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Keywords:

  • non-Hodgkin lymphoma;
  • translocation;
  • quantitative polymerase chain reaction;
  • B cells;
  • immunophenotype

Summary

  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest disclosure
  7. Acknowledgements
  8. References
  9. Supporting Information

The t(14;18)(q32;q21) is the characteristic chromosomal translocation of follicular lymphoma (FL). Highly sensitive polymerase chain reaction (PCR) techniques can also detect t(14;18)-sequences in the blood and lymphoid tissues of healthy individuals (HI). The aim of this study was to determine the immunophenotypic markers of t(14;18)-positive cells in HI and to relate these features to lymphocyte maturation. B cells from 10 subjects with t(14;18)-positive and three subjects with t(14;18)-negative peripheral blood mononuclear cells (PBMC) were fluorescence-activated cell sorted for antigen-naïve (CD27), immunoglobulin M (IgM) memory (IgM+CD27+) and switched memory (IgM CD27+) cells. t(14;18)-recombinations were detected by quantitative PCR. Among PBMC-positive subjects, t(14;18)-frequency was significantly higher in IgM memory (median: 380/106) than in antigen-naïve (median: 16/106) or switched memory (median: 5/106) B cells. All PBMC-negative subjects nevertheless had detectable t(14;18) in sorted B cells; levels were lower than in PBMC-positive subjects, but had the same relative predominance. These results suggest that t(14;18) is generated during early B-cell development in the bone marrow and that affected cells may mature and expand in germinal centres. t(14;18)-frequency was highest in IgM memory cells, a B-cell subset that shares immunophenotypic similarities with FL. The significance of these cells as lymphoma precursors or indicators of lymphoma risk remains to be established.

The t(14;18)(q32;q21) translocation is the characteristic chromosomal aberration of the most common subtype of non-Hodgkin lymphoma, follicular lymphoma (FL), and can be detected cytogenetically in about 90% of FL (Weiss et al, 1987; Horsman et al, 1995). The translocation juxtaposes the proto-oncogene BCL2 on chromosome 18 in cis with the regulatory sequences of the immunoglobulin (Ig) heavy chain locus on chromosome 14q. This abnormality leads to a constitutive overexpression of the anti-apoptotic bcl2 protein, and is believed to be one of the initiating events in the lymphomagenesis of FL (Graninger et al, 1987; Hockenberry et al, 1990). In mice carrying an IGH-BCL2 fusion transgene, the constitutive expression of BCL2 results in a three- to fourfold expansion of resting B cells which manifests as lymphatic hyperplasia (Vaux et al, 1988; McDonnell et al, 1989). After a median latency of 16 months, about 10% of these transgenic mice develop high-grade diffuse large-cell lymphomas, frequently exhibiting a re-arranged MYC (McDonnell et al, 1990). This model suggests that the progression from benign follicular hyperplasia to malignant lymphoma is due to secondary changes and that the t(14;18)-translocation alone is not sufficient to transform a normal B cell into a malignant lymphoma cell.

In humans, the detection of t(14;18)-positive cells is not restricted to patients with FL. Using polymerase chain reaction (PCR) techniques with a sensitivity of ≥10−5, several groups have shown that t(14;18)-positive cells are detectable in 30–60% of healthy individuals (HI) at frequencies of 1–200 translocation positive cells per 106 peripheral blood mononuclear cells (PBMC) (Schüler et al, 2003;Janz et al, 2003). The t(14;18) translocation in B-cell neoplasms is thought to result from an illegitimate recombination of variable, diversity and joining genes (VDJ) of the lg heavy chain locus (Tsujimoto et al, 1985), either during the initial VDJ recombination at the pro-B-cell stage or during receptor editing. The present study aimed to determine whether t(14;18) in HIs results from similar processes and whether the abnormality interferes with subsequent differentiation of the affected cells.

