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J Mol Diagn. Nov 2002; 4(4): 216–222.
PMCID: PMC1907354

Rapid and Accurate Detection of Monoclonal Immunoglobulin Heavy Chain Gene Rearrangement by DNA Melting Curve Analysis in the LightCycler System

Abstract

The detection of immunoglobulin heavy chain gene rearrangement (IgH-R) is a standard tool for distinguishing polyclonal from monoclonal B-cell populations. Current DNA-based polymerase chain reactions (PCR) strategies can diagnose monoclonal IgH-R either by measuring the length of the amplicon or by detecting gel mobility variations owing to sequence-dependent conformational changes. However, amplification and analysis remain sequential operations usually requiring manual transfer. We have developed a novel PCR strategy for detecting monoclonal IgH-R that monitors fluorescence of the specific double-stranded DNA binding dye SYBR Green I during melting curve analysis using the LightCycler System. We compared polyacrylamide gel electrophoresis (PAGE) versus melting curve analysis in 130 clinical DNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues (mostly skin biopsies) of 128 patients. The identical FR3 primers were used to amplify the IgH variable region for both analytic techniques. We detected IgH-R in 24 DNA samples from FFPE tissue of 22 patients. Melting curve analysis, compared to PAGE, revealed no false negative and no false positive results, yielding both sensitivity and specificity equal to 100%. We also compared Southern blot analysis versus melting curve analysis in 23 clinical DNA samples from fresh-frozen lymph nodes of 23 patients. We detected IgH-R by melting curve analysis in 7 DNA samples from fresh-frozen lymph nodes. Melting curve analysis, compared to Southern blot analysis, revealed sensitivity equal to 58.3% (7 of 12) and specificity equal to 100% (11 of 11). We conclude that continuous fluorescence monitoring of PCR products with DNA melting curve analysis can rapidly and reproducibly distinguish polyclonal from monoclonal B-cell populations.

Detecting antigen receptor gene rearrangements has become a standard laboratory test for distinguishing polyclonal from monoclonal B-cell populations. Both Southern blot and polymerase chain reaction (PCR) techniques work well. 1, 2 In recent years, PCR has become the method of choice because of its more rapid turn-around time, lower cost, and greater technical robustness in analyzing DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. 1, 2 Current DNA-based PCR strategies for detecting monoclonal immunoglobulin heavy chain (IgH) gene rearrangement (IgH-R) rely either on measuring the length of the amplicon 3, 4, 5 or on detecting gel mobility variations owing to sequence-dependent conformational changes. 6, 7, 8

The melting temperature (Tm) of duplex DNA reflects fundamental material properties that depend on length, sequence, G:C content, and Watson-Crick base pairing. 9, 10 DNA duplex melting has been used to assay DNA mutations, 11 single nucleotide polymorphism, 7 and variations in DNA length and sequence. 8 We hypothesized that a DNA-based PCR strategy for detecting monoclonal IgH-R, based on differences in internal melting domains corresponding to the IgH hypervariable region, could be developed using melting curve analysis. In this report, we demonstrate that amplicons derived from the IgH variable region yield highly reproducible DNA melting curves that can be used to rapidly and reliably diagnose monoclonal IgH-R.

Materials and Methods

Patient Samples

One-hundred-thirty-three consecutive FFPE tissue samples from 131 patients with suspected B-cell lymphoproliferative disorders (mostly skin biopsies) were received between 2000–2001 at Montefiore Medical Center, Department of Pathology, Bronx, NY. Three samples were excluded from this study: one had less than 2% B cells, and two skin biopsies inked with dibromohydroxymercurifluorescein produced a highly fluorescent background. Therefore, we retrospectively analyzed 130 samples from 128 patients. We compared two different analytical methods for identifying monoclonal IgH gene rearrangement: an established, standard method using polyacrylamide gel electrophoresis (PAGE) versus our proposed new strategy using DNA melting curve analysis in the LightCycler System (Roche Molecular Biochemicals, Mannheim, Germany).

In addition, 23 fresh-frozen lymph nodes suspected of lymphoma were studied for IgH-R between 1995–1996 using Southern blot analysis, as previously described. 12 These archival DNA samples were also studied for IgH-R using melting curve analysis in the LightCycler System.

DNA Extraction

DNA was extracted from FFPE tissue samples according to standard methods. Briefly, twenty 5 μm-thick sections were deparaffinized with xylene, washed twice with 100% ethanol, then vacuum-dried. DNA was reconstituted with PK buffer (100 mmol/L Tris-HCl, 4 mmol/L EDTA, pH 7.6) and digested with proteinase K at 56°C for 24 to 48 hours. Wizard PCR Preps DNA Purification System (Promega, Madison, WI) was used according to the manufacturer’s recommendation and the concentration of DNA was determined with a DYNA-Quant minifluorometer (Hoefer, San Francisco, CA).

