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Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2651-2658
NEOPLASIA
From the Department of Adult Oncology, Department of Medicine,
Harvard Medical School, and Department of Biostatistics, Dana-Farber
Cancer Institute, Boston, MA.
Tumor-related immunoglobulin heavy-chain (IgH) rearrangements are
markers for polymerase chain reaction (PCR) detection of minimal
residual disease (MRD) in B-cell malignancies. Nested PCR with patient
IgH allele-specific oligonucleotide primers can detect 1 tumor cell in
104 to 106 normal cells. In childhood acute
lymphoblastic leukemia (ALL), persistence of PCR-detectable disease is
associated with increased risk of relapse. The clinical significance of
qualitative PCR data can be limited, however, because patients can
harbor detectable MRD for prolonged periods without relapse. Recent
studies indicate that a quantitative rise in tumor burden identifies
patients who are at high risk for relapse. Therefore, an efficient and
reliable PCR method for MRD quantification is needed for ALL patients. We have developed a real-time PCR method to quantify MRD with IgH
VH gene family consensus fluorogenically labeled probes.
With this method, a small number of probes can be used to quantify MRD
in a large number of different patients. The assay was found to be both
accurate and reproducible over a wide range and capable of detecting
approximately 1 tumor cell in 5 × 104 normal cells. We
demonstrate that this methodology can discriminate between patients
with persistence of MRD who relapse and those who do not. This
technique is generally applicable to B-cell malignancies and is
currently being used to quantify MRD in a number of prospective clinical studies at our institution.
(Blood. 2000;95:2651-2658)
Although patients with B-cell malignancies can achieve
clinical complete remission, many ultimately relapse. These relapses result from residual cancer cells that persist in patients below the
limits of detection by standard techniques. Therefore, considerable effort has been directed at developing techniques that sensitively detect minimal residual disease (MRD). In B-cell malignancies, polymerase chain reaction (PCR) assays have been established that use
tumor-specific immunoglobulin heavy-chain (IgH) or T-cell receptor
rearrangements as markers of disease.1-4 These highly sensitive assays have the potential to detect 1 malignant cell in up to
106 normal cells. PCR detection of MRD holds the promise
not only of providing important prognostic information regarding
clinical outcome, but could also be a useful tool for monitoring
patients' response to therapy.5-8 If PCR methods are
capable of clearly identifying patients at risk for relapse, the goal
of therapy could be the achievement of a "molecular complete
remission" and the therapeutic endpoint for treatment could be
determined by PCR.9 Additionally, if MRD detection is
established as a surrogate marker of subsequent relapse, such assays
could be used to rapidly evaluate experimental treatment approaches for
the management of cancer.
The clinical significance of PCR-detectable MRD, however, is not always
clear. Patients with a variety of malignancies can have long-term
persistence of PCR-detectable MRD without relapse.10-12 Recent studies in acute lymphoblastic leukemia (ALL) have indicated that both MRD levels and the kinetics of tumor reduction early in
therapy may be more informative of a patient's risk of relapse than
simple detection.5,13,14 Thus, it is desirable to develop and use methods that can quantitatively assess the levels and dynamics
of MRD rather than simply its presence or absence. Several methods are
available that permit a quantitative or semiquantitative assessment of
MRD levels, including limiting dilutions and various competitive PCR
strategies.6,15-20 The application of these quantitative assays to large studies can be limited, however, by their low throughput because of their requirement for extensive post-PCR processing and relatively limited dynamic range.
Real-time quantitative PCR is a method that has been developed to
address deficiencies of traditional quantitative PCR
strategies.21-23 This method exploits the 5'-3'
nuclease activity associated with Taq polymerase and uses a
fluorogenically labeled target-specific DNA probe. This probe is
designed to anneal between the forward and reverse oligonucleotide
primers used for PCR amplification. The nuclease activity of Taq
polymerase cleaves the labeled probe during the extension phase of PCR
amplification, producing a fluorescent signal that can be detected in
solution. The amount of fluorescence produced in a reaction by this
method is proportional to the starting DNA target number during the
early phases of amplification. Thus, when this reaction is performed on
a combined thermal cycler/sequence detector such as the PE Biosystems
7700 (Foster City, CA), a quantitative assessment of input
target DNA copy number can be made in the tube as the reaction
proceeds. This method eliminates the need for post-PCR sample
processing and thereby greatly increases throughput. Real-time PCR also
reduces the potential for false positive results by adding the
additional level of specificity provided by the hybridization of a
probe to sequences internal to amplification primers and offering a
closed tube assay system. Significant for purposes of MRD studies,
real-time PCR has a relatively wide dynamic range. Thus, it is possible
to accurately quantify MRD in samples with greatly differing levels of
tumor contamination.
