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Journal of Clinical Oncology, Vol 21, Issue 7 (April), 2003: 1398-1403
© 2003 American Society for Clinical Oncology

PCR-Detectable Nonneoplastic Bcl-2/IgH Rearrangements Are Common in Normal Subjects and Cancer Patients at Diagnosis but Rare in Subjects Treated With Chemotherapy

Marco Ladetto, Daniela Drandi, Mara Compagno, Monica Astolfi, Federica Volpato, Claudia Voena, Anna Novarino, Berardino Pollio, Alfredo Addeo, Irene Ricca, Patrizia Falco, Federica Cavallo, Sonia Vallet, Paolo Corradini, Alessandro Pileri, Giacomo Tamponi, Antonio Palumbo, Oscar Bertetto, Mario Boccadoro, Corrado Tarella

From the Divisione di Ematologia, Dipartimento di Medicina ed Oncologia Sperimentale-Universita’ di Torino, and Divisione di Oncologia Medica-Azienda Ospedaliera San Giovanni Battista, Torino; Laboratorio di Ematologia Molecolare-Istituto Scientifico H.S. Raffaele, and Unità Trapianto Midollo Osseo, Istituto Nazionale per lo Studio e la Cura dei Tumori-Università di Milan, Italy.

Address reprint requests to Marco Ladetto, MD, Cattedra di Ematologia, Via Genova 3, 10126 Torino, Italy; email: marco.ladetto{at}unito.it.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: To assess whether nonneoplastic Bcl-2/IgH rearrangements act as a confounding factor in the setting of minimal residual disease analysis by evaluating their incidence in a panel of lymphoma-free subjects, including cancer-free donors and chemotherapy-naive and chemotherapy-treated cancer patients.

Patients and Methods: A total of 501 nonlymphoma subjects have been assessed: 258 cancer-free patients and 243 patients with malignancies other than lymphoma, 112 of whom were chemotherapy-naive. Patients were primarily assessed by nested polymerase chain reaction (PCR), followed by real-time quantitative PCR if they scored positive. In addition, six initially PCR-positive cancer-free donors were prospectively reassessed by qualitative and quantitative PCR after 30 and 60 days.

Results: The overall incidence of Bcl-2/IgH positivity was 9.6%, with a median number of 11 rearrangements per 1,000,000 diploid genomes (range, 0 to 2,845 rearrangements), as assessed by real-time PCR. The incidence was similar in healthy subjects and cancer patients at diagnosis (12% and 12.5%; P = not significant). In contrast, the incidence of this translocation was only 2.3% in chemotherapy-treated patients (P < .001). In addition, three initially PCR-positive cancer-free donors showed persistence of their rearrangements when assessed after 30 and 60 days.

Conclusion: The low incidence of nonneoplastic Bcl-2/IgH rearrangements following chemotherapy provides further evidence of the prognostic role of persistent PCR-positivity in the posttreatment molecular follow-up of follicular lymphoma patients.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE T(14;18) translocation involving the Bcl-2 proto-oncogene and the immunoglobulin heavy-chain (IgH) genes is commonly used as a polymerase chain reaction (PCR)-amplifiable tumor-specific marker for minimal residual disease (MRD) evaluation in follicular lymphoma (FL).1–11 Indeed, several studies have shown that persistent PCR-positivity after chemotherapy is associated with a high risk of relapse,1–11 indicating that patients failing to achieve PCR-negativity should receive additional treatment to increase cytoreduction and prolong disease-free survival.5

Despite the striking evidence of the prognostic value of MRD evaluation, physicians are reluctant to treat patients in complete remission on the strength of their PCR results alone. This is mostly because nonneoplastic Bcl-2/IgH rearrangements are common in the peripheral blood (PB), bone marrow, and other tissues of healthy donors12–18 and, hence, are likely to produce a high number of false-positive results, which may increase the risk of overtreating large groups of patients. However, healthy blood donors are not ideal controls for assessment of the confounding role of these rearrangements in the follow-up of FL patients after chemotherapy. It would be more appropriate to know whether these rearrangements also occur in cancer patients, particularly after chemotherapy. If these rearrangements also occur in chemotherapy-treated cancer patients, their use as a prognostic indicator would require careful reconsideration. However, if nonneoplastic Bcl-2/IgH positive cells are rare or absent in this population, the prognostic role of MRD analysis would persist, despite the high incidence of these rearrangements in the PB of healthy subjects.

