Clinical Chemistry 47: 2003-2011, 2001;
(Clinical Chemistry. 2001;47:2003-2011.)
© 2001 American Association for Clinical Chemistry, Inc.
Denaturing HPLC for Identification of Clonal T-Cell Receptor 
Rearrangements in Newly Diagnosed Acute Lymphoblastic Leukemia
Udo zur Stadt1,1a,
Johannes Rischewski1,1,
Reinhard Schneppenheim1 and
Hartmut Kabisch1
1
Department of Pediatric Hematology and Oncology, University Children’s Hospital, Martinistrasse 52, D-20246 Hamburg, Germany.
aAuthor for correspondence. Fax 49-40-42803-8931; e-mail zurstadt{at}uke.uni-hamburg.de.
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Abstract
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Background: Denaturing HPLC (DHPLC) can be used to screen DNA for known and unknown mutations. We describe a novel, HPLC-based method for discrimination among polyclonal, oligoclonal, and/or clonal T-cell receptor 
(TCR-) rearrangements in samples from children with newly diagnosed acute lymphoblastic leukemia.
Methods: TCR rearrangements were PCR amplified from initial leukemic samples and, after heteroduplex-induction, the clonality status of each product was evaluated. To attain this, we used DHPLC on a high-resolution micropellicular matrix. Running conditions were established by melting-curve analysis of known clonal and polyclonal products and melting-point prediction software. Elution profiles were studied at 50 °C (native) and, to achieve optimal separation, at different column temperatures between 56 and 64 °C.
Results: For VI-J1.3/2.3 rearrangements, an analysis temperature of 60 °C with a linear triethylammoniumacetate—acetonitrile gradient separated clonal bands from the polyclonal background amplification. Less than 15% clonal PCR product was detectable in mixtures of initial leukemic cell DNA and polyclonal DNA. Biallelic rearrangements produced two sharp peaks. Clonality of PCR products from 100 initial leukemic samples was completely identified in all investigated cases.
Conclusions: Heteroduplex analysis with standardized DHPLC conditions simplifies the detection of unknown clonal or polyclonal TCR rearrangements in newly diagnosed leukemias. Clonal targets for detection of minimal residual disease are available after a short, automated analysis of PCR amplified rearrangements.
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Introduction
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Initial diagnosis of acute lymphoblastic leukemia (ALL)
2
is established by routine cytomorphologic and immunphenotypical analysis. Important improvements to achieve better outcomes are based on the identification of new clinical and biologic markers. The initial response to remission-induction therapy is one of the most important prognostic factors in childhood ALL. For example, a slow response to induction therapy is associated with a high risk of relapse (1)(2). Complete remission is defined as a blast cell fraction of <5% in the bone marrow examined by light microscopy.
Detection of minimal residual disease (MRD) is a new practical tool for a more exact measurement of remission induction during therapy because leukemic blasts can be detected down to 10-4–10-6. In the future, this may be the way for a new definition of remission in childhood ALL (3). PCR-based MRD analysis uses clonal antigen receptor rearrangements detectable in 
90–95% of the investigated patient samples. Amplification of polyclonal products often leads to false-positive PCR amplicons not suitable for MRD analysis. Therefore, PCR analysis alone will only be sufficient if a more detailed analysis follows. Single-strand conformation analysis, heteroduplex analysis, temperature gradient gel electrophoresis, or gene scanning analysis have been described to circumvent this background problem (4)(5)(6)(7)(8)(9)(10).
