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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2871-2878
DNA Fiber Fluorescence In Situ Hybridization Analysis of
Immunoglobulin Class Switching in B-Cell Neoplasia: Aberrant
CH Gene Rearrangements in Follicle Center-Cell Lymphoma
By
Jan-Willem Vaandrager,
Ed Schuuring,
Hanneke C. Kluin-Nelemans,
Martin J.S. Dyer,
Anton K. Raap, and
Philip M. Kluin
From the Department of Pathology, the Department of Hematology, and
the Department of Cytometry and Cytochemistry, Leiden University
Medical Centre, Leiden, The Netherlands; and the Academic Department of
Hematology and Cytogenetics, ICR, Royal Marsden Hospital, Sutton, UK.
 |
ABSTRACT |
Immunoglobulin class switching usually involves deletion of part of
the immunoglobulin CH region. By DNA fiber fluorescence in
situ hybridization (FISH) with a barcode of probes covering the
DH, JH, and CH genes, the
configuration of the entire CH region can be visualized on
single DNA molecules. Using this technique, we have studied class
switching in three types of B-cell neoplasia, mantle-cell lymphoma
(MCL), follicular lymphoma (FL) and hairy cell leukemia (HCL),
representing B cells in, respectively, pregerminal center, germinal
center, and postgerminal center stages of development. In MCL and FL,
simultaneous detection of the t(11;14) and t(14;18) breakpoint with
probes for the BCL-1 and BCL-2 loci, respectively, allowed
differentiation between productive and nonproductive alleles. In none
of 10 MCL cases was class switching detected. In 21 HCL, all
nonimmunoglobulin M (IgM) cases had class-switch deletion consistent
with the expressed isotype on at least one allele. In FL, however, a
peculiar pattern of CH rearrangement was observed. In IgM
expressing FL, the translocated alleles had switched in 11 of 13 cases,
and the nontranslocated allele showed complex rearrangements downstream
from the Cµ-C genes in 9 of 13 cases. These downstream
rearrangements may reflect tumor-specific deregulation of the
class-switch machinery. All seven immunoglobulin G (IgG) expressing FL
showed class switching on both alleles. Fiber FISH analysis also showed
several polymorphisms. The most frequent one, present on 38% of all
analyzed alleles, consisted of an extra C gene or pseudogene in the
3 cluster.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
IMMUNOGLOBULIN HEAVY-CHAIN class
switching in B cells is generally mediated by looping out of genomic
DNA between the recombined variable-diversity joining
(VDJ) genes and a target-constant region (CH)
gene,1-3 as has been shown by the detection of circular, excised DNA fragments containing intervening CH sequences.
In mouse B-cell lines, the frequent presence of identical switch deletions on productive and nonproductive alleles indicate a certain specificity in the class-switch process.4,5 Results
obtained in human B-cell tumors and cell lines point to a less-specific mechanism,6-9 in which the nonproductive alleles frequently
remain unswitched or have switched to one of the neighboring
CH genes. However, the available information is limited,
and no comparative studies on class switching in different types of
B-cell neoplasia have been performed.
Methods for the detection of class-switch deletions include Southern
blotting,10 polymerase chain reaction (PCR),11
and inverse PCR.12 However, these techniques have a very
narrow detection window, viewing only the recombination between two
switch sites. If one would like to know the configuration of the entire immunoglobulin H (IgH) CH region on both alleles, fiber
fluorescence in situ hybridization (FISH)13,14 is a more
appropriate technique. We used fiber FISH with a barcode of probes
labeled in two colors covering the CH, the JH,
and part of the DH genes. By hybridization of this barcode,
the entire CH region could be visualized and rearrangements
could be analyzed on both alleles separately.