PCR Primers

The DNA from these samples was PCR-amplified using a commercial master mixture containing seven IgH variable gene framework region 3 (FR3) primers plus a joining region (JH) consensus primer (InVivoScribe Technologies, San Diego, CA). These primers have been designed to target conserved DNA sequences surrounding the unique hypervariable antigen-binding complementarity determining region 3 (CDR3). 13

Perkin-Elmer Thermalcycler PCR and PAGE Analysis

We used the IgH FR3 Master Mixture (InVivoScribe Technologies) to amplify the IgH variable region in a 9700 Perkin-Elmer thermalcycler, GeneAmp PCR System (Cetus, Norwalk, CT). Genomic DNA (500 ng) was added up to a final reaction volume of 50 μl. After an initial “hot start” using TaqStart Antibody (Clontech Laboratories, Inc., Palo Alto, CA) at 95°C for 10 minutes, PCR cycle parameters were: denaturing at 94°C for 30 seconds, followed by annealing at 55°C for 30 seconds, and extending at 72°C for 1 minute. This program was repeated for 35 cycles. A final extension was performed at 72°C for 10 minutes. For PAGE analysis, 20 μl of amplicon was electrophoresed on an 8% non-denaturing acrylamide gel (16 × 16 × 0.1 cm) using D Gene System (Bio-Rad Laboratories, Hercules, CA) at room temperature for 150 minutes at 200 volts. The gel was then stained with 0.5% ethidium bromide and photographed under ultraviolet light. The expected size range of the amplicon varied from 69 to 129 bp. The quality of the DNA in each sample was confirmed by amplifying a 324-bp fragment of β-globin. 14

LightCycler PCR and Melting Curve Analysis

The IgH variable region was amplified in the LightCycler System. The reaction mixture of each microcapillary contained 18 μl of IgH FR3 Master Mixture, 0.4 μl of TaqDNA polymerase (Promega), 0.2 μl of 1:1000 dilution of SYBR Green I dye (Sigma) in TE buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 7.6), 0.1 μl of bovine serum albumin (20 mg/ml; Sigma), and 0.8 μl of 25 mmol/L MgCl2 (Promega). Genomic DNA (100 ng) was added up to a final volume of 20.5 μl. After initial “hot start” using TaqStart Antibody (Clontech Laboratories, Inc.) at 95°C for 10 minutes, PCR cycle parameters were: denaturing at 95°C for 0 second, annealing at 55°C for 10 seconds and extension at 72°C for 15 seconds. After 40 PCR amplification cycles, the LightCycler System DNA melting curve analysis was performed. In this program, PCR products were denatured at 95°C for 30 seconds, allowed to anneal at 55°C for 30 seconds and quickly raised to 70°C at a transition rate of 20°C/second; then the temperature was increased slowly from 70°C to 95°C at a transition rate of 0.05°C/second during continuous fluorescence monitoring at 521 nm.

Results

Optimization of DNA Melting Curve Analysis

We first determined the optimal number of PCR cycles needed to amplify IgH in the LightCycler System. Samples containing 100 ng of DNA from 100% Namalwa B-cell line, a mixture of 50% Namalwa and 50% tonsil, 100% tonsil, and a negative control containing all reagents without DNA were amplified for 20, 30, 40, 50, and 60 cycles (Figure 1)1) . The ability to visualize the Tm inflection point of melted DNA was enhanced by plotting the negative first derivative (−dF/dT) versus T (F = fluorescence, T = temperature) as recommended by the manufacturer.

Figure 1.
Optimization of PCR cycles. DNA from 100% Namalwa B-cell line (N, top curve), a mixture of 50% Namalwa and 50% tonsil (0.5 N + 0.5 T, middle curve), and 100% tonsil (T, bottom curve) was amplified for 20 (A), 30 (B), 40 (C), 50 (D), and 60 (E ...

To select an optimal number of PCR cycles, we examined the −dF/dT peak height ratio between Namalwa B-cell line and tonsil. The 100% Namalwa/100% tonsil −dF/dT peak height ratio equaled 2.27 at 30 cycles and 3.88 at 40 cycles. Additional cycles did not significantly improve this ratio (Figure 11 and Table 11 ). Based on these observations, we set the criteria for diagnosing a monoclonal IgH-R DNA sample as having a −dF/dT peak height at least twice as high, and less than one-half as wide as a polyclonal tonsil DNA sample when measured at the base of the curve after 40 PCR cycles.

Table 1.
Optimization of LightCycler PCR Cycle Number

Precision of LightCycler System Melting Curve Analysis

The precision of the method was tested by repeating the LightCycler PCR and DNA melting curve assay 10 separate times using 50% Namalwa B-cell line DNA diluted into tonsil DNA. The coefficient of variation (CV) of the assay was 0.47% at mean Tm equal to 84.98°C.

