slightly positive at TP2 with TM and negative with LC, but the LC in this case was less sensitive. Of the 28 positive tests, 24 showed a difference between the two machines within a two- or three-fold range. Four rearrangements showed differences of a 1/2 log order (Table 3, panel a).
Using the conditions of test B (Table 2), 19 rearrangements (four TcR-δ, three TcR-γ, eight IgH, four Igκ) were tested for sensitivity. In 16 of 19 rearrangements, the sensitivity was the same. In one case, the TM had a one log higher sensitivity and in two cases the LC had higher sensitivity (Table 3, panel b).
In test B, only positive follow-up samples were compared. The difference was higher than two-fold in only six of 37 rearrangements. One case showed a 1/2 log difference (Table 3, panel b).
The same PCR mastermix was tested in samples at relapse for 10 rearrangements (one TcR-δ, three IgH and six Igκ) in both machines, one in each laboratory (Table 2). In four of 10 rearrangements the sensitivity was equal; in three cases the TM and in three cases the LC reached a sensitivity one logarithm higher (Table 3, panel c).
Using test C, all negative follow-up samples were consistently negative on both machines. Two of the seven positive samples showed a difference higher than two-fold (Table 3, panel c).
The aim of test D was to assess the reproducibility of the RQ-PCR assay using the same DNA, independent on the conditions used.
For 22 different rearrangements (seven TcR-δ, four TcR-γ, seven IgH, four Igκ), the sensitivity was compared between SDS 7700 and LC according to the conditions of test D in Table 2. Briefly, in two different laboratories, LC was used with PCR mix 1/probe set 1, and TM was used with PCR mix 2/probe set 2.
In 18 of 22 rearrangements, the same sensitivity was reached with both machines. In three cases, the TM was one log more sensitive, and in one case the LC had a 2 log higher sensitivity (Table 3, panel d).
We then compared the MRD levels early during therapy as detected by TM and LC, in 18 newly diagnosed ALL patients. Comparable results were obtained with both techniques in all negative follow-up samples. A difference within a two-fold range was reached in 13 of 17 positive follow-up samples, while a difference higher than 1/2 log step was found for four positive follow-up samples. In one case (#481, VH3JH4), a higher difference was obtained between TM and LC within a quantification range corresponding to 10−5 sensitivity, at the limit of the reproducibility (Table 3, panel d).
A linear regression analysis was performed to investigate the relationship between the quantitative data obtained by both technologies at the early TPs during front-line or relapse therapy (Figure 2). Concordance between TM and LC was very high for the positive follow-up samples for tests A, B and C, with a correlation coefficient higher than 0.9. The correlation coefficient for test D was 0.85.
Ongoing collaborative European childhood ALL treatment protocols are using MRD detection for stratification of patients to different chemotherapeutic regimens, with the aim of developing an individually tailored treatment for children with newly diagnosed or relapsed ALL. These multicenter studies must apply laborious specific and sensitive molecular methods for both the detection of clonal TcR/Ig gene rearrangements and the analysis of MRD during early follow-up TPs used for clinical decision. Moreover, standardized conditions, definitions and quality control are necessary for all molecular methods before clinical use. For this reason, to date, centralized specialized laboratories are required for MRD analysis. As a consequence, one of the major efforts for such an organization is how to manage the huge amount of work sent by multicenter centralized protocols to a single lab. In this context, several efforts are being made to simplify the procedure of MRD analysis and to speed up the process to be in time for the results to be applied in the clinical decision. An initial step in this direction has been the introduction of quantitative PCR analysis by RQ-PCR instead of standard semiquantitative and laborious dbh analysis. The RQ-PCR offers a highly accurate, reproducible and fast technology for quantification of molecular response to therapy in clinical trials.
As a step forward, the aim of this work was to compare the flexibility of the two most used real-time PCR instruments for MRD quantification, the SDS7700 and LC, in order to test the possibility of transferring the quantification assays from one detection system to the other in the same laboratory. Moreover, by using validated and standardized quantification methods tested in parallel, different RQ-PCR machines can be used within the same clinical trial in different laboratories.
It has been previously reported that the use of clone-specific primers is much more sensitive and cost-effective than clone-specific probes.13 In this study, quantitative PCR analyses of clonal TcR/Ig gene rearrangements were performed in the two different RQ-PCR instruments, by applying the same quantification format, germline TM probes with clone-specific primers. Two different PCR mixtures were tested, each either suggested by the manufacturer and/or originally established in one of the two laboratories taking part to this study.
