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© 2001 American Society for Clinical Oncology Frequency of the Bcl-2/IgH Rearrangement in Normal Individuals: Implications for the Monitoring of Disease in Patients With Follicular LymphomaFrom the Imperial Cancer Research Fund Medical Oncology Unit, St Bartholomew’s Hospital Medical College, London; and Division of Molecular and Genetic Medicine, Royal Hallamshire Hospital, Sheffield, United Kingdom. Address reprint requests to Jude Fitzgibbon, PhD, Imperial Cancer Research Fund Medical Oncology Unit, St Bartholomew’s Hospital Medical College, Charterhouse Square, London EC1M 6BQ, England, United Kingdom; email jfitzgib{at}hgmp.mrc.ac.uk
PURPOSE: To determine the incidence and frequency of the Bcl-2/IgH rearrangement in the peripheral blood of normal individuals to define the potential complication this may pose for the molecular monitoring of disease in patients with follicular lymphoma (FL). MATERIALS AND METHODS: The incidence and frequency of the major breakpoint cluster region rearrangement in DNA extracted from peripheral blood or lymphoblastoid cell lines from 481 normal individuals was determined using a TaqMan real-time polymerase chain reaction assay (PE Applied Biosystems, Foster City, CA). RESULTS: Twenty three percent of samples were positive for the Bcl-2/IgH rearrangement, with approximately 3% of these at levels of more than 1 in 104 cells. CONCLUSION: The presence of circulating Bcl-2/IgH+ cells, other than those derived from the malignant clone, could confound the detection and quantitation of minimal residual disease in patients with FL, particularly at low levels of tumor burden.
FOLLICULAR LYMPHOMA (FL) is characterized by the presence of the t(14;18)(q32;q21) (Bcl-2/IgH) chromosomal translocation that results in the rearrangement and upregulation of the Bcl-2 proto-oncogene. The majority of rearrangements in Bcl-2 occur at two distinct chromosomal regions, the major breakpoint cluster region (MBR) in 70% and the minor cluster region in 10% of patient’s tumors.1-3 These rearrangements are detected in the bone marrow and peripheral blood of patients with FL independent of stage at diagnosis,4,5 and may persist even in patients who have achieved a complete clinical response (CR) after treatment.6 Conventionally, response to therapy for lymphoma has been defined according to clinical and radiologic criteria and morphologic examination of the peripheral blood and bone marrow. Because CR in these terms is frequently followed by recurrence,7 it would be attractive to define the outcome of therapy more precisely according to the amount of molecular rearrangement detectable during remission. However, the occurrence of the Bcl-2/IgH rearrangement in the peripheral blood or tissues from patients with follicular hyperplasia,8,9 persistent polyclonal B cell lymphocytosis,10 and normal individuals11-14 suggests that circulating Bcl-2/IgH+ cells, other than those derived from the malignant clone, may pose a potential complication when monitoring minimal residual disease (MRD) in patients with this disease. Real-time polymerase chain reaction (PCR) is a rapid technique for quantitation and monitoring of tumor burden.15-18 In this study, the technique has been used to determine the incidence and frequency of PCR-detectable MBR Bcl-2/IgH rearrangement in the peripheral blood from 481 normal individuals. The implications for monitoring MRD are discussed.
DNA Samples DNA obtained from the peripheral blood of 481 normal individuals was analyzed in the study. Of these, 397 were obtained from the Blood Transfusion services in London (180 samples) and Sheffield (217 samples). The median age of the blood donors was 42.3 years (range, 21 to 69 years) with 184 males (46%) and 212 females (53%), with one donor unspecified. All blood donors were negative for hepatitis B and C, syphilis and human immunodeficiency virus. A further 84 DNA samples extracted from Epstein Barr virus–transformed lymphoblastoid cell lines derived from the peripheral blood of normal individuals were kindly provided by J. Bodmer, DSc (Imperial Cancer Research Fund Laboratories, Oxford, United Kingdom).