Previous reports indicated high sensitivity and specificity of denaturing HPLC (DHPLC) for mutation detection (11)(12)(13)(14)(15) and for detection of single-nucleotide polymorphisms (16). The detection principle is based on the instability of a PCR product’s helical structure at or close to the melting temperature (partially denatured PCR product). Sequence variations will increase or decrease the number and spatial positions of hydrogen bonds within a double-stranded (full-helical) PCR product. This causes an altered percentage of nonhelical, or partially single-stranded segments within the PCR product. The conformational changes become detectable because of the altered physico-chemical interaction of the negatively loaded surface of the PCR product and the reversed-phase column. The higher the single-stranded fraction, the earlier the PCR product will elute from the column as the negative loads on the DNA surface are further spaced. The interaction of negative surface loads of the PCR product and the lipophilic surface of the column is mediated by a slow linear decrease of a triethylammonium acetate (TEAA) buffer. At partial-denaturing conditions, this buffer decrease leads to different retention times of hetero- and homoduplex products [sequence-dependent separation; for a review see Ref. (17)]. In general, PCR products contain two alleles, A and B, and each allele has a sense (A, B) and an antisense (A*, B*) orientation. After heat denaturation and during heteroduplex induction, single strands are allowed to slowly reanneal, and homoduplexes (AA*, BB*) and heteroduplexes (AB*, BA*) will be formed. At partial-denaturing conditions, the percentage of nonhelical PCR product will be higher in the heteroduplex fraction, and this leads to an earlier elution of this fraction. The method uses an automated instrumentation setup, and each sample can be analyzed within 5–10 min.
The aim of our investigation was to establish a routine method for identification of clonal rearrangements in newly diagnosed leukemias with this highly automated system. We applied the DHPLC principle to the detection of a present clonal cell fraction within a polyclonal background. The polyclonal product contains a multitude of sequence variations regarding length and base composition in the junctional region. With partial-denaturing HPLC conditions, no detectable single clonal peak should be present because the given multitude of sequence variations should lead to an even higher amount of heteroduplexes. In contrast, a clonal PCR product should contain one highly predominant sequence and should therefore elute as one distinct homoduplex peak. To test this, we evaluated the sensitivity and reliability of this method for the most common VI-J1.3/2.3 rearrangement, using patient samples with a known or unknown clonality status. Here we describe the standard conditions for these targets and our evaluation of the method on 100 initial leukemic samples.
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Materials and Methods
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Bone marrow samples from the time of diagnosis were collected during the 1997 Co-operative Study Group for Childhood Acute Leukemia (COALL) trial. Ficoll-separated cells were frozen in 100 mL/L dimethyl sulfoxide–500 mL/L fetal bovine serum–RPMI 1640 and stored at -80 °C until use. DNA from 106–107 cells was isolated using the High Pure PCR Template Preparation Kit (Roche Molecular Diagnostics). Three different leukemic samples and a mixture of blood mononuclear cells from five different healthy donors (as a polyclonal control) were investigated.
pcr and heteroduplex induction
We performed PCR in 50-µL reactions containing 100–300 ng of DNA for each reaction. We identified PCR targets for detection of MRD from initial leukemic DNA using a standard set of primers (18)(19). The PCR product was subjected to heteroduplex induction as follows: preheating at 95 °C; denaturing for 5 min at 95 °C; gradually reannealing from 95 °C to 10 °C with 1 °C/min; and storage at 4 °C before analysis. Samples (5–10 µL) were injected in a preheated C18 reversed-phase column with nonporous poly-(styrene-divinybenzene) particles (DNASep®; Transgenomic). The injected sample was eluted within a linear acetonitrile gradient consisting of buffer A (0.1 mol/L TEAA) and buffer B (0.1 mol/L TEAA, 250 mL/L acetonitrile) with a 2% increase of buffer B per minute. PCR products were separated with a flow rate of 0.9 mL/min. Retention time was measured on-line via ultraviolet absorbance at 254 nm in the eluate. Result diagrams showed absorbance intensity in millivolts over the retention time in minutes (mV/min) after injection into the column.
analysis temperature and gradient estimation
We analyzed PCR products on the HPLC system, using the following analytical conditions for optimization strategies. (a) We predicted the melting-temperature area of a 532-bp VI-J1.3/2.3 product (resulting from a theoretically rearranged germline product without deleted or added nucleotides). This sequence serves as the basis for the melting-point prediction software analysis (WAVEMAKER; Transgenomic). (b) We estimated the melting profile by serial injections of PCR products at different column temperatures. The latter always included a run at 50 °C (complete double-stranded product), at 70 °C (complete single-stranded product), and at several temperatures (56–64 °C) close to the predicted melting temperature. Several PCR products were used to establish the method: a polyclonal, a clonal, a biclonal, and two mixed-clonal products.