This question has been investigated by determining the incidence of Bcl-2/IgH rearrangements on PB samples from nonlymphoma subjects. These were cancer-free subjects and patients with malignancies (hematologic and solid) other than lymphoma; some patients were chemotherapy-naive, and some had received intensive or conventional chemotherapy. In addition, prospective evaluation after 30 and 60 days of a panel of cancer-free donors who tested Bcl-2-positive at their first examination was performed to assess whether these lesions were transient or persistent. Our results show that nonneoplastic Bcl-2/IgH rearrangements are common in chemotherapy-naive subjects (with or without cancer) but are extremely rare after chemotherapy. In addition, three of six subjects who tested PCR-positive on day 0 reverted to PCR-negativity when assessed after 30 and 60 days. These results demonstrate that nonneoplastic Bcl-2/IgH-positive cells cannot act as major confounding factors for PCR analysis because of their low frequency in chemotherapy-treated subjects. This study further supports the clinical significance of persistent PCR-positivity in the posttreatment follow-up.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Population
A total of 501 subjects (> 98% whites of Italian nationality) were evaluated (Table 1Go): 258 subjects with no cancer history (166 healthy blood donors and 92 patients taking warfarin for cardiovascular disease; the latter were chosen to increase the number of elderly subjects) and 243 patients with cancer (hematologic disease in 136 patients, solid malignancies in 107 patients). Of these 243 patients, 112 patients were chemoradiotherapy-naive (surgery was allowed), whereas 131 patients had already received chemotherapy (37 patients underwent intensified intravenous [IV] chemotherapy with autologous transplantation, whereas 94 patients received conventional IV chemotherapy). Patients treated with mild oral regimens, such as melphalan plus prednisone, or with monoclonal antibodies were not included. Sixty percent of samples from chemotherapy-treated patients were taken during complete or very good partial remission, and 40% of samples were taken from patients with radiologic or histologic evidence of active disease. Median time from the last chemotherapy course was 11 months (range, 1 to 26 months). Sex and age distributions were similar among the three major subgroups (cancer-free subjects, chemotherapy-naive cancer subjects, and chemotherapy-treated cancer subjects). Informed consent was obtained from all subjects.


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  		Table 1. Characteristics of the Study Population
 
Samples and DNA Extraction
Mononuclear cells were separated by Ficoll-Hypaque (Axis-Skilo, Oslo, Norway) density gradient centrifugation. Genomic DNA was extracted with the DNAzol reagent (GIBCO BRL Life Technologies, Grand Island, NY) according to the manufacturer’s recommendations.

Nested PCR for the Bcl-2 Major Breakpoint Region Translocation
The major breakpoint region (MBR) was amplified by nested PCR on genomic DNA using oligonucleotide primers and amplification conditions as previously described.2,19 Amplified DNAs were analyzed by electrophoresis on 2% agarose gel containing ethidium bromide and were visualized with ultraviolet light. Sensitivity was one Bcl-2/IgH rearrangement in 100,000 normal cells on repeated dilution assays. Samples were always tested twice on separate amplifications performed on different days, and both negative and weakly positive controls were added in each reaction. Twice PCR-positive samples were scored as PCR-positive, and twice PCR-negative samples were scored as PCR-negative. When one amplification was PCR-positive and one was PCR-negative, the amplification was repeated four times and samples were scored as positive if at least two of the five reactions were PCR-positive. Standard procedures to avoid contamination were carried out as previously reported.20

