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Long-term clonal persistence and evolution of t(14;18)-bearing B cells in healthy individuals

Leukemia volume 20, pages 158–162 (2006)Cite this article

Chromosomal translocations between the immunoglobulin heavy chain locus (IGH) and different oncogenes, are a genetic hallmark of human B-cell malignancies.1 The development of sensitive PCR methods has recently revealed that such translocations can be also recurrently found at low frequencies in peripheral blood mononuclear cells (PBMNC) from many healthy individuals in absence of clinical manifestation.2 Particular interest focused on the t(14;18)(q32;q21) translocation, which is strongly associated with 85–90% of follicular lymphoma (FL) and represents one of the most common abnormalities in non-Hodgkin's lymphoma (NHL). The t(14;18) translocation, arising from the illegitimate joining between the BCL2 proto-oncogene and the IGH locus, and the subsequent block of apoptotic cell death are believed to constitute early events in the pathogenesis of FL. The t(14;18) translocation has been reported in PBMNC of healthy adults with a prevalence going from 8 to 80% mainly depending on the PCR technology used.2 The frequency of circulating t(14;18)-bearing cells usually vary between 1 in 105 and 1 in 107 normal cells and multiple translocations can co-exist in single individuals. However, the relationship between t(14;18) in healthy individuals and progression to disease is still unclear. Particularly, the long-term evolution of t(14;18)-carrying cells remains largely unknown. The detailed understanding of its evolution in healthy individuals is the key both to gain insights into the pathogenic steps involved in tumor progression, and to adequate usage as a potential early biomarker for lymphoma. Current available data is limited to short-term follow-up studies with a persistence of the t(14;18) for few months in only some individuals.3 We recently complemented those data by analyzing a larger population of healthy individuals sampled twice on a 4-month interval reporting both a stable frequency of t(14;18)-positive cells and the maintenance of identical BCL2-IGH clones.4 Therefore, to address the question of the long-term follow-up, we have performed a longitudinal study on the same healthy population after a three-year period, and investigated the evolution and features of the t(14;18) translocation in peripheral blood from 26 individuals (mean: 42.5, age: 27–59 years). To detect and quantify the t(14;18)-bearing cells, a sensitive fluctuation nested PCR analysis of the IGH/BCL2 der(14) breakpoint (30 repetitions per sample) was performed for each time point on DNA extracted from PBMNC, and the frequency was estimated using a Poisson's model, as previously described.4 Most PCR products were sequenced and sequences were compared with the GenBank database using BLAST program to identify BCL2 and IGH breakpoint positions, DH, JH regions, and the intervening region sequence.