First a fiber FISH map of the germline CH region was
constructed, using probes specific for Cµ, C, and C genes. We
then analyzed the CH region configuration in three types of
human B-cell malignancies: mantle-cell lymphoma (MCL), follicular
lymphoma (FL), and hairy cell leukemia (HCL). Each of these tumor-types is thought to be derived from B cells in a particular stage of development; MCL from immature follicle mantle cells, FL from follicle-center cells that have encountered antigen and are in the
process of affinity maturation and immunoglobulin class switching, and
HCL from mature B cells, probably preplasma cells. To be able to
differentiate between functional and nonfunctional alleles, MCL and FL
were selected for the presence of a BCL-1 or BCL-2 translocation
breakpoint, respectively. These translocations could be detected by
hybridizing IgH probes together with probes from the BCL-1 or BCL-2
region14 (Vaandrager et al, in preparation). Juxtaposition of BCL-1 or BCL-2 probe signals to IgH probe signals indicated that this IgH allele was translocated and thus could not be
the functional allele.
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MATERIALS AND METHODS |
Tissue samples and cell line.
The foreskin fibroblast cell line VH25 was obtained from the Department
of Radiation Genetics, Leiden University Medical Centre ([LUMC]
Leiden, The Netherlands). Peripheral blood leukocytes (including
granulocytes) of 10 normal donors were obtained from the Blood Bank,
LUMC. Frozen tissue of 10 MCL and 23 FL, selected for the presence of a
BCL-1 or BCL-2 translocation breakpoint, respectively, was obtained
from the Department of Pathology, LUMC. The tumor involved a lymph node
in all but two cases (thyroid, salivary gland). By estimation in
hematoxylin and eosin-stained paraffin sections, the
percentage of tumor cells varied between 10% and 50% for FLs and
higher than 50% for MCLs. Mononuclear cell suspensions, isolated from
spleen (n = 15) or blood (n = 6) of 21 HCL patients by Ficoll
density-gradient centrifugation, were obtained from the Department of
Hematology, LUMC. All samples containing greater than 80% hairy
cells15 were directly used. Samples initially containing
less hairy cells were T-cell depleted before analysis, using anti-CD3
monoclonal antibodies (MoAbs) (anti-leu4; Becton Dickinson, Mountain
View, CA) and sheep-antimouse-coated magnetic beads (Dynabeads; Dynal,
Oslo, Norway). All HCL patients except HCL20 have been karyotyped
before, and no 14q32 abnormalities have been found.16 For
all tumors Ig expression was assessed by two-color immunofluorescence
on cell suspensions or frozen tissue sections, as described
before.15 The IgH subclasses were detected with the
following MoAbs: anti-IgG1 (MH 161-1-MO2; CLB, Amsterdam, The
Netherlands), anti-IgG2 (MH 162-1-MO3; CLB), anti-IgG3 (MH 163-1-MO3;
CLB), anti-IgG4 (MH 164-1-MO4; CLB), and anti-IgA1 (NI 69-11; Nordic,
Tilburg, The Netherlands), followed by fluorescein isothiocyanate
(FITC)-conjugated goat-antimouse immunoglobulins (GAM-FITC; CLB). To
obtain T-cell-enriched fractions of HCL samples, samples were cultured
for 72 hours in RPMI medium with 10% human serum, 180 µg/mL
phytohemagglutinin (PHA) (PHA HA 15; Murex, Dartford, UK), and 120 U/mL
IL-2. After 1 more week of culture with IL-2 but without PHA, cells
were harvested. The percentage of T cells before and after PHA/IL-2
stimulation was determined by immunostaining with OKT3 mouse-anti-CD3
MoAb and goat-antimouse-FITC (DAKO, Glostrup, Denmark) and subsequent
FACS analysis, using a FACSCAN (Becton Dickinson, San Jose,
CA).
DNA fiber preparation.