Real-Time PCR for the Bcl-2 MBR Translocation
Real-time PCR for the Bcl-2 MBR translocation was carried out as previously described.21 Briefly, real-time PCR was performed by amplifying triplicate samples containing 600 ng of target DNA in a 25-µL volume using the TaqMan PCR Core Reagent Kit reagents (PE Applied Biosystems, Foster City, CA). The following conditions were used: 1 x PCR Core Reagent Kit Buffer, 2.5 mmol/L MgCl2, 200 µmol/L of deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, 400 µmol/L of deoxyuracil triphosphate, adding 10 pmol of each primer, 5 pmol of reporting probe derived from the sense strand of the Bcl-2 gene (5'-FAM-ctgtttcaacacagacccacccagag-TAMRA-3'), 0.5 U of AmpErase uracil-N-glycosylase, and 1.25 U of Taq Platinum DNA polymerase (GIBCO BRL, Gaithersburg, MD). After an incubation of 2 minutes at 50°C for optimal activity of AmpErase uracil-N-glycosylase and an incubation of 10 minutes at 95°C to activate Taq Platinum, the following reaction was carried out: 42 cycles of denaturation at 95°C for 15 seconds and annealing at 62°C for 1 minute. Reactions were performed in an AbiPrism model 7700 sequence detector system (PE Applied Biosystems). The GAPDH gene was chosen as reference standard to normalize patient samples for DNA quality and quantity, as previously described.22

Sequencing Analysis of Bcl-2/IgH Rearrangements
The presence of Bcl-2/IgH rearrangements was confirmed by direct sequencing of both forward and reverse DNA strands of PCR products obtained from nested PCR. For direct sequencing, PCR products were run on 2% low melting point (LMP) agarose gels, excised and purified using the QIAquick Gel Extraction Kit (QIAGEN SpA, Milan, Italy). Samples were then sent to the Genenco M-Medical sequencing facility laboratories (Rome, Italy) for sequencing reactions and runs. Sequences were then compared to the published germline Bcl-2 and IgH gene sequences23,24 to identify breakpoints, JH gene usage, and N insertions.

Statistical Analysis
Fisher’s exact test was used to compare the incidence of rearrangements among different subgroups of subjects.25


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Incidence of Bcl-2 Rearrangements in Nonlymphoma Subjects
A PCR-amplifiable Bcl-2 translocation was observed in 9.6% of samples. Incidence of Bcl-2 rearrangement was 12% in cancer-free subjects, 12.5% in chemotherapy-naive cancer patients, and only 2.3% in chemotherapy-treated cancer patients (P < .001; Fig 1Go). The translocation was never amplified from patients treated with intensified chemotherapy with autologous transplantation (Fig 1Go), indicating that its occurrence decreases as a function of the intensity of chemotherapy. The three cancer patients who tested PCR-positive following chemotherapy were Pt-316B, a 76-year-female with breast cancer treated with four courses of carboplatin 400 mg plus etoposide 100 mg/m2; Pt-347GI, a 79-year-old male with rectal cancer treated with two courses of fluorouracil 425 mg/m2 days 1 to 5 plus levamisole 10 mg/m2 days 1 to 5; and Pt-380CL, a 65-year-old female with chronic lymphocytic leukemia treated with four courses of fludarabine 25 mg/m2 days 1 to 5. Among cancer-free and chemotherapy-naive cancer subjects, the incidence of Bcl-2/IgH rearrangements was approximately 12% for both groups. There were no significant differences in incidence according to sex (13% in males v 12% females; P = not significant [NS]) or age (13.2% in subjects aged < 40 years, 11.4% in subjects aged between 40 and 60 years, and 12.5% in subjects aged > 60 years; P = NS) as also reported by others.12 No clustering according to tumor histology was noted; the incidence of this translocation was 9.7% in chemotherapy-naive solid tumors (one lung, one colorectal, and one ovarian cancer) and 13% in chemotherapy-naive hematologic tumors (nine multiple myeloma patients and two chronic lymphocytic leukemia patients; P = NS).