Real-time quantitative PCR has been applied to the assessment of MRD
levels in several diseases in which chromosomal translocations are the
targeted tumor marker.24-27 Under these circumstances, 1 or
several probes are suitable for MRD quantitation of a large number of
different patients. Its application to MRD quantitation when IgH
rearrangements are the targeted marker, however, is not as simple. The
assembly of IgH genes from their constituent variable (VH),
diversity (DH), and joining (JH) regions
creates a DNA target that is complex and unique to each patient.
Although the use of patient-specific probes is possible,28
this approach would be prohibitively expensive for large studies
because of the high cost of producing an individual fluorogenically
labeled probe specific for each patient.
In the present report, we describe an assay that uses a limited number
of probes designed to consensus sequences in the framework region 3 (FR3) of the VH gene families. These probes are used together with patient IgH allele-specific oligonucleotides (ASO) for
real-time PCR reactions to detect and quantify MRD. This assay allows
the application of an accurate and reliable quantitative method to the
study of the clinical significance of MRD in large patient studies and,
in addition, can be applied to a number of B-cell malignancies.
Patients, samples, and cell lines
DNA sample preparation
Polymerase chain reaction To identify patient tumor-related and cell-line IgH rearrangements, genomic DNA extracted from cell lines or patient diagnostic samples was PCR-amplified using a series of 7 VH family FR1 consensus primers and a JH consensus primer in a modification of a method previously described.2 Individual VH family consensus primers were used to ensure that all sequences from patients with oligoclonal rearrangements were identified at diagnosis for subsequent use in MRD analyses. PCR amplifications were performed in 100 µL with 1 to 2 µg of genomic DNA, 10 µL of 10 × PCR Buffer II: 100 mM of Tris-HCl, pH 8.3; 500 mM of KCl (PE Biosystems); 10 µL of 25 mM MgCl2 (PE Biosystems); 200 µM each of dATP, dCTP, dTTP, and dGTP; 1.25 units of AmpliTaq DNA polymerase (PE Biosystems); and 20 pmol of VH and JH consensus primers. Amplifications were performed on a Perkin Elmer DNA Thermal Cycler 9600 as follows: 94°C for 1 minute, 62°C for 30 seconds, and 72°C for 30 seconds for 30 cycles, followed by a 7-minute final extension at 72°C before cooling to 4°C. "Hot start," to increase specificity, was achieved either by the use of AmpliTaq Gold (PE Biosystems) or the addition of Ampliwax PCR Gem wax beads (PE Biosystems). A total of 15 µL of the completed reactions were electrophoresed on 3% agarose gels with ethidium bromide and visualized under UV light. Amplification of clonal rearrangements gave a sharp band of the 300 to 350 base pairs. Well-established precautions were taken to prevent carryover contamination of PCR reactions.30Patient standard and GAPDH plasmid construction Patient tumor and cell-line IgH sequences were PCR-amplified as described above and cloned using TA Cloning Kits (Invitrogen, Carlsbad, CA) following recommended procedures. Briefly, about 10 ng of fresh PCR product was ligated to 20 ng of pCR2.1 overnight at 16°C. TOP10F'-competent Escherichia coli were transformed with 20% of the ligation reaction and plated on LB agar with 100 µg/mL of ampicillin, which were spread with 40 µL of 40 mg/mL X-gal and 40 µL of 100-mM IPTG for blue/white selection. A small number (8 to 16) of white colonies were analyzed by PCR to identify clones bearing recombinant plasmids. Recombinant plasmids were recovered with Wizard Plus Miniprep DNA purification system (Promega) and sequenced as described above to confirm their identity.TaqMan probes TaqMan probes were synthesized by PE Biosystems. The IgH probes were labeled at the 5' end with 6-carboxy fluorescein (FAM) and 6-carboxy-tetramethyl rhodamine (TAMRA) at the 3' end. The GAPDH probe was labeled at the 5' end with 2,7-dimethoxy-4,5-dichloro-6-carboxy-fluorescein (JOE) and TAMRA at the 3' end. Probes were aliquoted to 20 µL and stored at 20°C until use.
