Follicular lymphoma-like B cells in healthy individuals: a novel intermediate step in early lymphomagenesis

Abstract

Follicular lymphoma is one of the most common adult lymphoma, and remains virtually incurable despite its relatively indolent nature. t(14;18)(q32;q21) translocation, the genetic hallmark and early initiating event of follicular lymphoma (FL) pathogenesis, is also present at low frequency in the peripheral blood of healthy individuals. It has long been assumed that in healthy individuals t(14;18) is carried by circulating quiescent naive B cells, where its oncogenic potential would be restrained. Here, we question this current view and demonstrate that in healthy individuals, t(14;18) is actually carried by an expanding population of atypical B cells issued from germinal centers, displaying genotypic and phenotypic features of FL, and prone to constitute potent premalignant FL niches. These findings strongly impact both on the current understanding of disease progression and on the proper handling of t(14;18) frequency in blood as a potential early biomarker for lymphoma.

Figure 1.

Most t(14;18) translocations in HI are associated with CSR. The translocation region (here with a mbr/J_H6 breakpoint) on the der(14) chromosome is depicted with either an unswitched (A) or a switched (B) configuration. PCR primers are indicated. Reverse 3′C/Sγ primers are consensus for the 4 γ isotypic subclass regions (Cγ_1-4/S γ_1-4). The sizes of the BCL2/Sμ amplicons are expected to range from 7.5 to 8 kb, depending on BCL2 breakpoints. As CSR is imprecise and can occur anywhere in the ∼3 kb Sμ and Sγ recombination sites (29), BCL2/Sγ amplicon sizes are expected to range from 4 to 11 kb. N, N nucleotides present in the BCL2/J_H junction; ovals, switch regions upstream of each constant region (except Cδ). (C) BCL2/Sμ (top) and BCL2/Sγ (bottom) LR-PCR from two of the six HI (#127, #103; left) and from 8 of the 30 FL patients (right) are shown. 23 out of 30 FL samples underwent CSR to Cγ (not depicted). Given the low t(14;18) frequency level in HI, a two-step fluctuation nested LR-PCR assay was used, consisting of at least five parallel replicates per sample (left, lanes 1–5). In the fluctuation range, at most one target molecule is present per PCR replicate, and if so will give rise to a detectable amplicon. The ratio of positive versus negative replicates allows the estimation of the frequency of the target. A standard single round LR-PCR was used for the FL samples (right). The t(14;18)⁺ RL7 cell line harbors a Sγ1-associated BCL2/J_H6 der(14), and was used as positive control for the BCL2/Sγ amplification (C+) and as negative control for the BCL2/Sμ (C−); PCR amplifications of the β-globin gene (β-glob, 7.5 kb) and of a Dβ1-Jβ2.7 germline fragment of the human TCR-β locus (TCR-β, 11.5 kb) were performed on each sample as controls for LR-PCR. In case #127, BCL2/Sγ amplification gave rise to two different sizes of amplicons. Cloning/sequencing revealed that the two amplicons corresponded to two distinct translocations, indicating the presence of oligo/polyclonality in the switched population. In FL, the differences in PCR intensities are related to the individual t(14;18) frequency, the origin (lymph node, bone marrow, PBMCs), and the conditioning (e.g., paraffin) of the particular samples (28). All positive PCR products were confirmed by cloning/sequencing (unpublished data).

Figure 2.

t(14;18)⁺ B cells in the peripheral blood of HI are not naive B cells and constitute an expanding population of FL-like clones. (A) Human PBMCs from 10 consecutive blood samples were collected from anonymous healthy donors, and IgD⁺/CD27⁻ naive, IgD⁺/CD27⁺ memory, and IgD⁻/CD27⁺ switched memory B cell populations were isolated by cell sorting. Results from a representative cell-sorting experiment are shown. (B) Genomic DNA from the three purified subsets was tested for the presence of t(14;18) using the short-range BCL2/J_H PCR assay (mbr20A/21A and J_HcoB/cointB nested primers; see Fig. 1 A). A two-step “fluctuation” nested PCR assay was used, consisting of at least five parallel replicates performed for each sample (lanes 1–5). Results from sample #20 using 100 ng per replicate is depicted. (C) The contribution of naive (CD27⁻, white circles) and memory (CD27⁺, black triangles) B cells to the total t(14;18)⁺ frequency (CD19⁺, gray squares) in the healthy donors is shown. The translocation frequency in each subset was plotted for each donor (Table II). The 10 samples are ordered according to increasing CD19⁺ total B cell translocation frequencies (gray squares). (D) PCR fragments from CD27⁺ memory B cells were cloned and sequenced, and clonality was identified on the basis of the BCL2/J_H junction, which is unique for a given translocation. The frequency of each independent BCL2/J_H clone was calculated, and the contribution of each clone (vertical bars, each color represents a given clone in a given sample) was superimposed on the t(14;18) frequency from the CD27⁺ subset (black triangles). All positive amplicons were sequenced except one in sample #12, three in #15, and two in #24 (percentage on top of histograms corresponds to the number of sequenced amplicons on the total number of positive replicates; Table II). For #99, which required DNA dilution for the calculation of the frequency, 100% corresponds to the sequencing of 10 out of 10 clones obtained from amplifications performed on diluted DNA (1:5; Table II) and undiluted DNA (9:9; not depicted). (E) BCL2-Sγ (top) and BCL2/Sμ LR-PCR (bottom) were performed in the 3 IgD⁺/CD27⁻ naive, IgD⁺/CD27⁺ memory, and IgD⁻/CD27⁺ switched memory B cell subsets for four HI (#15, #20, #21, and #24). Results from sample #24 are shown. β-globin/TCR-β controls were performed as control for long-range amplifications (unpublished data). Most positive amplicons were confirmed by cloning/sequencing and alignment to germline BCL2, Sμ, and Sγ loci (not depicted). Samples presenting multiple size bands revealed oligo/polyclonality. (F) The overall distribution of switched (gray histograms) versus unswitched (white histograms) translocated alleles in the three B cell subsets from samples #15, #20, #21, and #24 is represented. The ratio of positive replicates is indicated at the top of the bars.