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Results
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software-predicted melting profile
The percentage of helical (double-stranded) PCR product within a temperature range of 50–70 °C is shown in Fig. 1B
. The melting point was defined as the temperature at which 50% of the PCR product had a helical structure. To achieve optimal separation of homo- and heteroduplexes, DHPLC analysis had to be performed close to the melting point of the PCR product. The theoretical percentage of helical, i.e., fully double-stranded, product vs the expected analysis temperature is demonstrated in Fig. 1
. The software demonstrated that the PCR product stayed fully double stranded up to 55 °C. Thereafter, a two-step melting profile was observed, giving 56 and 59 °C as the points with the steepest decline from double stranded to partially single stranded. At 65 °C, all product was predicted to be melted (fully single stranded). Different junctional regions led to a different temperature stability of the helical configuration. These differences had the most influence on the retention patterns 1 or 2 °C below the points of steepest decline (56 and 59 °C).
The melting behavior of the PCR products depends on the base-pair composition of a given sequence. The different interval domains of the PCR product with respect to their local melting point are demonstrated in Fig. 1C
. An AT-rich region in the first part of the product leads to an earlier melting domain at 56 °C. Our region of interest (the junctional region of rearranged TCR genes; Fig. 1A
) has a relatively higher GC content, leading to a higher temperature stability, and is expected to melt at 59 °C. Typically, melting-curve prediction software visualizes the melting behavior of each target; it does not replace optimization runs to define analysis conditions. In the following experiments we compared these theoretical predictions to the practical approaches.
dhplc analysis conditions
All optimization runs were performed with gradients according to Tables 1
and 2
. After heteroduplex induction, 8–10-µL samples were injected into the preheated column. A polyclonal VI-J1.3/2.3 PCR product was analyzed at 50, 56, 57, 58, 59, 60, 61, 62, 63, 64, and 70 °C (Fig. 2
). At 50 °C, the polyclonal products elute as a single peak. This is in accordance with the literature (17), which describes a length-discrimination performance of 1–3% at nondenaturing conditions. A 530-bp product with a length variation (gaussian distribution) of ± 20 nucleotides should not be discernable. With higher temperatures, this peak "melts" and a broader peak elutes earlier from the column. At 60 °C, a plateau-like peak elutes between 10 and 12 min. In contrast, the resulting clonal VI-J 1.3 product from a T-cell ALL (T-ALL) sample at the time of diagnosis showed a distinct (clonal) peak at this temperature (Fig. 2A
). This peak corresponds to a sequence with seven deleted and six randomly added nucleotides in the junctional region (Table 3
).
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Figure 2. Optimal temperature selection.
DHPLC melting profiles of four VI-J1.3/2.3 products. Melting curves are drawn as intensity (mV) over retention time (min) at 11 different column oven temperatures. At 50 °C, all products show a single elution peak between 14 and 15 min (peak 1). Denatured products (at 70 °C) are eluted after 6–7 min (peak 3), whereas partially denatured products (56–64 °C) with different helical fractions are eluted later (peak 2). Panel A shows a polyclonal (left) and a clonal (right) product, and panel B, a biclonal (left) product from one patient sample and two mixed-clonal (right) products from two patients, respectively. The most discriminating temperature for this target is represented at 60 °C. The elution profiles of clonal and biclonal products are mainly influenced by the base composition of the junctional region and are nearly independent of product length.
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In two other experiments, a biclonal patient sample and two mixed-clonal products were analyzed under the same conditions (Fig. 2B
). The aim was to test the effect of sequence variations only and in combination with length differences. The biclonal product (amplified from one patient sample) was cloned and sequenced and showed a length difference of 10 nucleotides (germlines 1a and 1b in Table 3
). The clonal products from two known samples that were mixed at equal parts after PCR amplification are shown in Fig. 2
. The two products showed no length difference but a different junctional region (germlines 2 and 3 in Table 3
). The elution profiles from the DHPLC analysis are highly sequence specific and have a distinct retention time. Length separation plays only a minor role at partial-denaturing conditions.
