Follicular lymphoma with a novel t(14;18) breakpoint involving the immunoglobulin heavy chain switch mu region indicates an origin from germinal center B cells

Juxtaposition of the BCL2 gene to theIGH locus is a result of the t(14;18)(q32;q21) and is observed in more than 90% of follicular lymphomas (FLs).1The composition of the breakpoints indicate an origin from aberrant RAG1/RAG2-mediated VDJ recombination.2 Most breakpoints are located in the major and minor breakpoint regions (MBRs and mcrs) 3′ of BCL2. In consequence, BCL2 is deregulated by juxtaposition to the centromeric part of theIGH complex containing the IGH enhancers. In contrast, 5′BCL2 breakpoints commonly show juxtaposition to immunoglobulin light chain loci.3,4 Using DNA-fiber fluorescent in situ hybridization (FISH), we recently described the configuration of t(14;18) breakpoints in 40 FLs, including 2 cases with a double 5′ and 3′ breakpoint and insertion in the IGHlocus.5 One other lymphoma contained a single5′BCL2 breakpoint joined to IGHsequences.3 In this paper, we describe this breakpoint in detail. DNA and RNA analysis revealed genomic breakpoints atIGH-S_μ and BCL2 intron 1 and expression of a chimeric I_μ-BCL2 fusion product. Furthermore, we investigated the nontranslocated, functionalIGH allele to find out whether the tumor cells were still able to undergo hypermutations in the V_H region. These data indicate that in sporadic but otherwise classical FL, the t(14;18), and in consequence the BCL2 activation, can occur as a late event during germinal center B-cell development.

Both FL samples (FL5094 and FL10444) were classified according to the Revised European-American classification of Lymphoid Neoplasms.6 Immunophenotyping and fiber FISH have been previously described.3 Polymerase chain reaction (PCR) and reverse-transcriptase PCR (RT-PCR) were performed under standard conditions; the sequences of the primers are described in the figure legends. PCR products were cloned, sequenced, and analyzed as previously described.7,IGH V_Hgene hypermutations were analyzed according to Aarts et al.8 

The original inguinal lymph node biopsy (FL5094) from a woman age 71 years taken in 1996, showed a CD20, CD79a, CD10, BCL2, and BCL6 protein positive follicle center cell lymphoma6 with a partially follicular growth pattern, grade 1 according to the Berard system.9 Interfollicular plasmacytoid cells weakly expressed IgAκ. Clinical staging revealed stage I. A relapse (FL10444) in 1997 showed a completely diffuse follicle center cell lymphoma, grade 1. The patient was treated with chlorambucil and died in 1999 because of sepsis.

Fiber FISH analysis of FL5094 with IGH and BCL2probes identified a 5′BCL2 breakpoint. Both derivatives, the putative der(14) and der(18), were visualized, suggesting a reciprocal translocation.3 The BCL2 and IGHgenes were in the same orientation as seen in regular FL with a 3′ MBR/mcr BCL2 breakpoint. In consequence, the IGHconstant-region genes (C_H) were not juxtaposed to the BCL2 gene but rather to its derivative 5′ flanking region. To investigate the position of the Eμ enhancer, additional fiber FISH with a PCR-generated E_μ probe was performed. This revealed juxtaposition of E_μ to the 5′ side ofBCL2, suggesting a breakpoint at S_μ. The der(14) consisted of a large 5′ part and probably also the first (noncoding) exon of BCL2 and a part of the IGH C_H locus, suggesting a class-switch event with deletion of some C_H genes. To confirm an S_μ/BCL2 fusion at der(18), PCR was performed with primers for the region 5′ of S_μ and for the first intron of BCL2 (Figure 1A). Sequence analysis of this 500–base pair (bp) patient-specific product showed a BCL2 breakpoint in intron 1 and an IGHbreakpoint 5′ of S_μ (Figure 1B). This part of S_μ is commonly used in normal switch recombination events10 and in t(8;14) translocations with a breakpoint at S_μ/MYC in Burkitt lymphoma.11 