The main aim was to test the different machines and the transfer of PCR conditions from one detection system to the other: PCR-mix 2/primer-probe set 2 from TM to LC (test A), and PCR-mix 1/primer-probe set 1 from LC to the TM (test B). In test B, PCR-mix 1/primer-probe set 1 was tested at the LC and TM in two different laboratories. The two machines on different laboratories were assayed on test C, using the same PCR conditions. Furthermore, different consensus TM probes and primers and PCR mixes were tested in comparison D.
The comparison of the sensitivity in all four tests A, B, C and D showed quite comparable results. The sensitivity was better on the TM in 11 cases, and better on the LC in six cases (Table 3). Both machines have the potential for sensitive results when the quantification format uses germline TM probe with clone-specific primer and a hot-start Taq polymerase (TaqGold/ Platinum Taq).
RQ-PCR proved to be at least as sensitive as the conventional dbh, whereas in most cases the sensitivity could be improved by real-time PCR, confirming the tendency observed in more extensive analyses (G Cazzaniga 2002, unpublished observation).
Both machines produced similar results and proved equally suitable for quantitative analysis of MRD. The manufacturers indicate that the RQ-PCR allows the detection of two-fold differences between samples when a well-established RQ-PCR system is used. In MRD studies using TcR and Ig gene rearrangements, in order to have a sequence-based clone-specific amplification, a patient-/clone-specific primer has always to be designed, representing a permanent change of an essential PCR condition, dependent on the junctional region of TcR/Ig gene rearrangements. Therefore, the reproducibility of the PCR is not always optimal, and differences can be observed between two different PCR runs or two different PCR machines. However, the variations have to be in the range required for clinical decision-making within ongoing therapeutic protocols, meaning that differences should be not higher than 1/2 log, optimally within a two-fold range.
For the rearrangements used for quantification of positive follow-up samples (n=89), in nine cases a difference higher than 1/2 log was seen. Three of those nine cases derived from the comparison test D, which had several variable conditions, as the probe set (except IgH), PCR conditions and the laboratories. In 23 of the 89 rearrangements, the difference was higher than two-fold, but lower than five-fold. A variance in this range of an RQ-PCR assay is marginal, but acceptable for an assay with very strong limits for one primer by the length and composition of the junctional region. Again, the largest proportion of patients with these differences derived from test D.
Overall, both real-time PCR systems provide reproducible, specific and sensitive results for MRD quantification, in the range required for clinical decision-making within ongoing therapeutic protocols.
Considering the application of both instruments for clinical MRD trials using TcR and Ig gene rearrangements as clonal markers, both machines have advantages and disadvantages. The SDS7700 offers the possibility of the simultaneous analysis of 96 samples, thus allowing the run of target and reference gene together, in replicates. By contrast, one LC run takes only 32 samples, representing a disadvantage, in particular when the analysis requires a contemporary amplification of an adequate number of control samples. However, in the LC the cycle steps can be shortened to a minimum time and one complete run can be performed in 30 min. Furthermore, the machine can process a smaller number of samples and perform a wide range of diagnostic assays. The handling of glass capillaries or a 96-well plate actually can be operator dependent. However, it should be considered that the 96 glass capillaries are clearly more expensive than a 96-well plate.
In conclusion, we were able to demonstrate that the SDS 7700 and LC real-time PCR technology produce similar MRD quantification results and the quantification assays can be easily transferred from one detection system to the other just by adapting the PCR conditions (addition of BSA to the LC reaction mixture and ROX to the TM reaction). Using the same detection format, both techniques can be applied in combination for a standardized MRD quantification in multicenter studies. One possibility could be the combined use of the LC for testing the sensitivity of several patient-specific primers on the same run and the use of the TM for the real quantification of follow-up TPs, running on the same plate replicates of TPs for both target and reference genes.
Considering the variable use of different PCR formats in different laboratories participating in large multicenter studies, it would be interesting to investigate the influence on the quantification results of using different detection formats, that is, replacing the hydrolyzation probe by hybridization probes or using SYBR Green I as the detection dye.
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This study was kindly supported by Deutsche Kinderkrebsstiftung, Germany, Fondazione Tettamanti, Associazione Italiana per la Ricerca sul Cancro (AIRC), MIUR cofin 40%, CNR Progetto Oncologia and Fondazione Cariplo.
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Eckert, C., Scrideli, C., Taube, T. et al. Comparison between TaqMan and LightCycler technologies for quantification of minimal residual disease by using immunoglobulin and T-cell receptor genes consensus probes. Leukemia 17, 2517–2524 (2003). https://doi.org/10.1038/sj.leu.2403103
- MRD quantification
- TcR/Ig gene rearrangements