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  		Fig 1. Incidence of nonneoplastic Bcl-2 rearrangements in nonlymphoma subjects. They are common in cancer-free subjects and in chemotherapy-naive cancer patients but are rare after chemotherapy, particularly if delivered at myeloablative doses.

 
Real-Time Quantitative PCR Analysis of NonneoplasticBcl-2 Rearrangements
Real-time quantitative PCR was performed in triplicate on all samples testing positive by qualitative nested-PCR analysis, with the exception of two patients with insufficient DNA for real-time PCR. A representative example of real-time PCR analysis of nonneoplastic Bcl-2/IgH rearrangements is shown in Fig 2Go. The median number of Bcl-2/IgH rearrangements was 11 (range, 0 to 2,845 rearrangements). In nine samples, real-time PCR tested negative despite PCR-positivity by nested PCR. This probably reflects a small number of rearrangements, because our real-time PCR assay employs less DNA than nested PCR and is thus slightly less sensitive.18 Only two of the three chemotherapy-treated cancer patients were analyzed by real-time PCR because of an insufficient amount of DNA from Pt-316B. Pt-380CL tested PCR-negative by quantitative PCR, whereas Pt-347GI had 22 copies/1,000,000 diploid genomes.



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  		Fig 2. Real-time polymerase chain reaction for the Bcl-2/IgH rearrangement. (A) Ten-fold dilutions of cloned Bcl-2/IgH rearrangement. A no-template control is also included. (B) A standard curve of the experiment represented in (A). (C) Amplification of two samples with different amounts of Bcl-2/IgH rearrangements.

 
Prospective Monitoring of Nonneoplastic Bcl-2 Rearrangements in Healthy Subjects
Because PCR analysis is usually performed serially and poor prognosis is usually associated with the presence of repeatedly PCR-positive results, we assessed prospectively a panel of cancer-free donors that scored PCR-positive at their first analysis. Six donors (who were all receiving warfarin therapy and had no cancer history) were reassessed after 30 and 60 days. Persistence at 30 and 60 days was observed only in three subjects, whereas the others promptly reverted to PCR-negativity. Results are plotted in Fig 3Go. Interestingly, the three subjects with persistent PCR-positivity at 30 and 60 days (Pt-123, a 71-year-old male; Pt-130, a 74-year-old female; and Pt-178, a 60-years-old female) appeared to have a higher number of Bcl-2-positive rearrangements compared with those reverting to PCR-negativity (Fig 3Go). Real-time PCR was performed also on follow-up samples. The number of Bcl-2 rearrangements remained the same during follow-up examinations.



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  		Fig 3. Graphic representation of the amount of Bcl-2/IgH rearrangements measured by real-time polymerase chain reaction (PCR; expressed as Bcl-2 rearrangements/1,000,000 diploid genomes) observed on day 0, 30, and 60 in six initially PCR-positive cancer-free subjects scoring PCR-positive during their first assessment.

 
Sequencing Analysis of Bcl-2/IgH Rearrangements
Rearrangements from 30 Bcl-2/IgH-positive subjects were analyzed by direct sequencing. All evaluated subjects had different rearrangements, thus excluding cross-contamination between samples (data not shown). Rearrangements contained N insertions in 96% of cases. Interestingly, one patient displayed two rearrangements within the same sample (two different agarose electrophoresis bands), which allowed direct sequencing of two different rearrangements (data not shown). The two rearrangements employed different JH genes (JH4 and JH6) and contained different N insertions and, thus, appeared completely unrelated. The three patients testing PCR-positive at follow-up examinations on days 30 and 60 displayed the same rearrangements on basal and follow-up samples. This demonstrates that PCR-positivity was related to persistence of the same clone and not to the occurrence of novel unrelated rearrangements.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study evaluated the presence of nonneoplastic Bcl-2/IgH rearrangements in a large panel of nonlymphoma subjects. These rearrangements are frequently detected in the PB of chemotherapy-naive subjects (with or without cancer), but are extremely rare among chemotherapy-treated patients. Sequencing analysis of some of these lesions confirmed their clonal nature. Prospective evaluation of Bcl-2/IgH-positive subjects at day 30 and 60 showed persistence of the same Bcl-2/IgH rearrangement only in three subjects, whereas the other subjects promptly reverted to PCR-negativity. These results indicate that it is unlikely that the adverse prognostic value of persistent PCR-positivity in the follow-up after treatment could be reduced by the presence of nonneoplastic Bcl-2/IgH rearrangements.