Real-Time Quantitative PCR A 25-µL reaction mix was used incorporating TaqMan Universal PCR Master Mix (PE Applied Biosystems), 300 nmol/L of primers, a 200 nmol/L probe, and between 0.25 and 0.50 µg of DNA. After a 2-minute incubation at 50°C (to eliminate contamination from previous PCR products via the action of uracil-N-glycosylase) and denaturation for 10 minutes at 95°C, the DNA was subjected to 40 cycles of 15 seconds at 95°C and a 1-minute combined annealing/extension step at 60°C. Standard curves were constructed with the DoHH2 cell line that has a t(8;14;18) rearrangement.20 Tenfold dilutions were made in water to generate standards ranging from 10 to 105 DoHH2 cells. These standards were used to generate standard curves for both the Bcl-2/IgH rearrangement and the endogenous reference genes. Quantitation of the rearrangement, however, was similar whether DoHH2 was diluted into water or genomic DNA. The quantity of target (Bcl-2/IgH, beta-actin, or beta-2 microglobulin) in a test sample was derived from where it lay on the DoHH2 standard curve. Approximately 1.5 µg of DNA in 3 x 0.5-µg reactions (equivalent to 2.5 x 105 cells; 1 cell = 6 pg) from each of the 84 lymphoblastoid cell lines was screened for the presence of the Bcl-2/IgH rearrangement and beta-actin, with standard curves and six no-template controls for each 96 x 25 µL experiment. The 397 DNA samples extracted from peripheral blood were first analyzed using a two-step strategy for time and cost considerations. Initially, 1.5 µg of DNA (0.5 µg x three reactions) of each sample was screened to identify samples having the Bcl-2/IgH rearrangement, using real-time PCR, without standard curves. A control beta-2 microglobulin amplification of 0.5 µg of DNA was carried out for each sample and nine no-template controls were included in each 96-well experiment. Samples that were positive were rescreened using a further 2 µg of DNA and full standard curves for quantitation.
Sequence Analysis of Bcl-2/IgH Products
Validation of the Bcl-2/IgH MBR Real-Time PCR Assay The t(8;14;18)-bearing FL cell line DoHH2 was used to generate standard curves for real-time PCR with Bcl-2/IgH MBR and two endogenous reference genes, beta-actin and beta-2 microglobulin. Figure 1 shows typical standard curves obtained with beta-2 microglobulin (Figs 1A and 1B) and Bcl-2/IgH (Figs 1C and 1D). The slope value of the standard curve represents the number of cycles corresponding to one log difference in starting copy number. The higher threshold cycle values in the Bcl-2/IgH amplification plot reflect the presence of a single copy of the rearrangement in each DoHH2 cell, compared with two copies of beta-2 microglobulin per cell. The variable slope values are indicative of the difference in efficiencies of the PCR reactions, which may reflect the disparity in amplicon product size (variable Bcl-2/IgH product size v beta-2 microglobulin, 79 base pairs).
The Bcl-2/IgH MBR real-time reaction had a sensitivity of one in 105 cells. However, to obtain highly reproducible results, the presence of a minimum of 10 target copies of the rearrangement was necessary. Because the upper limit of DNA used per reaction was approximately 24 ng/µL or approximately 105 cells/25 µL reaction, this is equivalent to the reproducible detection of one copy of the rearrangement in 104 cells (one rearranged allele/cell). In contrast, detection of beta-2 microglobulin required a minimum of five target copies equivalent to a reproducible detection of one in 4 x 104 cells (two normal alleles/cell; representative experiment shown in Fig 1).