The characteristic profiles of four clonal and biclonal products together with a polyclonal control are summarized in Fig. 3
. Sequence variations in the junctional region have a dramatic effect on the separation power of these fragments. Elution times differed by >0.6 min (last three elution profiles in Fig. 3
). Therefore, separation was observed only with different helical fragments, i.e., induction of heteroduplexes via different junctional regions. The sequences of the analyzed products are summarized in Table 3
. We cloned and sequenced the biclonal rearrangement. Two different clones corresponding to the biclonal target were analyzed at 50 and 60 °C. To reduce analysis time, the TEAA gradient was shortened to the discriminating area according to Table 2
. New conditions corresponded to the gradient between 3 min and 8 min of the previous runs. DHPLC analysis with this shortened, optimized gradient indicated that the broad biclonal peak was a consequence of two overlapping single peaks (Fig. 3
, lower right panel at 60 °C). No genomic artifacts or heteroduplex peaks were observed.
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Figure 3. Partial-denaturing HPLC analysis (DHPLC).
Summary of five optimized DHPLC runs at a column oven temperature of 60 °C. A polyclonal PCR product was compared with biclonal and clonal amplicons. Retention profiles are highly specific for a distinct product as demonstrated by the artificially mixed biclonal product originating from two clonal patient samples. Each peak from the mixed elution profile is identical to the peaks from single amplified patient samples a and b. The lower panels show the two cloned fragments from the biclonal patient sample analyzed at 50 °C (left) and at 60 °C (right).
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detection limit for identification of clonal targets at diagnosis
Because most of the initial leukemic cell samples contained variable numbers of nonpathologic hematopoietic cells, sensitivity testing for identification of clonal targets was performed. Because bone marrow or peripheral blood usually contains at least >20% leukemic blasts at diagnosis, a detection limit of 15% in the mononuclear fraction should be sufficient to identify clonal targets. With the buffer gradient described above at 60 °C, several dilution steps were analyzed for evaluation of the minimal amount of leukemic cells necessary for detection of clonality. We serially diluted leukemic cell DNA in 12.5% steps into unrelated polyclonal DNA, PCR amplified the VI-J1.3/2.3 fragment, and subsequently induced heteroduplex formation. Down to a dilution of 12.5%, a clonal-leukemia-specific elution peak was detectable (Fig. 4
).
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Figure 4. Detection-limit testing.
Leukemic DNA from the time of diagnosis was serially diluted in unrelated polyclonal peripheral blood lymphocyte DNA from five independent healthy donors. After heteroduplex induction, PCR products were subjected to the DHPLC analysis. DHPLC was performed at the optimized column oven temperature at 60 °C. The detection limit of the clonal product is 12.5% in a polyclonal background.
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mixing experiments
We tested the discrimination power of the method, using two targets with known sequences. We mixed different percentages of genomic DNA from two patient samples, amplified them by PCR, and analyzed the products at 50 and 60 °C. At 50 °C, all sample mixtures showed weak heteroduplex peaks during elution from the column. Nevertheless, only one main elution peak was observed with this condition. On the other hand, the biclonal products were separated at 60 °C with different admixtures of genomic DNA from each patient before PCR (Fig. 5
).
screening of initial patient samples
DNA samples from 100 ALL samples at diagnosis [56 common ALL (C-ALL), 16 PRE-B-cell ALL (-B-ALL), 2 PRO-B-ALL, and 26 T-ALL] were analyzed for the presence of clonal rearrangements at the VI-J1.3/2.3 locus. In T-ALL, 24 of 26 samples showed clonal rearrangements, 7 of the 24 positive samples were rearranged on both alleles (29%). In 74 B-cell precursor ALL 36 clonal VI-J1.3/2.3 rearrangements were identified, 6 of these 36 patients samples were biclonal at this locus (17%). A more detailed description of the rearrangement pattern according to the immunologic subgroups is listed in Table 4
.