Preparations of DNA fibers were made according to the halo
technique13,14 with some modifications. Cells were
suspended in phosphate-buffered saline (PBS) with 1% bovine serum
albumin (BSA). Fifty µL of suspension was applied to
3-aminopropyltrietoxysilane-treated object slides. Nuclei were allowed
to adhere for 2 minutes. Slides were dipped for 30 seconds in the
following ice-cold solutions: solution 2 (25 mmol/L Tris.HCl pH 8.0, 0.2 mmol/L MgCl2, 2 mol/L NaCl) and solution 3 (solution 2 + 40 µg/mL propidium iodide). Slides were irradiated with ultraviolet
light (UV) light (254 nm, 7000 µW/cm2) for 7 minutes,
dipped in ice-cold solution 4 (25 mmol/L Tris.H Cl pH 8.0, 0.2 mmol/L
MgCl2, 0.2 mol/L NaCl), solution 5 (25 mmol/L Tris.HCl pH
8.0, 0.2 mmol/L MgCl2), and twice in H2O, and
air-dried. When cells were not sufficiently lysed using this protocol,
the dip in solution 2 was preceded by a dip in 0.25% sodium dodecyl sulfate (SDS) and a dip in solution 1 (25 mmol/L Tris.HCl pH8.0, 10 mmol/L MgCl2, 0.5 mmol/L CaCl2, 0.5% Nonidet
NP-40).
Probes and in situ hybridization.
IgH cosmids U2-2 and 3/64 have been described.17 Cosmid
U2-2 contains the JH and part of the DH region,
cosmid 3/64 contains the JH, Cµ, and C genes. Cosmid
cosIg6,18 containing the C3 gene and surrounding
sequences, was provided by T.H. Rabbitts (MRC Centre, Cambridge, UK).
The C probe, a 16 kb BamHI-BamHI fragment19 containing switch 1 (S1) and C1, and
the C4 probe, a 6.2 kb HindIII-BamHI
fragment20 containing the C4 gene, were provided by P. Leder (NIH, Bethesda, MD). Probes for the detection of 11q13/BCL-1
translocation breakpoints have been described previously.14 A contig of seven cosmid probes for detection of 18q21/BCL-2
breakpoints was made by subcloning YAC YA153A6 (provided by G.A.
Silverman, Washington University School of Medicine, St Louis, MO).
This contig is described elsewhere in detail (Vaandrager et al, in preparation). Probes were labeled by standard nick-translation with
biotin-16-deoxyuridintriphosphate or
digoxygenin-11-deoxyuridintriphosphate (Boehringer Mannheim, Mannheim,
Germany). The hybridization solution consisted of 30%
formamide, 10% dextransulphate, 50 mmol/L sodiumphosphate pH 7.0, 2 × SCC, 3 ng/µL of each probe, and a 50-fold excess of human
Cot-1 DNA. Hybridization and immunofluorescence detection were
performed as described previously.13,14
Fluorescence microscopy.
Images were captured using a COHU 4910 series monochrome CCD camera
(COHU, San Diego, CA) attached to a DM fluorescence microscope (Leica,
Wetzlar, Germany) equipped with a PL Fluotar 100×, NA 1.30 to
0.60 objective and I3 and N2.1 filters (Leica), and Leica QFISH
software (Leica Imaging Systems, Cambridge, UK). Images were processed
with Paintshop Pro (JASC Inc, Eden Prairy, CA) and Harvard Graphics
(Software Publishing Corp, Santa Clara, CA).
| |
RESULTS |
Mapping of the germline immunoglobulin CH region.
Several combinations of biotin- or digoxigenin-labeled probes were
hybridized on DNA fibers from the fibroblast cell line VH25. From these
experiments, the relative positions of the probes were derived. Cosmid
cosIg6 gave two long hybridization signals, each almost twice as long
as the insert size. Although the cosmid was cloned from the C3
region, it hybridized with all CH genes and surrounding
sequences. This cross-hybridization reflects the evolutionary
duplications that have led to the current CH complex containing 4 highly homologous C genes and 1 C pseudogene. The C probe hybridized to both C genes and S sequences, and also to switch µ (Sµ), which is homologous to switch S19.
