Abstract

With the use of DNA-fiber fluorescent in situ hybridization, a BCL2 protein positive follicular lymphoma with a novel BCL2 breakpoint involving the immunoglobulin heavy chain (IGH) switch mu (Sμ) region instead of the JH orDH gene segments was identified. Sequence analysis showed that the genomic breakpoint is localized between the Sμ region of the IGH complex and the first intron of BCL2. Reverse-transcriptase polymerase chain reaction showed expression of a unique hybrid IGH-BCL2 transcript involving the transcription initiation site Iμ. Sequence analysis of the VH region of the functional nontranslocatedIGH allele showed multiple shared somatic mutations but also a high intraclonal variation (53 differences in 15 clones), compatible with the lymphoma cells staying in or re-entering the germinal center. This is the first example of a t(14;18) translocation that results from an illegitimate IGH class-switch recombination during the germinal center B-cell stage.

Introduction

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-Sμ and BCL2 intron 1 and expression of a chimeric Iμ-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 VH 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.

Study design

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 VHgene hypermutations were analyzed according to Aarts et al.8 

Results and discussion

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 (CH) 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 Eμ probe was performed. This revealed juxtaposition of Eμ to the 5′ side ofBCL2, suggesting a breakpoint at Sμ. The der(14) consisted of a large 5′ part and probably also the first (noncoding) exon of BCL2 and a part of the IGH CH locus, suggesting a class-switch event with deletion of some CH genes. To confirm an Sμ/BCL2 fusion at der(18), PCR was performed with primers for the region 5′ of Sμ 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 Sμ (Figure 1B). This part of Sμ is commonly used in normal switch recombination events10 and in t(8;14) translocations with a breakpoint at Sμ/MYC in Burkitt lymphoma.11 

Fig. 1.

Genomic organization of the

IGH-BCL2 breakpoint. (A) Schematic demonstration of part of the germline IGH locus (IGH), the germline 5′ part of the BCL2 gene (BCL2), and the fusion product, der(18), observed in FL5094 (Fl5094). The 2 arrows indicate the position of primers used for PCR and genomic sequence analysis. (B) The sequence of the breakpoint junction and alignment with BCL2 intron 1 and germline Sμ sequences. The sequence shown is part of a 500–base pair (bp) product obtained with primers 5′-GGCAATGAGATGGCTTTAGCTG (5′Sμ forward, bp 66 through 87 Genbank X54713) and 5′-CATACACACACTACAAGTAACACGG (BCL2intron 1 reverse, bp 1072 through 1094, Genbank M13994.1). Nucleotides 1 through 38 are homologous to bases 442 through 485 of human Sμ sequence (X54713), and nucleotides 41 through 72 are homologous to bases 1001 through 1039 of BCL2 sequence (M13994.1) The genomic chromosomal breakpoint is located at nucleotides 41 through 42 (ct), which are common to both sequences.

Fig. 1.

Genomic organization of the

IGH-BCL2 breakpoint. (A) Schematic demonstration of part of the germline IGH locus (IGH), the germline 5′ part of the BCL2 gene (BCL2), and the fusion product, der(18), observed in FL5094 (Fl5094). The 2 arrows indicate the position of primers used for PCR and genomic sequence analysis. (B) The sequence of the breakpoint junction and alignment with BCL2 intron 1 and germline Sμ sequences. The sequence shown is part of a 500–base pair (bp) product obtained with primers 5′-GGCAATGAGATGGCTTTAGCTG (5′Sμ forward, bp 66 through 87 Genbank X54713) and 5′-CATACACACACTACAAGTAACACGG (BCL2intron 1 reverse, bp 1072 through 1094, Genbank M13994.1). Nucleotides 1 through 38 are homologous to bases 442 through 485 of human Sμ sequence (X54713), and nucleotides 41 through 72 are homologous to bases 1001 through 1039 of BCL2 sequence (M13994.1) The genomic chromosomal breakpoint is located at nucleotides 41 through 42 (ct), which are common to both sequences.