Persistence of a PCR-detectable Bcl-2/IgH rearrangement after chemotherapy has been repeatedly reported as an extremely powerful prognostic indicator of FL recurrence,1–11 as opposed to what might be expected on the basis of the high incidence of this rearrangement in the PB of cancer-free subjects. Indeed, if the incidence of nonneoplastic Bcl-2 rearrangements were similar in both chemotherapy-treated and healthy subjects, one would expect to see a high proportion of patients with no evidence of relapse despite their persistent PCR-positivity during the molecular follow-up after treatment. To explain this contradiction, we hypothesized that these rearrangements are less common in chemotherapy-treated cancer patients than in cancer-free subjects and chemotherapy-naive cancer patients.12,18 Our data on 501 subjects clearly show that these rearrangements are common in chemotherapy-naive subjects, but are rare following chemotherapy. This explains why the predictive value of PCR analysis in the follow-up after chemotherapy is not strongly influenced by the presence of nonneoplastic Bcl-2/IgH rearrangements, as postulated by studies investigating their incidence in PB taken only from healthy donors.12

Even in chemotherapy-free subjects, the incidence of nonneoplastic Bcl-2 rearrangements was lower than that observed in other reports, particularly the large screening recently published by Summers et al.12 There is no clear explanation for these differences, although ethnic or environmental differences may be involved.18,26,27 Indeed, a recent report has shown substantial differences between the incidence of these rearrangements in Asian and white subjects from Northern Europe.18 It is possible that Mediterranean subjects might have a lower incidence of nonneoplastic Bcl-2/IgH rearrangements compared with people from northern Europe. Although this hypothesis is worthy of consideration, additional comparative studies need to be performed to rule out the possibility that these differences are the result of variations of sensitivity among different laboratories.

Another major difference that characterizes at least a proportion of nonneoplastic Bcl-2 rearrangements is their lack of persistence. In contrast, the most prominent molecular feature associated with poor outcome in the posttreatment follow-up of FL patients is persistent PCR-positivity, usually defined as the presence of two or more PCR-positive results in repeated analyses. Thus, performance of a tandem PCR analysis on days 0 and 30 will further minimize the risk of incorrectly classing a patient as one with a high risk of relapse. Finally, if an expensive or potentially life-threatening option (such as allogeneic transplantation) is the planned treatment for persistently PCR-positive, direct sequencing of the posttreatment rearrangement and comparison with the sequence observed at diagnosis will clearly differentiate nonneoplastic Bcl-2/IgH-positive cells and malignant FL cells.

Our study also shows that the disappearance of nonneoplastic Bcl-2/IgH rearrangements is not related to the presence of cancer but, instead, to the effect of chemotherapy. One explanation is that these cells belong to a highly chemosensitive population that may be markedly reduced or even eradicated by chemotherapy. Although this hypothesis is attractive, it does not take into account that cells carrying these rearrangements also were short-lived in several chemotherapy-free subjects. Chemotherapy-induced perturbations of B-cell development, which impair genetic events, leading to nonphysiologic rearrangements or strongly reduce their occurrence, may also be responsible.