Real-time quantitative PCR Quantitative PCR analysis was performed using the TaqMan PCR Core Reagent Kit (PE Biosystems). Reactions for patient tumor-related IgH quantitation were performed in 50 µL with about 250 ng of DNA; 5 µL of 10 × TaqMan Buffer A (500 mM of KCl; 100 mM of Tris-HCl, pH 8.3); 5 µL of 25 mM MgCl2; 200 µM each of dATP, dCTP, dTTP, dGTP; 0.5 U uracil-N-glucosidase (UNG); 1.25 U of Platinum Taq DNA polymerase (Gibco BRL); 100 nM of TaqMan probe; and 400 nM of VH consensus and patient ASO primers. Wherever possible, the following FR3 VH consensus primers were used to limit the size of amplicons: VH1Q 5'-GAAG- TTYCAGGGCAGRGTCAC-3', VH2Q 5'-CATCTCTGAAGAGCAGG- CTC-3', VH3Q 5'-GGCCGRTTCACCATCTCC-3', VH4Q 5'-CCCTCAAG- AGTCGAGTYACC-3', VH5Q 5'-GTCCTTCCAAGGCCAGGTC-3', and VH6Q 5'GTCGAATAACCATCAACCCAG-3'. The amplification conditions for quantitation were an initial 2 minutes of incubation at 50°C (to allow UNG to destroy any contaminating templates), 10 minutes at 95°C (to activate the enzyme), 3 cycles of denaturation at 97°C for 15 seconds, annealing/extension at 60°C for 1 minute (to ensure optimal denaturation of genomic DNA), followed by 37 cycles of denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 1 minute. The amplifications were performed on an ABI Prism 7700 sequence detector equipped with a 96-well thermal cycler. Data were collected and analyzed with Sequence Detector v1.6 software (PE Biosystems). Reactions for quantifying GAPDH copy number were performed exactly as described above except for the use of the forward primer 5'-caaagctggtgtgggagg-3', reverse primer 5'-ctcctggaagatggtgatgg-3', and the GAPDH probe described above. IgH values were corrected for the values obtained for GAPDH from the same DNA samples to obtain the values reported. Briefly, the mean of experimentally obtained GAPDH copy numbers obtained from a sample run in triplicate was divided by the expected value based on the amount of DNA added to a reaction to obtain a GAPDH normalizing value. Triplicate experimental IgH copy number values were then individually divided by this normalizing value and the mean reported as IgH/GAPDH normalized.Statistical analysis For each dilution, Fisher's sign test was used to evaluate accuracy and test the null hypothesis that the observed IgH copies equals the expected IgH copies (in log 10 scale).32 Corresponding 95% confidence intervals for each dilution were constructed. Wilcoxon rank-sum test was used to determine the reproducibility between days by testing the difference observed between days. Nonparametric 95% confidence intervals for the difference of each dilution were also constructed. All P values were 2-sided.
Consensus probes A major factor limiting the use of TaqMan real-time quantitative PCR for MRD studies in B-cell malignancies is the high cost of producing probes unique to each patient's tumor-specific IgH rearrangement. We therefore sought to design probes to consensus sequences in the variable gene (VH) region of IgH rearrangements to reduce the number of probes required for MRD studies in large numbers of patients. Although it is possible to design probes to JH genes,33 we felt that deletions in this region, which can at times accompany VDJ recombination, might limit their broad use as consensus probes. To identify sequences within each of the major VH gene families appropriate for the design of consensus TaqMan probes, 151 clonal IgH rearrangements from pediatric ALL patients were PCR-amplified and sequenced. These sequences were characterized, grouped by VH gene family, and aligned by GeneJockey II software (data not shown). Sequences were aligned from 20 VH1, 11 VH2, 76 VH3, 23 VH4, 4 VH5, and 17 VH6 patient sequences. Short regions of sequence identity or homology in the FR3 were selected that met the criteria established by PE Biosystems for TaqMan probes (no 5' G residue, no runs of more than 3 consecutive G residues, and a melting temperature of about 70°C). Figure 1 illustrates the design of probes for both VH1 and VH3 IgH gene families. While short regions of sequence identity appropriate for the design of a single probe were found for VH1, VH2, VH4, VH5, and VH6, this was not possible for the large and diverse VH3 gene family. Therefore, 2 probes were designed for patients with VH3 IgH rearrangements in regions of sequence homology. These probes appear to be functional for most ALL patients with VH3 rearrangements, although additional probes may be needed to quantify all possible VH3 rearrangements. Antisense probes were designed in all cases so that PCR extension from the antisense patient ASO primers would result in cleavage of the fluorescent-labeled probe (Figure 2A). The sequences of the consensus IgH probes are shown in Figure 2B. These probes were tested in quantitation reactions on serially diluted patient IgH plasmids, and each gave a characteristic sigmoid amplification plot (representative data shown in Figure 2C). Here, it can be seen that each dilution has a unique threshold cycle number (Ct) where increasing fluorescence produced by cleavage of the probe during PCR amplification rises above the background level of fluorescence and that this number has an inverse relationship with plasmid copy number.