Very likely, and similarly to FL with a regular t(14;18), the observed juxtaposition of Eμ 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 Iμ. Therefore, we performed RT-PCR with primers for the region immediately 3′ of Iμ 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 Iμ 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 Sμ 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.

Fig. 2.

An IGH-BCL2 hybrid transcript resulting from the t(14;18) translocation results in.

(A) The top diagram shows the organization of der(18) transcript as result of the t(14;18) translocation. The transcription initiates from Iμ and splices to an acceptor site at exon 2 ofBCL2. There is also splicing from exon 2 to exon 3. Donor and acceptor sites between Iμ and BCL2 exon 2 boundary are shown within the boxes. The orientation of primers used for RT-PCR is shown with arrows. (B) The complementary DNA (cDNA) sequence of the breakpoint junction and alignment with germline 14q32 sequence preceeding Sμ and BCL2 cDNA sequences are shown. The sequence is part of a 1-kb product obtained with primers 5′-AGCCCTTGTTAATGGACTTGGAGG (5′Iμ forward, Genbank X97051, bp 91629 through 91652) and 5′-CAGATAGGCACCCAGGGTGAT (BCL2 exon 3, reverse Genbank M13994.1, bp 2146 through 2167). The relative location of these primers is represented by arrows in panel A. Nucleotides 1 through 78 are homologous to bases 91629-91706 of 14q32 sequence (X97051) located 5′ of Sμ in germline DNA. Nucleotides 78 through 166 are homologous to bases 1172 through 1260 of BCL2 sequence (M13944.1). The breakpoint on 14q32 is located 30 bp beyond a known HindIII site (shown) preceding the Sμ sequence. The remaining parts of the splice donor and acceptor sites are underlined.

Fig. 2.

An IGH-BCL2 hybrid transcript resulting from the t(14;18) translocation results in.

(A) The top diagram shows the organization of der(18) transcript as result of the t(14;18) translocation. The transcription initiates from Iμ and splices to an acceptor site at exon 2 ofBCL2. There is also splicing from exon 2 to exon 3. Donor and acceptor sites between Iμ and BCL2 exon 2 boundary are shown within the boxes. The orientation of primers used for RT-PCR is shown with arrows. (B) The complementary DNA (cDNA) sequence of the breakpoint junction and alignment with germline 14q32 sequence preceeding Sμ and BCL2 cDNA sequences are shown. The sequence is part of a 1-kb product obtained with primers 5′-AGCCCTTGTTAATGGACTTGGAGG (5′Iμ forward, Genbank X97051, bp 91629 through 91652) and 5′-CAGATAGGCACCCAGGGTGAT (BCL2 exon 3, reverse Genbank M13994.1, bp 2146 through 2167). The relative location of these primers is represented by arrows in panel A. Nucleotides 1 through 78 are homologous to bases 91629-91706 of 14q32 sequence (X97051) located 5′ of Sμ in germline DNA. Nucleotides 78 through 166 are homologous to bases 1172 through 1260 of BCL2 sequence (M13944.1). The breakpoint on 14q32 is located 30 bp beyond a known HindIII site (shown) preceding the Sμ sequence. The remaining parts of the splice donor and acceptor sites are underlined.

Our data show transcription over the translocation breakpoint into exons 2 and 3 of BCL2, suggesting transcription from Iμ or more upstream promoters.12Interestingly, Iμ 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 Sμ 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 atJH or DH 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 VHfamily–specific primers, VH4/Cα 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.

Supported by grants from the Dutch Cancer Society and Yorkshire Cancer Research.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

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Author notes

Philip M. Kluin, Professor in Oncological Pathology, Department of Pathology and Laboratory Medicine, Academic Hospital Groningen, Rm U1-109, PO Box 30001, 9700 RB Groningen, The Netherlands; e-mail: p.m.kluin@path.azg.nl.