Material and methods

  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest disclosure
  7. Acknowledgements
  8. References
  9. Supporting Information

After informed consent, lymphapheresis was performed on healthy volunteers at the Department of Transfusion Medicine of the NIH Clinical Center. Collected lymphocytes were aliquoted and stored in 50% fetal calf serum/10% dimethyl sulphoxide at −80° C. Between 3·0 and 6·0 × 108 cells were thawed and washed, and dead cells were removed by DNase I incubation. B-cell enrichment was performed by positive selection with CD19 microbeads and LS separation columns (http://www.miltenybiotec.com). CD19-positive cells were incubated with anti-CD20-phycoerythrin (PE), anti-CD27-fluorescein isothiocyanate and anti-IgM-allophycocyanin antibodies (BD PharMingen, http://www.bdbiosciences.com) and then sorted on a FACSVantage (BD Biosciences, http://www.bdbiosciences.com) Cell Sorter. CD20-positive lymphocytes were divided into three separate populations of antigen-naïve (CD27), IgM memory (IgM+CD27+) and switched memory (IgMCD27+) B cells (Fig 1). DNA was extracted with use of the PuregeneTM Kit (http://www.gentra.com). Real-time quantitative PCR for the t(14;18) translocation was performed on a ABI 7500 Sequence Detection System (http://www.applied-biosystems.com), as previously described (Dölken et al, 1998). Briefly, 5–10 aliquots of 1 μg DNA were tested for t(14;18) sequences and for the reference gene KRAS (2 copies/cell). t(14;18) frequency was expressed as t(14;18) copies per 106 cells. Standard curves were generated by using a cloned PCR fragment (186 bp) from the t(14;18)-positive cell line Karpas 422; standard curves using cloned PCR fragments of 102 and 260 bp gave essentially similar results for samples with low copy numbers. The fractional contribution of t(14;18)-positive cells in each subset to the frequency in total B cells was estimated as the product of the t(14;18)-frequency times the percentage of B cells in a given subset, divided by the sum of these products for all three subsets. Size of t(14;18) PCR fragments was determined by electrophoresis on 2% agarose gels. All PCR fragments were isolated by using WAVE 4500 denaturing high-performance liquid chromatography system (Transgenomics, http://www.transgenomic.com) and sequenced with a BigDye® Terminator cycle sequencing kit on a Applied Biosystems 310 Genetic Analyzer (both Applied Biosystems, http://www.appliedbiosystems.com).

image

Figure 1.  Representative example for the settings of the fluorescence-activated cell sorting. The first gate was set on lymphocytes in the forward-sideward-scatter plot (top left), from which B cells were identified by analysis of CD20 expression (bottom left). The population of CD20+ lymphocytes was then used for the sorting of naive (CD27), IgM memory (IgM+ CD27+) and switched memory B cells (IgM CD27+) (right).

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Results

  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest disclosure
  7. Acknowledgements
  8. References
  9. Supporting Information

Twenty-six of 56 (45%) healthy volunteers were t(14;18)-positive on initial PBMC PCR screening. Ten t(14;18)-positive and three t(14;18)-negative subjects were selected for further study, based on the availability of sufficient cell numbers in lymphapheresis samples. The percentage of B cells (CD19+) in lymphapheresis samples from the 10 t(14;18)-positive subjects tended to be higher (median: 29%, range: 13–72%) than in samples from the 3 t(14;18)-negative subjects (median: 19%, range: 17–19%), but the difference did not reach the statistical significance (defined as P < 0·05) (Mann–Whitney's U-test, P = 0·11). For all 13 subjects combined, the median percentages for B-cell subsets of antigen-naive, IgM memory and switched memory cells were 60%, 12% and 17% respectively and did not significantly differ between t(14;18)-positive and t(14;18)-negative subjects.