Frequency of MBR in Lymphoblastoid Cell Line DNA
Frequency of MBR in Peripheral-Blood DNA
DNA (2 µg) from 74 of these 101 positive samples was reanalyzed using standard curves to quantify the frequency of the Bcl-2/IgH rearrangement (Fig 2B). DNA was not available for analysis for the remaining 27 individuals. Twenty eight samples (28 of 74; 38%) remained positive in this study in the range of one in 2.6 x 103 to 6.6 x 104 cells. Twelve of 28 of these were positive at frequencies of more than one in 104 cells, including six of seven samples that had previously been positive in all three reactions. Of the remaining six samples, five had previously been positive in two of three reactions and one sample had been positive in one of three reactions. Samples previously positive in only a single reaction were less likely to be PCR-positive on retesting, supporting the finding that highly reproducible results are limited to a minimum target copy number of 10.
Age and Incidence of Bcl-2/IgH
Confirmation of the Bcl-2/IgH Rearrangements in Normal DNA
The Bcl-2/IgH rearrangement can be used as a marker to monitor the course of the disease in patients with FL. However, the finding that the rearrangement is detected in normal individuals poses a potential complication when used to quantitate MRD. In this study, the incidence and frequency of the Bcl-2/IgH rearrangement was determined in 481 DNA samples from normal individuals using real-time PCR. The rearrangement was detected in 23% (112) of the 481 samples analyzed. In at least 3% of normal samples (two of 84 lymphoblastoid lines, 12 of 397 blood DNA samples), the MBR rearrangement was present at frequencies of more than one in 104 cells, which is higher than that observed in many patients. The lower incidence of the rearrangement compared with that observed in other studies13 probably reflects the smaller amount of DNA analyzed for each normal individual. Furthermore, the real-time PCR assay used in this study requires a minimum of 10 target copies for the reproducible detection of the rearrangement, increasing the likelihood of false-negative results. The occurrence of the Bcl-2/IgH rearrangement in 23% of normal peripheral-blood samples is likely therefore to represent a lower limit for the incidence of the rearrangement in normal individuals. Previous studies have shown an increasing incidence of the Bcl-2/IgH rearrangement with age that suggested a link between the incidence of FL and the occurrence of the Bcl-2/IgH rearrangement.13 The normal individuals described here exhibit no apparent age-associated increase in the incidence and frequency of circulating Bcl-2/IgH-bearing cells. The high detection rate of the rearrangement in the peripheral blood of normal individuals and the low incidence of FL21 would also seem to rule out the possibility that these cells represent a subclinical form of the disease.12 This high incidence of the Bcl-2/IgH rearrangement in normal individuals will make it difficult to quantify the frequency of the malignant clone in patients with FL who may similarly carry a background of Bcl-2/IgH+ cells unrelated to their disease. The difficulty involved in discriminating between the malignant clone and other Bcl-2/IgH-bearing cells, usually determined by sequence comparison with the known clonal rearrangement, can also be overcome using clone-specific primers designed to breakpoint and junctional region N-nucleotides.22 This may be a necessary approach to confirm the quantitation of the malignant clone in patients, particularly those having low levels of detectable Bcl-2/IgH+ cells. As reproducible detection/quantitation of the rearrangement is only obtained with a minimum initial copy number of five to 10 Bcl-2/IgH+ cells,16,19 the current real-time PCR protocols seem to be inappropriate as the sole technique for monitoring MRD in FL. However, efforts to further optimize the reproducibility of this real-time PCR assay will be worthwhile, as the technique offers a powerful and simple method of quantifying the Bcl-2/IgH rearrangement when compared with conventional semiquantitative nested PCR approaches. In conclusion, this study quantifies the presence of Bcl-2/IgH MBR rearrangement in a proportion of normal individuals at levels comparable to or greater than that found in patients with FL. It is likely that the presence of a background of Bcl-2/IgH+ cells in patients could confound the detection and quantitation of MRD, particularly at low levels of tumor burden.
We thank John Anson for excellent technical assistance, Bryan Young and Louise Jones for critical reading of the manuscript, and Vivienne Weller for secretarial assistance.
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Copyright © 2001 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