Standard curve A standard curve is required to quantify MRD levels in patient follow-up samples by real-time PCR. This is typically produced by serially diluting a known amount of target DNA and performing TaqMan PCR on the series alongside patient samples. Although a dilution series of patient sample DNA with a well-characterized tumor involvement could be used, this is frequently not possible because availability of pure leukemia cell samples can be limited. We therefore molecularly cloned patient IgH rearrangements into plasmids for our standard curves. After construction (see "Materials and methods"), patient IgH plasmids were quantified by spectrophotometry and serially diluted 10-fold into normal donor peripheral blood mononuclear cell DNA (plasmid copy numbers ranging from 106 to 100). This was done to mimic a patient sample that would contain tumor cells and their clonal IgH rearrangement within the polyclonal rearrangements of normal B cells. The patient IgH plasmid dilution series were PCR-amplified by TaqMan real-time PCR as described above, using as primers the patient ASO together with the appropriate VH consensus primer and TaqMan probe (Figure 2A). Standard curves with correlation coefficients greater than 0.970 were produced from the data collected for each patient plasmid dilution. Results obtained on serial dilutions of a patient's cloned IgH rearrangement using the VH5 probe are shown in Figure 3. Similar standard curves were produced using each of the other consensus TaqMan probes, demonstrating that this approach can be used for each of the VH families (data not shown).
Internal control To ensure that quantitation of MRD in serial samples was not affected by differences in the amount of DNA added to PCR reactions or sample-to-sample differences in levels of PCR inhibition, an internal control reaction was run alongside the IgH reactions. We targeted the GAPDH locus for which a well-characterized GAPDH TaqMan probe is commercially available (PE Biosystems). Oligonucleotide primers were designed to amplify a small region spanning intron C and exon 4 to eliminate the possibility of cross-reactions with processed pseudo genes elsewhere in the genome. The primers were tested by standard endpoint PCR, and the single band that was obtained was sequenced directly to ensure its identity (data not shown). The standard curve for GAPDH quantitation was produced by serially diluting the GAPDH plasmid 10-fold into water (copy numbers ranged from 106 to 100) and performing real-time PCR as described. Standard curves from the data collected in triplicate reproducibly resulted in correlation coefficients of more than 0.970 (data not shown). The GAPDH values were used to normalize IgH values as described in "Materials and Methods." Attempts to multiplex the IgH and GAPDH reactions consistently resulted in a loss of sensitivity for both reactions (data not shown). This problem was not significantly corrected by limiting the concentrations of either IgH or GAPDH primers (data not shown). Therefore, in all experiments reported, these reactions were performed in separate tubes to ensure maximum sensitivity.Sensitivity and accuracy Important to MRD analysis in hematologic malignancies is that the assay is both accurate and sufficiently sensitive to detect tumor cell contamination of blood or bone marrow samples over a wide range. To determine both the sensitivity and accuracy of real-time quantitation using consensus IgH probes and standard curves produced from serially diluted patient IgH plasmids, we performed quantitative PCR on dilution series of known amounts of cells with characterized IgH rearrangements. The ALL cell lines NALM-6 and 697 were serially diluted in 7 10-fold increments into normal peripheral blood mononuclear cells and the DNA prepared as described. These dilutions resulted in DNA samples with ratios of malignant:normal lymphocytes ranging from 100% to 0.001% tumor. Standard curves for quantifying these cell lines were made by serially diluting plasmids that contained cell line-associated IgH rearrangements into normal peripheral blood lymphocyte DNA as described. For each reaction, 0.5 µg of DNA, or the equivalent of 165,000 cells total, was subjected to quantitative analysis. Representative results from 1 experiment performed in triplicate for each cell line is shown in Figure 4. These data demonstrate that this real-time method can consistently detect 1 tumor cell in 104 to 105 normal cells. While sensitivity of detection could vary from patient to patient due to the use of unique patient primers and different consensus probes, the results from NALM-6 and 697 (which use VH1 and VH4 probes respectively and unique ASOs) show that similar sensitivities are possible. Cell-line dilution experiments were repeated 5 times and the data pooled together for statistical analysis of accuracy (data not shown). For each concentration, there was no statistical difference (P values ranged from .14 to .5) between expected and calculated number of IgH copies.