Persistence of the same Bcl-2/IgH rearrangements after 60 days is an interesting finding, especially because these patients displayed a high and fairly stable number of rearrangements. Indeed, it can be hypothesized that these cells may be more closely related to FL cells than short-living populations that promptly disappear within a few weeks. To address the possibility that Bcl-2/IgH-positive clones have even a broader life span heterogeneity spectrum, we plan to reassess the PCR status of a panel of Bcl-2/IgH-positive subjects monthly over a long period.

In addition to these considerations, several aspects still need to be clarified to fully characterize the biologic role of these rearrangements. First, it is unknown whether nonneoplastic Bcl-2/IgH cells occur periodically in every subject or only in a subgroup of individuals because of specific genetic or immunologic defects. Second, we do not know whether cells with these rearrangements are fully benign or whether, at least in some cases, they should be considered as a preneoplastic population. The indication that they are particularly common among ethnic groups with a high incidence of FL strongly indicates that they are linked to its pathogenesis.18 Third, we do not know whether the occurrence of these lesions might appear in association with some specific immunologic stresses such as viral infection or reactivation. Finally, we do not know whether the immune system can recognize Bcl-2/IgH-positive cells and possibly act to ensure their prompt eradication. Our study and other reports have defined the epidemiology and the potential confounding role of this rearrangement in the setting of MRD analysis.1–11 Additional studies are needed to fully characterize the biology and the role in lymphomagenesis of this frequently observed, although misunderstood, genetic lesion.


   ACKNOWLEDGMENTS
 
We thank Massimo Massaia, MD, for helpful suggestions.


   NOTES
 
Supported by Associazione Italiana Ricerca sul Cancro (AIRC), Milan, and Compagnia di San Paolo, Torino, Italy. F.V. and M.C. are recipients of a fellowship from Fondazione Angela Bossolasco. D.D. is the recipient of a fellowship from AIRC.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Gribben JG, Freedman AS, Neuberg D, et al: Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma. N Engl J Med 325:1525–1533, 1991[Abstract]

2. Corradini P, Astolfi M, Cherasco C, et al: Molecular monitoring of minimal residual disease in follicular and mantle cell non-Hodgkin’s lymphomas treated with high-dose chemotherapy and peripheral blood progenitor cell autografting. Blood 89:724–731, 1997[Abstract/Free Full Text]

3. Moos M, Schulz R, Martin S, et al: The remission status before and the PCR status after high-dose therapy with peripheral blood stem cell support are prognostic factors for relapse-free survival in patients with follicular non-Hodgkin’s lymphoma. Leukemia 12:1971–1976, 1998[CrossRef][Medline]

4. López-Guillermo A, Cabanillas F, McLaughlin P, et al: The clinical significance of molecular response in indolent follicular lymphomas. Blood 91:2955–2960, 1998[Abstract/Free Full Text]

5. Corradini P, Ladetto M, Pileri A, et al: Clinical relevance of minimal residual disease monitoring in non-Hodgkin’s lymphomas: A critical reappraisal of molecular strategies. Leukemia 13:1691–1695, 1999[CrossRef][Medline]

6. Freedman AS, Neuberg D, Mauch P, et al: Long-term follow-up of autologous bone marrow transplantation in patients with relapsed follicular lymphoma. Blood 94:3325–3333, 1999[Abstract/Free Full Text]

7. Rambaldi A, Lazzari M, Manzoni C, et al: Monitoring of minimal residual disease after CHOP and rituximab in previously untreated patients with follicular lymphoma. Blood 99:856–862, 2002[Abstract/Free Full Text]

8. Ladetto M, Corradini P, Vallet S, et al: High rates of clinical and molecular remissions in follicular lymphoma patients receiving high-dose sequential chemotherapy and autografting at diagnosis: A multicenter, prospective study of the Gruppo Italiano Trapianto di Midollo Osseo (GITMO). Blood 100:1559–1565, 2002[Abstract/Free Full Text]

9. Gribben JG, Freedman A, Woo SD, et al: All advanced stage non-Hodgkin’s lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment. Blood 78:3275–3280, 1991[Abstract/Free Full Text]