Very likely, and similarly to FL with a regular t(14;18), the observed juxtaposition of E_μ to the 5′BCL2 region resulted in deregulation of BCL2. However, the observed configuration might also have led to the formation of a chimeric IGH-BCL2 transcript starting from the transcription initiation site I_μ. Therefore, we performed RT-PCR with primers for the region immediately 3′ of I_μ and exon 3 of BCL2(Figure 2A). Sequence analysis of the 5′ end of this product of approximately 1 kilobase (kb) revealed 78 bp of the I_μ sequence directly followed by the 5′ nontranslated region of the second exon of BCL2 (Figure 2B). Thus, splicing events at regular splice acceptor and donor sites removed most of the S_μ region and all BCL2 intronic sequences. In addition, there was normal splicing of the second intron of BCL2.12 Of note, the first exon is untranslated, and in consequence a breakpoint in the first intron will not lead to structural abnormalities of the BCL2 protein.

Our data show transcription over the translocation breakpoint into exons 2 and 3 of BCL2, suggesting transcription from I_μ or more upstream promoters.12Interestingly, I_μ is implicated in germline transcription necessary for class switching in normal B cells.13 In our case, such germline transcription may have preceded an illegitimate class-switch event at S_μ leading to this particular translocation.

Normal B cells undergo a series of gene recombinations starting with RAG1/RAG2–mediated V(D)J rearrangements of theIGH and immunoglobulin light chain genes in bone marrow precursor B cells, followed by somatic hypermutations in the early phase and immunoglobulin gene class-switch recombinations in a later phase of the follicle center cell reaction.14,15 In all FLs analyzed so far, t(14;18) breakpoint sequences point to a RAG1/RAG2–mediated illegitimate recombination atJ_H or D_H sites. Furthermore, although this has been debated since the observations that RAG1/RAG2 can be expressed in mature B cells,16-20 both the presence of N-regions and the occasional occurrence of a terminal transferase positive blast crisis21 support the origin from precursor B cells. Thus, the t(14;18) in FL is considered to occur in precursor B cells while the tumor cells undergo modifications, including somatic hypermutations of the functional IGHgenes, much later, when they have entered the follicle center cell compartment. To our knowledge, the present case is the first FL with anIGH-BCL2 breakpoint within a switch site.14,15We also did not identify in the literature any t(14;18) breakpoint that might have originated from aberrant somatic hypermutation. Thus, although we cannot formally exclude that this particular class switching–mediated translocation had occurred in a precursor B cell,22 the configuration supports a late origin during the follicle center cell reaction. Translocations involvingIGH switch regions (or sites of hypermutations) are by contrast regularly found in sporadic Burkitt lymphoma,11myeloma,23 and diffuse large cell lymphoma.24 

To study other aspects of B-cell maturation that might shed light on the ontogeny of this lymphoma, we investigated possible somatic mutations on its functional IGH allele. Using V_Hfamily–specific primers, V_H4/C_α transcripts were amplified by RT-PCR from both tumor samples. Sequence analyses of the crude RT-PCR products as well as, respectively, 15 and 8 subclones, showed the highest homology to V4-34. In comparison with this gene segment, the individual clones of both biopsies showed 40 and 39 shared mutations, respectively, indicating clonal expansion from an already heavily mutated B cell. In addition, the 15 clones of the first biopsy contained 53 nonshared mutations (intraclonal variation, 3.5 mutations per clone) corroborating the “ongoing mutations” as seen in normal FL.8 The follow-up biopsy showed a much lower intraclonal variation of fewer than 0.4 mutations per clone, which was most likely due to a selective outgrowth of tumor cells.8 Thus, we propose that in this particular FL, a normal mature B cell acquired the t(14;18) during IGH class switching. Just like normal FL cells, its daughter cells could re-enter the follicle center cell reaction elsewhere in the lymphoid tissues where they underwent additional rounds of somatic mutations in the functional IGHallele.