Reproducibility We examined the reproducibility of the quantitation method by comparing results obtained from replicate samples both during the same reaction run (intra-run variability) and those obtained on different days (inter-run variability). Intra-run variability analysis was performed on 697 cell-line dilution samples that were 100%, 1%, and 0.01% tumor cells in normal peripheral blood lymphocytes. For this experiment, 7 replicates for each dilution were amplified. Results are shown in Figure 5. There was little variability in the Ct of the 7 replicates at each concentration of tumor cell (Figure 5A). Of note, the amount of DNA in each sample analyzed was constant as assessed by quantification of GAPDH copy number where Ct values are nearly identical (Figure 5B). We assessed inter-run variability by performing quantitative PCR as described above on a series of 6 10-fold dilution samples of NALM-6 cells on 4 separate days (Figure 6). When the means of 3 replicate measures at each dilution level from each day are compared, no pair of results had a difference of more than 1 log. Although some measurement variability can be seen, we believe this variability would not be clinically relevant because it is likely that only log differences in tumor burden (and thus MRD levels) would reflect success or failure of therapy in patients.
Quantitation of acute leukemia follow-up samples An important objective for quantitative analysis of MRD is to discriminate between patients who are persistently PCR-positive and will subsequently relapse and those who will not relapse. To address whether real-time PCR analysis would be helpful in discriminating between these patient groups, we quantified samples obtained from the time of diagnosis to last follow-up from 16 patients with pediatric ALL. From patients who were persistently PCR-positive using standard qualitative seminested PCR, at least 1 representing each of the VH gene families was analyzed to ensure that each probe was capable of quantifying unknown samples. Each patient's tumor-related IgH sequences had previously been characterized (unpublished results). Individual standard curves were produced as described above. Bone marrow or peripheral blood samples obtained from each patient during the course of treatment were analyzed by both qualitative and quantitative PCR analysis. Patients 14, 43, 67, 79, 85, 111, 127, 146, 155, and 166, who remained in remission, had starting levels of MRD between 5 × 103 to 5 × 105 copies IgH/normalized GAPDH, which then dropped to levels that were undetectable (Figure 7A). As indicated, these patients were persistently PCR-positive by seminested PCR, which has a level of sensitivity greater than the single amplification of this quantitative method. This demonstrates that the level of tumor contamination in these patients is below 10-4 (the level of MRD that can be detected by real-time PCR) but more than 10-6 (the level of MRD that can be detected by seminested qualitative PCR). In contrast, although patients 27, 86, 94, 105, 178, and 263 appeared to have an initial decrease in tumor burden, subsequent samples showed quantitative increase in tumor burden. These patients all relapsed at the time of the last sample analyzed (Figure 7B).
We have developed a real-time PCR method that uses tumor-related IgH rearrangements for quantitative assessment of MRD in B-cell malignancies. The assay employs consensus V region probes in addition to patient-specific ASO and VH gene consensus primers. The advantage of this approach is that only a limited number of probes is required to quantify MRD for a large number of patients. This PCR method is accurate, reproducible over a wide range, and allows a high throughput of samples. We demonstrate that this method can be used in patient samples to quantify MRD during and after therapy. We are currently examining the clinical significance of MRD dynamics in a large cohort of patients undergoing therapy for ALL in a prospective study, but we are encouraged to find that this method allowed discrimination of patients who had persistence of MRD and have not relapsed and patients who subsequently relapsed. Our finding that patients with persistence of detectable disease might be further risk-stratified demonstrates the importance of accurate quantification of MRD in this patient population.
We thank Stephen Sallan, MD, and members of the Dana-Farber Cancer
Institute ALL consortium for contributing patient samples for the
study. We thank Stacy Waters for sample procurement and Edie Weller for
help with biostatistics.
Submitted April 29, 1999; accepted December 16, 1999.
J.G.G. supported by P01 CA68484 from the National Cancer Institute,
Bethesda, MD.
Reprints: John W. Donovan, Dana Farber Cancer Institute, 44 Binney St, Boston, MA 02115; e-mail:
john_william_donovan{at}dfci.harvard.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Top
Abstract
Introduction
Rn) detected through the final 37 cycles of a 40-cycle PCR
amplification reaction on a 10-fold serial dilution of a
plasmid-encoded tumor-related VH5 IgH sequence with the
VH5 consensus probe (IgH copy numbers ranging from
106 to 0). Ct represents the threshold cycle at which
fluorescence is first detected above background.
cycle number at
which fluorescence first rises above background) to create a standard
curve for quantification of unknown samples.
and 
T-cell receptor genes.
Blood.
1994;83:1892
3' exonuclease activity of Thermus aquaticus DNA polymerase.
Proc Natl Acad Sci U S A.
1991;88:7276
