Blood, 15 January 2002, Vol. 99, No. 2, pp. 716-718
BRIEF REPORT
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
James A. L. Fenton,
Jan-Willem Vaandrager,
Wilhelmina M. Aarts,
Richard J. Bende,
Karel Heering,
Martin van
Dijk,
Gareth Morgan,
Carel J. M. van Noesel,
Ed Schuuring, and
Philip M. Kluin
From the Department of Pathology, Leiden University
Medical Center, The Netherlands; Department of Haematology, University
of Leeds, United Kingdom; Department of Pathology, University of
Amsterdam, The Netherlands; Departments of Internal Medicine and
Clinical Pathology, Groene Hart Hospital, Gouda, The Netherlands.
 |
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 or
DH 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 nontranslocated
IGH 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.
(Blood. 2002;99:716-718)
© 2002 by The American Society of Hematology.
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Introduction |
Juxtaposition of the BCL2 gene to the
IGH locus is a result of the t(14;18)(q32;q21) and is
observed in more than 90% of follicular lymphomas (FLs).1
The 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 the
IGH 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 IGH
locus.5 One other lymphoma contained a single
5'BCL2 breakpoint joined to IGH
sequences.3 In this paper, we describe this breakpoint in
detail. DNA and RNA analysis revealed genomic breakpoints at
IGH-Sµ and BCL2 intron 1 and
expression of a chimeric Iµ-BCL2 fusion product.
Furthermore, we investigated the nontranslocated, functional
IGH 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 VH
gene 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 IgA
. 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 BCL2
probes identified a 5'BCL2 breakpoint. Both derivatives, the
putative der(14) and der(18), were visualized, suggesting a reciprocal
translocation.3 The BCL2 and IGH
genes were in the same orientation as seen in regular FL with a 3'
MBR/mcr BCL2 breakpoint. In consequence, the IGH
constant-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 of
BCL2, 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 IGH
breakpoint 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
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| Figure 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 (BCL2
intron 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.
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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.
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| Figure 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 of
BCL2. 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.
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Our data show transcription over the translocation breakpoint into
exons 2 and 3 of BCL2, suggesting transcription from
Iµ or more upstream promoters.12
Interestingly, 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 the
IGH 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 at
JH 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 IGH
genes, much later, when they have entered the follicle center cell
compartment. To our knowledge, the present case is the first FL with an
IGH-BCL2 breakpoint within a switch site.14,15
We 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 involving
IGH switch regions (or sites of hypermutations) are by
contrast regularly found in sporadic Burkitt lymphoma,11
myeloma,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 VH
family-specific primers, VH4/C
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 IGH allele.
| |
Footnotes |
Submitted February 8, 2001; accepted September 21, 2001.
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.
Reprints: 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{at}path.azg.nl.
| |
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Top
Abstract
Introduction