The C4 probe hybridized to all C genes and C . One of the alleles of VH25 contained the expected number of 5 C probe signals, grouped in two clusters that colocalized with the two cosIg6 signals (haplotype A, Fig 1). Because
we could not identify the different C and C genes on basis of the
hybridization, the order of genes was inferred from a previously
published map of the CH region.21 The other
allele, which is shown in Fig 2, contained
an extra C signal in the 3 CH cluster (haplotype
B). Haplotype A was used to make a physical map (Fig 1). On five
straight and optimally stretched signals, relative distances were
measured. These measurements were averaged, and the relative distances
were converted to kilobasepairs, using the distance between C3 and
C1 as an internal standard of 26 kb.18
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| Fig 1.
The upper part is a fiber FISH map of the
immunoglobulin CH region, based on the hybridization
pattern of the probes indicated as red (biotin-labeled) and green
(digoxigenin-labeled) bars. Although cosmid probes U2-2 and 3/64 each
showed a single, continuous signal, cosmid cosIg6 hybidized to two
stretches of genomic DNA, each twice as long as a normal cosmid and
separated by a gap. These two stretches represent the two evolutionary
duplication units of the human CH region, illustrating the
high degree of homology between and within these units. The 16 kb C
probe hybridized to both C genes and S regions and also gave a
small signal at the site of Sµ, because Sµ is highly homologous to
S. The C4 probe hybridized to all C genes as well as to the
C gene. Below the scale bar a fiber representing the germline
configuration of the most common haplotype (A) is shown. The second
fiber is an allele that has undergone VDJ recombination, resulting in
loss of almost the whole cosmid U2-2 probe signal. The four fibers
below are alleles, derived from HCL cases that have also recombined
their V, D, and J genes, but in addition have undergone class-switch
deletion to, from top to bottom, the C, C3, C1, and C1
genes. In each case this deletion has led to loss of all probe signals
between VDJ and the target CH gene. In the case with C
switching this deletion is recognized by shortening of cosmid 3/64
signal combined with absence of the Sµ signal. The probe signals
representing the remains of cosmids U2-2 and 3/64 containing the
JH-5 Sµ region are often too small to be observed.
In the C3-and C1-switched fibers they are visible, however, as a
red and a green dot at the left end of the fiber (arrow). To facilitate
interpretation, only fibers of haplotype A were selected for this
figure, and the lengths of the class-switched fibers were normalized to
the germline fiber. This was only done for Fig 1, allowing the
inclusion of a scale bar. In the other figures no scale bars were given
because the fibers have different degrees of magnification and
stretching.
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| Fig 2.
Examples of CH duplication and deletion
polymorphisms. The upper fiber shows haplotype B, which has an extra
C signal in the 3 cluster as compared with haplotype A (Fig
1), and was present at a frequency of 37.5% in our series. The second
fiber has two extra C signals in the 3 cluster. The third
fiber shows an example of the second most frequent type of
polymorphism, appearing as a multigene duplication. The lower two
fibers are the two germline alleles of one HCL patient, allele 1 having
a multigene deletion, allele 2 having a more complex pattern, probably
the result of multiple deletion/duplication events. On the alleles in
this figure most C and C genes could not be identified and are
indicated only with and .
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The probe combination described above was hybridized on DNA fibers
prepared from peripheral blood leukocytes of 10 normal donors, spleen
or blood samples of 21 HCL patients, and frozen tumor samples of 10 MCL
and 23 FL. For the MCL the IgH probe set was supplemented with probes
for the BCL-1 locus, for FL with probes for the BCL-2 locus. Using
these probe sets we identified three different groups of IgH
rearrangements: complete or almost complete deletion of the U2-2 cosmid
signal, deletion of downstream probe signals consistent with
class-switch deletion, and juxtaposition of IgH to BCL-1 or BCL-2
signals. Deletion of the cosmid U2-2 signal that covers the DJ region
was exclusively observed in samples containing either polyclonal or
monoclonal B cells, and hence was ascribed to VDJ or nonfunctional DJ
rearrangement. Fibers without cosmid U2-2 deletion should be derived
from non-B cells or occasional B cells with monoallelic rearrangement
(with a possible exception for nonfunctional DJ rearrangement involving
DQ52, which might be missed by this analysis). For the analysis of
polymorphisms we exclusively tested DNA fibers without deletion of the
U2-2 signal (Fig 2). Among 128 alleles analyzed, 69 (54%) had
haplotype A, 48 (38%) had haplotype B, and 11 alleles (9%) had other
haplotypes. Most of the latter could be matched with previously
described duplications within the 3 part of the CH
region.22,23 In cases HCL3, HCL5, and HCL12, a
T-cell-enriched fraction was prepared by PHA-stimulation. This
fraction showed the same polymorphism as found in crude spleen samples,
further proving that these patterns represented real polymorphisms, and
not B-cell-related rearrangements.