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Discussion
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The aim of this study was to establish a simplified method for discrimination between clonal and polyclonal TCR- rearrangements in newly diagnosed ALL. To achieve this, we examined the specificity and sensitivity of DHPLC for the detection of clonality in newly diagnosed ALLs. PCR amplification of antigen receptor rearrangements followed by agarose gel electrophoresis does not discriminate exactly between clonal and polyclonal cases. For an exact clonality analysis, further labor-intensive methods are often necessary. In addition, both alleles are often rearranged and not distinguishable by conventional length separation. Heteroduplex analysis followed by acrylamide gel electrophoresis separate monoclonal bands from polyclonal background smears (4). This method is time-consuming and has to be established for each laboratory.
Here we demonstrate the potential of an automated HPLC-based system for clonality assessment in newly diagnosed leukemic samples with unknown rearrangements. Polyclonal samples together with known and unknown V-J rearrangements were tested for eligibility in routine diagnosis. For prediction of the optimal column oven temperature, we used a prediction software program (WAVEMAKER; Transgenomic) and tested it empirically. Fragments were run at multiple temperatures to determine the temperature at which the clonal or polyclonal targets were optimally separated. Both methods demonstrated similar results regarding the best column oven temperature. Whereas the software predicted 59.4 °C, the best practical temperature was 60 °C (20).
After identification of optimal separation conditions (buffer gradient and column oven temperature), distinct mixed-clonal products of known length and known junctional regions were separated. We showed that this separation greatly depends on the heteroduplex conformation of the analyzed product and the applied column oven temperature conditions. Length independence is one of the major advantages of this type of separation. Targets with minor length differences but large junctional differences in homogeneity were separated with a high resolution (Fig. 1B
).
Because a gaussian length distribution is often observed in samples with a high percentage of polyclonal lymphocytes, clonal sequences may be undetectable by length separation only. DHPLC analysis distinguishes mainly on the basis of the sequence pattern around the junctional region of each target. Therefore, even alleles with identical length can be discriminated from each other. In contrast, we always found a substantially reduced retention time of polyclonal sequences, ensuring a highly specific and reproducible separation of clonal and polyclonal targets. The found sensitivity is high enough for the identification of clonal targets in samples from the time of diagnosis.
The evaluation of 100 initial leukemic samples gave the expected results compared with published frequencies of clonal TCR- rearrangements in B-cell precursor and T-ALL (21)(22).
Because of a limited junctional region diversity in TCR- rearrangements, a high-resolution separation technique is needed to clearly identify clonality. Because nonpathologic complete VJ-rearrangements are often present in the peripheral blood, background amplification is a major pitfall during MRD target identification. These cases require cloning with subsequent sequencing, which makes the analysis extremely laborious and expensive, but often not successful for target identification. DHPLC is a practical approach for identification of clonality that uses the native PCR product and can be automated. After initial PCR, the time required for DHPLC analysis and target identification is 12 min. The method needs no further pre- or postanalytical hands-on time (e.g., gel preparation or gel staining). The results achieved are highly reproducible and interpretation is easy. The versatility of DHPLC analysis (14), the minimal preanalytical preparation time, and the minimal cost of approximately 1 Euro per sample makes the described principle applicable for routine molecular laboratories.
Analysis conditions for these targets are now available for all users and can easily be adapted without major changes. In addition, other MRD targets can be established by applying the described optimization strategy. Because of the automated setup, results are highly reproducible even in different laboratories.
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Acknowledgments
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This work was supported by the Fördergemeinschaft Kinderkrebszentrum Hamburg.
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Footnotes
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1 These authors contributed equally to the work. 
2 Nonstandard abbreviations: ALL, acute lymphoblastic leukemia; MRD, minimal residual disease; DHPLC, denaturing HPLC; TEAA, triethylammonium acetate; T-ALL, T-cell ALL; and B-ALL, B-cell ALL.
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Abstract
Introduction
). This curve is used to select the temperature for the optimization runs. (C), local melting behavior of the TCR 
). In DHPLC at optimized temperature and buffer gradient, all clonal and biclonal products were identified with the specific retention profile and unique retention times (lower panel,