10. Luthra R, McBride JA, Cabanillas F, et al: Novel 5' exonuclease-based real-time PCR assay for the detection of t(14;18)(q32;q21) in patients with follicular lymphoma. Am J Pathol 153:63–68, 1998[Abstract/Free Full Text]

11. Olsson K, Gerard CJ, Zehnder J, et al: Real-time t(11;14) and t(14;18) PCR assays provide sensitive and quantitative assessments of minimal residual disease (MRD). Leukemia 13:1833–1842, 1999[CrossRef][Medline]

12. Summers KE, Goff LK, Wilson AG, et al: Frequency of the Bcl-2/IgH rearrangement in normal individuals: Implications for the monitoring of disease in patients with follicular lymphoma. J Clin Oncol 19:420–424, 2001[Abstract/Free Full Text]

13. Aster JC, Kobayashi Y, Shiota M, et al: Detection of t(14;18) at similar frequencies in hyperplastic lymphoid tissues from American and Japanese patients. Am J Pathol 141:291–299, 1992[Abstract]

14. Liu Y, Hernandez AM, Shibata D, et al: BCL2 translocation frequency rises with age in humans. Proc Natl Acad Sci U S A 91:8910–8914, 1994[Abstract/Free Full Text]

15. Limpens J, Stad R, Vos C, et al: Lymphoma-associated translocation t(14;18) in blood B cells of normal individuals. Blood 85:2528–2536, 1995[Abstract/Free Full Text]

16. Ji W, Qu GZ, Ye P, et al: Frequent detection of bcl-2/JH translocations in human blood and organ samples by a quantitative polymerase chain reaction assay. Cancer Res 55:2876–2882, 1995[Abstract/Free Full Text]

17. Dolken G, Illerhaus G, Hirt C, et al: BCL-2/JH rearrangements in circulating B cells of healthy blood donors and patients with nonmalignant diseases. J Clin Oncol 14:1333–1344, 1996[Abstract/Free Full Text]

18. Yasukawa M, Bando S, Dolken G, et al: Low frequency of BCL-2/J(H) translocation in peripheral blood lymphocytes of healthy Japanese individuals. Blood 98:486–488, 2001[Abstract/Free Full Text]

19. Gribben JG, Neuberg D, Freedman AS, et al: Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma. Blood 81:3449–3457, 1993[Abstract/Free Full Text]

20. Kwok S, Higuchi R: Avoiding false positives with PCR. Nature 339:237–238, 1989[CrossRef][Medline]

21. Ladetto M, Sametti S, Donovan JW, et al: A validated real time quantitative PCR approach shows a correlation between tumor burden and successful ex vivo purging in follicular lymphoma patients. Exp Hematol 29:183–193, 2001[CrossRef][Medline]

22. Donovan JW, Ladetto M, Zou G, et al: Immunoglobulin heavy-chain consensus probes for real time PCR quantification of residual disease in acute lymphoblastic leukemia. Blood 95:2651–2658, 2000[Abstract/Free Full Text]

23. Bakhshi A, Wright JJ, Graninger W, et al: Mechanism of the t(14;18) chromosomal translocation: Structural analysis of both derivative 14 and 18 reciprocal partners. Proc Natl Acad Sci U S A 84:2396–2400, 1987[Abstract/Free Full Text]

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26. Fuscoe JC, Setzer RW, Collard DD, et al: Quantification of t(14;18) in the lymphocytes of healthy adult humans as a possible biomarker for environmental exposures to carcinogens. Carcinogenesis 17:1013–1020, 1996[Abstract/Free Full Text]

27. Paltiel O, Zelenetz A, Sverdlin I, et al: Translocation t(14;18) in healthy individuals: preliminary study of its association with family history and agricultural exposure. Ann Oncol 11:75–80, 2000 (suppl)

Submitted June 12, 2002; accepted December 20, 2002.


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