Absence of class-switch deletions in MCL.
In agreement with literature data,24 all MCL expressed
IgM(D). In each MCL sample at least 10 fibers with juxtaposition of
11q13/BCL-1 probes were analyzed, and no deletions or other rearrangements in the CH region were found. Similarly, 10 to 20 fibers with deletion of the U2-2 signal but without juxtaposition of 11q13/BCL-1 probes were analyzed, and these fibers also lacked CH rearrangements. Because these fibers were found at
approximately the same frequency as fibers with BCL-1 translocation,
the majority should represent the functional IgH allele of the tumor
cells. From these data we conclude that MCL lack class-switch
recombination on both alleles.
Class-switch deletions in HCL.
Fiber FISH analysis of 21 cases of HCL showed several types of
class-switch deletion (Fig 1). On basis of (V)DJ recombination, class-switch deletion, and polymorphisms, the two HCL-derived IgH
alleles could be distinguished in all 21 cases. The fiber FISH results
are shown in Table 1, and are aligned with
IgH isotype expression as determined by immunofluorescence and the
presence of class-switch deletions as previously determined by Southern blotting with JH and Cµ probes.25 In contrast
to previous immunophenotyping15, we found in none of these
21 HCL cases expression of multiple isotypes. Among the IgM cases, only
one of eight (HCL5) showed monoallelic class-switch deletion. In all
non-IgM cases, at least one allele had class-switch deletion concordant
with the expressed IgH isotype. These included two cases with
expression of IgD only (HCL9 and HCL10).25
Class-switch deletions and other CH gene rearrangements
in follicular lymphoma.
Fiber preparations of 23 cases of FL were hybridized with a combination
of IgH probes and BCL-2 probes. The translocated, nonproductive allele
could be identified in all cases on basis of juxtaposition of BCL-2 to
IgH probe signals (Fig 3). In
3 of 23 FL (FL2, FL8, and FL21) the translocated IgH allele had
retained the Cµ-C region. In 2 of these cases the entire
CH region was in the germline configuration, whereas in the
third case (FL21) rearrangements downstream from C were present. In
the remaining 20 cases the translocated IgH allele had undergone class
switching to one of the C or C genes (Table
2 and Fig 3). In one of these cases (FL16),
in addition to a switch deletion between Sµ and S1, a downstream
deletion in the 3 CH cluster had occurred.
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| Fig 3.
Examples of class-switch rearrangements on the
translocated allele of FLs, detected with a combination of IgH and
BCL-2 probes. The red signal on the left side of each fiber is a BCL-2
cosmid probe located about 20 kb 5 from the major breakpoint
cluster (MBR) and 50 kb 5 from the minor cluster region (MCR) of
BCL-2. The location of the translocation breakpoint (derived from
hybridizations with multiple BCL-2 probes, data not shown) is indicated
with a white line. Originally, a more proximal (3) BCL-2 cosmid
was used that covers the breakpoint in case of a major breakpoint
translocation. The more 5 cosmid in this figure was used to
create a gap between BCL-2 and IgH signals and hence facilitate
interpretation of the IgH configuration. The upper 2 fibers show
translocations with an MBR-breakpoint and with class switching to C4
and C2. The lower 3 fibers have translocation breakpoints in the
MCR. Two of these have class switching to C1 and C1,
respectively. The bottom one has retained the Cµ-C region but has
downstream rearrangements, apparently consisting of deletion of a
single C gene from both the 5 and the 3 CH
cluster.