Reproducibility of Melting Curve

Repeated melting curve analyses of the same B-cell line and patient clonal DNA samples reproducibly yielded identical −dF/dT versus T curves (low SD), indicating specific homoduplex formation. For example, triplicate repeat assays of two different monoclonal rearranged DNA samples yielded Tm values (mean ± SD) of 86.54 ± 0.06 and 85.68 ± 0.05°C. On the other hand, repeated melting curve analysis of the same tonsil and patient polyclonal DNA samples produced different −dF/dT versus T curves each time (high SD), indicating random formation of heteroduplexes. For example, triplicate repeat assays of two different patient polyclonal DNA samples yielded Tm values (mean ± SD) of 85.99 ± 0.79 and 86.12 ± 0.54°C. Thus, the SD of a polyclonal DNA sample is approximately tenfold greater than that of a monoclonal DNA sample.

Lower Limit for Detecting Monoclonal IgH Gene Rearrangement

To determine the minimum detectable percent clonal B-cell DNA with Tm analysis, we serially diluted Namalwa B-cell line (50%, 25%, 12.5%, 6.25%, 3.125%, and 1.56%) into tonsil. After performing PCR amplification for 40 cycles in the LightCycler, we could still detect a distinct peak by melting curve analysis with the expected Tm of the original B-cell clone at the 12.5% level (Figure 2)2) .

Figure 2.
Minimal detection of B-cell clonality. Namalwa B-cell line DNA (50%, 25%, 12.5%, 6.25%, 3.13%, and 1.56%) were serially diluted into tonsil DNA. After 40 cycles of amplification in the LightCycler System, a distinct peak with the expected Tm of Namalwa ...

Resolution of Melting Curve Analysis

To test whether melting curve analysis could discriminate the presence of two separate B-cell clones, equal amounts of two different DNA samples were mixed together, PCR-amplified and analyzed using melting curve analysis in the LightCycler System (Table 2)2) . We were able to resolve two peaks with a Tm difference equal to 1.78°C or greater. We could not resolve peaks with Tm difference of 0.49°C or 1.41°C. These data suggest that two closely related biallelic populations with one or only a few base differences could not be resolved by the current assay. However, even unresolved mixed DNA samples (Table 22 , experiments 1 and 2) yielded a single sharp peak with an intermediate Tm value that was clearly distinguishable from a broad polyclonal result.

Table 2.
Melting Curve Analysis of Two Different DNA Samples

Comparison of PAGE and Melting Curve Analysis

We compared PAGE versus melting curve analysis for detecting monoclonal IgH-R in 130 DNA samples from 128 patients with suspected B-cell lymphoproliferative disorders. The identical, commercially available, IgH FR3 and JH primers were used to amplify the DNA for both analytic techniques (see Materials and Methods). In the gel electrophoresis method, a clonal IgH-R was defined as a sharp band on an ethidium bromide-stained acrylamide gel. In the melting curve analysis, we used SYBR Green I fluorescent dye, which intercalates into double-stranded DNA, for measuring a sharp decrease in fluorescence as the sample is heated through its Tm to form single-stranded DNA. A clonal IgH-R was defined as a narrow DNA melting curve (plotted as −dF/dT versus T) that was greater than twice the peak height, but less than one-half the width, of a polyclonal B-cell tonsil sample (Figure 1)1) .

Among 130 DNA samples tested, we detected monoclonal IgH-R in 24 samples from 22 patients (Table 3)3) . These cases included 20 skin biopsies, 2 lymph nodes, 1 tonsil and 1 parotid gland. The morphological diagnoses were: extranodal marginal zone B-cell lymphoma (MZL) (N = 8); follicular lymphoma (N = 7); diffuse large B-cell lymphoma (N = 3); lymphoplasmacytic lymphoma (N = 2); nodal MZL (N = 1); lymphomatoid granulomatosis (N = 1); mixed cellularity Hodgkin lymphoma (MCHL) (N = 1); and B-cell chronic lymphocytic leukemia (N = 1). The single case of MCHL had numerous (greater than 5%) Reed-Sternberg cells. Melting curve analysis using the LightCycler System, compared to PAGE, revealed no false negative and no false positive results, yielding both sensitivity and specificity equal to 100%.

Table 3.
Summary of Cases Analyzed by PAGE and LightCycler

In two patients, DNA samples from two different biopsies yielded identical band patterns by PAGE and identical Tms by DNA melting curve analysis (Figure 33 and Table 33 ). We interpreted these results as reflecting persistence of the same B-cell clone. The Tms from 24 B-cell clonal clinical samples plus two B-cell lines (Namalwa and Ramos) ranged between 82°C to 88°C, with median Tm equal to 84°C (Table 3)3) . Since the primer-dimer Tm was always less than 70°C, it did not interfere with determining monoclonal IgH-R in any case.