Is this overall evolution mainly due to the occurrence of new translocations or to the persistence and/or expansion of pre-existing clones? In our previous report, we observed the presence within most individuals of multiple different BCL2-IGH junctions.4 To analyze their clonal evolution, we performed a systematic sequencing of the positive PCR fragments at P3s. An exhaustive set of sequence data at the P0s/w and the P3s was generated for eight available matched-samples (Table 2). Of a total of 79 sequences analyzed on these eight subjects at P3s, 15 independent BCL2-IGH rearrangements were obtained. Of these, 14 had been already detected at least once either at enrolment (P0w) or at the second assessment (P0s) (indicated by a+in Table 2). Conversely, only one new rearrangement (N in Table 2) was observed. Importantly, the clonal persistence phenomena was independent of the global t(14;18) frequency level of a given individual. Eventually, eight clones which were found at P0w or P0s were not detected anymore at the 3-year follow-up (D in Table 2). It is worth noting that most of these were relatively minor clones at enrolment, and might still be present after 3 years (P3s), but now falling under the detection threshold used in this study (see Supplementary Information). This is consistent with the low occurrence of new emerging clones which might also be present under the detection level (#111). Conversely, as shown in Table 2, the dominance of most major clones remained unchanged since the initial time point. Remarkably, one individual (#120) displayed a 100-fold increase of t(14;18) frequency after 3-years (Figure 1) due to an accumulation of a unique and conserved BCL2-IGH clone (Table 2). It is worth noting that this individual had received a polyvaccination combining hepatitis C, B, typhoid fever 3 months before the P+3s time point. Altogether, our data indicate that most BCL2-IGH clones are ‘long-lived’ and persist over several years in the periphery of healthy individuals. To our knowledge, the persistence of identical BCL2-IGH clones over such a long period constitutes an interesting finding which has been described so far in only one individual over a 5-year period. This persistence of clones in healthy individuals is well beyond the average life span of the normal naïve B-lymphocyte pool, which usually display a rapid turnover from few days until few months. Two main, albeit not exclusive, hypotheses can be considered: first, the ectopic level of BCL2 expression conferred by the translocation may contribute to this long life span; this is supported by the extended B-cell survival observed in vivo and in vitro from BCL2-Eμ transgenic mice.6 However, the lymphoid hyperplasia described in these mice concerns primarily the splenic B-lineage lymphocytes, and there is no evidence that BCL2 overexpression prolongs the life span of peripheral B-cells, which already physiologically express BCL2 at that stage of differentiation. Thus, the role of a translocation-deregulated BCL2 expression on the long-term persistence of peripheral naïve B-cells remains to be clarified. A second possibility would be that at least some t(14;18)-bearing cells in healthy individuals are ‘long-lived’ memory, rather than ‘naïve’ B-cells. Memory B cells are long-lived and proliferating in response to antigen. The recent findings that chronic hepatitis C virus (HCV) infection could promote either the occurrence and/or the maintenance of t(14;18)+ clones in lymphoma-free HCV patients argue toward this possibility.7 Importantly, an increased prevalence of t(14;18) among HCV-infected patients has been repeatedly observed compared to normal subjects. Moreover, the regression of such t(14;18)-bearing clones was strongly associated with the decrease of viral load in HCV infected patients treated with effective antiviral therapy.7 This leads to the concept that HCV-related t(14;18) clones need the persistence of an antigenic stimulus to be maintained. Although this issue clearly needs to be confirmed by additional cases, this is in line with our observation of a 100-fold increase of t(14;18) frequency in one individual who experienced a poly-vaccine therapy before blood sampling. In such a scenario, chronic stimulation by low doses of antigens might well account for more subtle maintenance and/or expansion of t(14;18) clones overtime and throughout ageing.

Table 2 Molecular analysis and evolution of BCL2-IGH junctions in healthy individuals during the 3-year follow-up
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Acknowledgements

The authors thank Maryline Duval (Pôle de Recherche Biologie médicale et Epidémiologie) for DNA sequencing, the staff of the laboratory of clinical biology for performing hematological parameters and nurses from the centralized unit of blood sampling (CLCC François BACLESSE). We are also grateful to Dr Michel Henry-Amar and Dr Didier Pottier for helpful discussions. Supported by grants from the Ligue Nationale contre le Cancer (Comite de la Manche), Association pour la Recherche sur le Cancer (ARC), Conseil Régional de Basse-Normandie, Conseil Général du Calvados, Université de Caen Basse-Normandie, Union des Industries de la Protection des Plantes and the Fondation Weisbren-Benenson. SR was a recipient of a fellowship from the Ligue Nationale contre le Cancer (Comité de la Manche) and the Conseil Régional de Basse-Normandie.

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  1. Groupe Régional d'Etudes sur le Cancer EA1772, Université de CAEN Basse-Normandie, CLCC François Baclesse, Caen, France

    S Roulland, P Lebailly, Y Lecluse & P Gauduchon

  2. Registre général des tumeurs du Calvados, CLCC François Baclesse, Caen, France

    P Lebailly

  3. Laboratoire de Mathématiques Nicolas Oresme CNRS UMR 6139, Caen, France

    N Heutte

  4. Centre d'Immunologie de Marseille-Luminy (CIML), CNRS-INSERM-Université de la Méditerranée, Marseille, France

    S Roulland & B Nadel

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Correspondence to S Roulland.

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Roulland, S., Lebailly, P., Lecluse, Y. et al. Long-term clonal persistence and evolution of t(14;18)-bearing B cells in healthy individuals. Leukemia 20, 158–162 (2006). https://doi.org/10.1038/sj.leu.2404035

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