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In all seven IgG expressing FL as well as in two nonexpressing cases,
one type of fiber with both (V)DJ recombination and class-switch
deletion was observed at a frequency comparable to the translocation
(5% to 30%). We concluded that these fibers represented the
functional tumor alleles. Switching had taken place to C1 in four FL
and to C2 or C4 in five FL (in some cases differentiation between
C2 and C4 was not possible due to polymorphisms). One
C1-switched allele (in FL20) had only two C genes in the 3
cluster. Because the germline alleles had in their 3 duplication
units three and four C genes, respectively, a downstream deletion
must have occurred on this functional tumor allele. In 4 of the 13 IgM(D) expressing FL (FL1-4), no class-switch rearrangements were
detected among the nontranslocated IgH fibers with deletion of cosmid
U2-2. In contrast, in each of the nine other IgM(D) FL (FL5-13) and in
one Ig-negative FL (FL14), an IgH allele was identified retaining its
Cµ-C region but with a rearrangement downstream of C (Fig
4). Because these downstream rearrangements
were present exclusively in combination with (V)DJ recombination, we
concluded that these IgH alleles represented somatic rearrangements in
tumor cells. In most cases, the rearrangement consisted of a deletion
of part of the CH locus. In cases FL5, FL8, and FL14, the
configuration was more complex and was probably the result of multiple
events including deletions and inversions. In some cases, for example
cases FL6, FL10, FL12, and FL13, the CH configuration could
not be reconstructed if the assumption was held that breakpoints occur
solely in switch regions. For example, in FL10 the entire C probe
signal was absent, though there is no known switch region located
3 from C2. In the Ig-negative case FL14 the rearrangements
also involved the JH-Cµ region, thereby probably
disrupting the VDJ-Cµ transcription unit. In one case of FL (FL10),
two different recurrent nontranslocated alleles were observed: one
allele with retention of Cµ-C and a downstream deletion (see Fig
4), and one allele containing one C and one C gene (not shown)
suggesting a C4-switched allele. Because the tumor exclusively
expressed IgM, the latter allele may represent a remnant of a
switch-circle that has been reintegrated somewhere in the
genome,26 or the reciprocal product of a translocation in a
S site. Alternatively, it may represent a second tumor subclone that
has lost IgH expression.
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| Fig 4.
Class-switch rearrangements with retention of the
Cµ-C region on the nontranslocated, functional alleles of nine IgM
expressing FL and one IgH-negative FL (FL14). The positions of Sµ and
the C and C plasmid probe signals are indicated. The
differentiation between C and C probe signals has been made by
sequentially hybridizing probe mixes with and without the C probe.
The position of the U2-2 cosmid in FL14 was determined by sequentially
hybridizing the IgH probe mix with and without U2-2. Its orientation
could be determined by the presence of a Sµ signal derived from the
C probe (indicated with an arrow).
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| |
DISCUSSION |
We have analyzed somatic rearrangements in B-cell malignancies using a
technique that allows structural characterization of a large genomic
region on single DNA molecules. We studied three types of B-cell
malignancies that are supposed to represent three different stages in
B-cell differentiation: MCL (pregerminal center B cells), FL (germinal
center B cells), and HCL (postgerminal center B cells). Because
germinal centers are major sites of class switching, switched IgH genes
are expected to be present in FL and HCL but not in MCL. MCL is thought
to be derived from follicle mantle B cells, always expresses IgM(D),
and does not show hypermutation of the Ig genes.27 Indeed,
in none of 10 MCL cases class-switch rearrangements were detected by
fiber FISH on functional or nonfunctional alleles.