Figure 3.
Detecting B-cell clonality in representative clinical DNA samples using melting curve analysis. Sharp peaks above dashed line (−dF/dT = 0.35) indicate clonally rearranged IgH: Namalwa B-cell line and four-patient DNA samples 1 2 5 19 ...

Comparison of Southern Blot Analysis and Melting Curve Analysis

We also compared Southern blot versus melting curve analysis for detecting IgH-R in 23 clinical DNA samples from fresh-frozen lymph nodes of 23 patients. We detected IgH-R by melting curve analysis in 7 DNA samples (Table 4)4) . Melting curve analysis, compared to Southern blot analysis, revealed sensitivity equal to 58.3% (7 of 12) and specificity equal to 100% (11 of 11) (Table 5)5) . The relatively low sensitivity is not surprising given the high proportion of follicular lymphomas in our samples, which are known to be hypermutated. However, our results closely match the sensitivity achieved by PCR in a recent multi-center study. 15

Table 4.
Summary of Cases Analyzed by Southern Blot and LightCycler
Table 5.
IgH Gene Rearrangement Test Results Obtained Using Southern Blot and LightCycler

Discussion

The IgH variable (V), diversity (D), and joining (J) gene segments recombine during early B-cell development: nucleotides may be randomly deleted or inserted in the V-D and D-J joining sites. 16 Later, when the B cell enters the germinal center, the somatic mutation mechanism further enhances diversity. 16 The resulting unique DNA sequence, CDR3, creates a B-cell signature that has been exploited to detect clonal IgH gene rearrangements. 16, 17, 18 Current DNA-based PCR strategies for detecting monoclonal IgH gene rearrangement measure either the length of the DNA product, such as PAGE and capillary gel electrophoresis (CGE), 4, 5, 15 or gel mobility variations arising from conformational changes, such as single-stranded conformation polymorphism analysis (SSCP) and denaturing gradient gel electrophoresis (DGGE). 3, 6, 7

We hypothesized that internal melting domains found in CDR3 could be used as a fingerprint for identifying B-cell clones. IgH FR3 and JH consensus primers yield PCR products with similar 5′ and 3′ ends but unique internal sequences, particularly across the V-D and D-J junctions and the D segment that define CDR3. 17, 18 Since mismatches at the internal melting domains are more destabilizing than mismatches at the ends, one expects that heteroduplex amplicons to melt at a lower Tm than homoduplex amplicons. 19, 20, 21 Although there has been a recent report of applying an analogous method for detecting clonal T-cell receptor γ gene rearrangement, 22 to our knowledge, we are the first to experimentally demonstrate that PCR amplicons from monoclonal B cells have a higher and sharper −dF/dT peak, reflecting homoduplex formation, compared with polyclonal B cells, which tend to have a much lower and broader −dF/dT peak, reflecting heteroduplex formation (Figures 11 22 3)3) .

The general method that we have described using consensus FR3 and JH primers is clearly not suitable for detecting minimal residual disease (MRD) since our sensitivity limit was only 12.5%. Further, small samples partially involved by monoclonal B-cell proliferations would also likely be missed. Other investigators have used either clone-specific primers or allele-specific oligonucleotide probes in a LightCycler format for quantifying MRD, with sensitivity yielding between 10−4 and 10−6 leukemic cells. 23, 24

When we compared Southern blot analysis with the LightCycler method for IgH-R from lymph node DNA samples (mostly follicular lymphomas), we achieved sensitivity equal to 58.3%, which is a typical sensitivity for PCR-based assays. 15 An additional set of FR primers would likely improve these results, as recently demonstrated using capillary electrophoresis. 5

DNA melting curve analysis in the LightCycler System has several advantages for determining monoclonal IgH-R: monoclonal versus polyclonal B cells are distinguished based on a fundamental DNA characteristic; 9 very fast temperature transition rates give rapid PCR results (approximately 40 minutes); 25 precise temperature control produces accurate and reproducible Tms; 25 combined PCR and DNA melting curve analysis in a closed system reduce cross-contamination risk; and 100% sensitivity and 100% specificity, compared to PAGE, in 130 clinical FFPE tissue samples (mostly skin biopsies) and 58.3% sensitivity and 100% specificity, compared to Southern blot, in 23 fresh-frozen lymph nodes (mostly follicular lymphomas) strongly suggest that DNA melting curve analysis in the LightCycler System has potential for evaluating monoclonal IgH-R in a clinical environment.

Notes

Address reprint requests to Dr. Howard Ratech, Montefiore Medical Center/Albert Einstein College of Medicine, Department of Pathology, North 4, 111 East 210th Street, Bronx, NY 10467. E-mail: .ude.uy.mocea@hcetar

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