For the 10 subjects with t(14;18)-positive PBMC, the median t(14;18) frequency in B cells (CD19+) was 46 per 106 cells (range: 2–14 000 per million). t(14;18) frequency was significantly higher in IgM memory B cells (median: 380/106 cells, range: 7–270 000/106 cells) than in antigen-naïve cells (median: 16/106 cells, range: PCR negative – 1700/106 cells, Wilcoxon's signed rank test: P = 0·006). With only one exception, the levels of IgM memory were also higher than in switched memory B cells. t(14;18) frequency was generally lowest in switched memory B cells (median: 5/106 cells, range: PCR negative – 1400/106 cells, P = 0·001) with negative t(14;18) PCR results for this subset in 5/10 subjects (Fig 2A). One subject with 4% IgM memory B cells had an extremely high frequency of t(14;18)-positive cells of 14 per 1000 B cells and 270 per 1000 IgM memory cells.

image

Figure 2.  Comparison of t(14;18) frequency in different B-cell subpopulations. Panel A shows the absolute t(14;18) frequency per million cells of all 13 healthy volunteers. Panel B depicts the relative frequency of the respective subpopulation compared with CD19+ cells (relative frequency = 1) for nine subjects with t(14;18)-positive B cells. The horizontal bar depicts the median of each subgroup.

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t(14;18)-positive cells were detected in sorted B-cell subsets from all three healthy volunteers who were t(14;18)-negative in PBMC. Levels were lower than in subjects with t(14;18)-positive PBMC, but had the same relative predominance with medians of 9 (range: 3 – 27)/106 cells in IgM memory, 5 (range: 0–10)/106 cells in antigen-naive, and 1 (0 – 3)/106 cells in switched memory B cells. B cells from two of the three subjects were also t(14;18)-negative; B cells for the third subject were not tested.

Similar results were obtained in comparison of t(14;18) frequency relative to the translocation frequency of unsorted B cells. The relative t(14;18) frequency was significantly elevated in IgM memory B cells (median: 3·8-fold, range: 1·6- to 19-fold, Wilcoxon's signed rank test, P < 0·001 versus unsorted B cells). Compared with IgM memory B cells, relative t(14;18) frequency was significantly lower in naïve (P = 0·05) or switched memory (P = 0·008) B cells (Fig 2B). Even though IgM memory B cells were the smallest of all three subsets (median 12%), this subset contributed most to t(14;18)-positive B cells: a median of 62% (range: 2–100) of t(14;18)-positive B cells were IgM memory cells, 38% (range: 0–91%) naïve and 3% (range: 0–13%) switched memory B cells.

Two t(14;18)-negative and one t(14;18)-positive subject had insufficient PCR amplicon products for size determination by gel electrophoresis or sequencing. All other PCR fragments were sequenced (Tables I and SI). In all cases, the sequences of t(14;18) fragments from different subsets of a donor were identical, indicating that the t(14;18)-positive cells from different subsets belonged to the same clone (Table I).

Table I.   t(14;18) Fragment distribution in different B-cell subsets.
Subject numbert(14;18) fragment length (bp)
CD19+CD27IgM+CD27+IgMCD27+
  1. NA, not available; ND, no band detected on agarose gel electrophoresis; Neg, PCR negative.

1312312312312
2NA137137137
3107107107107
4ND198198198
5261261261261
6181181181Neg
7214214214Neg
8176ND176Neg
9210Neg210Neg
11Neg199199ND

Discussion

  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest disclosure
  7. Acknowledgements
  8. References
  9. Supporting Information

Lymphomagenesis in FL is thought to be a multi-step process with the t(14;18) translocation being one of the first genetic aberrations (Dölken, 2001). The detection of cells with t(14;18) translocation is not restricted to FL patients. Limpens et al (1991) first demonstrated that t(14;18)-positive cells can be found in lymphoid tissues with follicular hyperplasia. Several groups have confirmed this observation and found that these cells were detectable in about 50% of subjects without lymphoma, depending on the sensitivity of the translocation detection assay (for review see Schüler et al, 2003). Although the prevalence and frequency of t(14;18)-positive cells in HIs is associated with FL risk factors, including age (Liu et al, 1994; Ji et al, 1995), pesticide exposure (Roulland et al, 2004) and ethnicity (Yasukawa et al, 2001), an association between t(14;18)-positive cells and subsequent FL has not been established. Moreover, the observation that t(14;18)-positive cells can be detected in most HIs with a sufficiently sensitive assay (Fuscoe et al, 1996) has called into question their identity as putative lymphoma precursor cells and the usefulness of t(14;18)-positivity as a biomarker of lymphoma risk.

A small percentage of HIs have >100 t(14;18)-positive cells per million PBMC and frequencies are even higher in patients with persistent polyclonal B-cell lymphocytosis (PPBL), a benign disorder with a polyclonal expansion of IgM+ IgD+ memory B cells (Himmelmann et al, 2001). Our subject with the very high frequency of t(14;18)-positive cells had only 4% IgM memory B cells, making the diagnosis of PPBL highly unlikely. Whether the presence of elevated numbers of circulating t(14;18)-positive cells is associated with increased risk for FL is unclear.

Similar to PPBL patients, in whom a greatly enlarged IgM memory B-cell subset is associated with significantly elevated t(14;18) frequencies, we found that in HIs most t(14;18)-positive B cells have an IgM memory B-cell immunophenotype. This is noteworthy as IgM memory cells were the smallest of the three subsets.

Follicular lymphoma harbours heavily mutated Ig genes that show signs of antigen selection and are hence thought to be derived from germinal centre (GC) B cells (Bahler & Levy, 1992; Stamatopoulos et al, 1997). Our observation that most circulating t(14;18)-positive cells in HIs similarly have a phenotype of post-GC memory B cells strengthens their relevance as putative precursors of FL.

The t(14;18) translocation is thought to be generated as a ‘mistake’ during VDJ recombination at the pro-B-cell stage in the marrow or during receptor editing in the process of affinity maturation in the GC. In most subjects of this study, the translocation was already detectable in pre-GC antigen-naïve B cells and therefore must have been generated in the bone marrow. The finding that the t(14;18) frequency was highest in IgM memory and lowest in switched memory B cells paralleled the observation of a predominant expansion of resting IgM+IgD+ cells in E μ-BCL2 transgenic mice (McDonnell et al, 1990) and the predominance of the IgM isotype among FL (Vaandrager et al, 1998).

Our finding of clonotypic sequences in different B-cell subsets indicates that the translocation arises during early B-cell development, but does not abrogate further maturation. As the few progeny cells of a single translocated pre-B cell would not be detectable by PCR, we concluded that a clonal proliferation must occur at or before the naïve B-cell stage. Some of these t(14;18)-positive naïve cells then enter the GC, where they are protected from apoptosis by their constitutive BCL2 overexpression and where they further proliferate, resulting in the high t(14;18) frequency in the post-GC subset of IgM memory B cells. This model would explain the results of Roulland et al (2006), who found that t(14;18)-positive cells underwent class-switch recombination of the non-productive translocated IGH allele.

In summary, the present study indicates that the t(14;18) translocation is generated in early B cells in the bone marrow and that the aberration permits further maturation of the affected cell up to the switched memory stage. t(14;18) may be more prevalent among HIs than generally appreciated, as even individuals with PCR-negative PBMC may harbour t(14;18)-positive IgM memory B cells. The frequency of t(14;18)-positive cells is highest in IgM memory B cells, a peripheral B-cell subset that shares many immunophenotypic markers with FL. However, the significance of these cells as clonal lymphoma precursors or indicators of risk remains to be established.

Conflict of interest disclosure

  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest disclosure
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors declare that they have no competing financial interests.

Acknowledgements

  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest disclosure
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to the Department of Transfusion Medicine of the NIH Clinical Center for providing leukapharesis samples from healthy blood donors. We are thankful to Barbara Taylor, Center for Cancer Research FACS Core, NCI, NIH, for cell sorting the B-cell subsets.

This study was supported by the Intramural Research Program of the NIH, NCI.

References

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  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest disclosure
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Material and methods
  4. Results
  5. Discussion
  6. Conflict of interest disclosure
  7. Acknowledgements
  8. References
  9. Supporting Information

Table SI Nucleotide Sequence And Breakpoints of t(14;18) Fragments Amplified from B Cell Subsets of Healthy Donors.

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BJH6671TableSI.doc25KSupporting info item

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