The immunophenotype of HCL, with relatively frequent expression of IgG
or IgA, indicates an origin from postgerminal center B
cells.15,28,29 Most (8 of 13) non-IgM expressing HCL had biallelic switch deletions. Although class switching to C1 dominated when both alleles were taken into account, the expression pattern of
HCL showed a strong preference for IgG3.15 Assuming that both alleles are subject to the same switch-regulating mechanism, the
large proportion of IgG3 expressing HCL may well be the result of a
selection process rather than induction of specific C3 switching. In
all HCL cases switching only involved CH genes in the
5 cluster (C3, C1, C1) or C. Within this region,
distribution of switching on both alleles seems to be a stochastic
process, with a high probability of switching to genes located in the
middle of this region (C1 and C3) and a lower probability of
switching to genes at the border (C and C1). These results
support a model in which probability of switching is determined by
long-range activation of part of the CH
region.30 The distribution of switch target genes in HCL
may be due to the influence of switch-inducing cytokines. For example
IL-10, which has been shown to induce preferential switching to C1
and C3,31 is highly expressed in HCL
cells.32
Follicular lymphoma is derived from follicle center cells and is
characterized by ongoing hypermutation.33-35 Most cases
express IgM and only a minority expresses IgG, IgA, or no IgH. However, at the ±2 kb resolution of fiber FISH, the translocated allele had
switched normally in almost all IgG and IgM expressing cases, as has
been observed before with Southern blotting, pulsed-field gel
electrophoresis and reverse transcription-PCR
techniques.36,37 On the functional alleles we found switch
deletions correlating with IgH protein expression in the IgG expressing
cases and retention of the Cµ-C gene region in the IgM cases.
However, a considerable proportion of the IgM(D) FL as well as one
Ig-negative case had undergone CH rearrangement downstream
from this Cµ-C gene region on their functional allele.
Deletion of multiple CH genes with retention of the Cµ
gene has been reported only once before,8 also in a case of
follicular lymphoma. An obvious hypothesis to explain rearrangement of
CH genes without involvement of Sµ is that Sµ is
inactivated on this particular allele. This inactivation may be due to
deletion of Sµ itself or to inactivation of parts of the
Eµ-enhancer that are required for expression of Sµ germline
transcripts. In mice with heterozygous deletion of JH-Eµ
sequences, stimulation with LPS and IL-4 can induce rearrangement of
S1 without involvement of Sµ.38 To investigate in our
FL with downstream rearrangements the presence of small genomic
abnormalities that might be undetectable by fiber FISH, we recently
performed Southern blotting on eight cases. On hybridization with a
probe for Cµ, all cases showed a rearranged band in Bgl II,
HindIII, and Xba I digests. In all cases but one
(FL14), the rearranged fragment was 1 to 2kb smaller than the germline
fragment in all 3 digests, strongly suggesting deletion of part of the
Sµ sequences (data not shown).
A remaining question is why this Sµ inactivation occurs so frequently
on the functional alleles of FL, whereas the translocated allele mostly
shows normal class switching. This discrepancy between the two
alleles would be compatible with selective pressure in favor of
IgM or against IgG expression on a B-cell population that is at
the same time permanently driven to switch. Such a process would
lead to a high chance of developing a clone with either a functional
IgM allele with downstream rearrangements, or a nonproductive allele
like in FL14.
Using the fiber FISH technique we have obtained an unprecedentedly
complete view of class switching in B-cell malignancies. We have found
no evidence for class-switch mechanisms other than deletion of
intervening sequences between Cµ and a downstream CH
gene. In IgM expressing FL, we have found frequent CH
rearrangements with retention of Cµ and C. The lack of Sµ
involvement in these cases is probably caused by deletion of part of
the Sµ sequence. Fiber FISH analysis of the CH region in
other types of B-cell malignancies and in subsets of normal B cells may
tell whether this phenomenon is unique to follicular lymphoma.
| |
FOOTNOTES |
Submitted February 26, 1998;
accepted June 8, 1998.
Supported by grant NKB95-1047 from the Dutch Cancer Society.
Address correspondence to Jan-Willem Vaandrager, Department of
Pathology, Leiden University Medical Centre, Bld 1, L1-Q, PO Box 9600, 2300 RC Leiden, The Netherlands;
e-mail:Jvaandrage{at}Path_1.MedFac.LeidenUniv.NL.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract