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Institute of Cancer Genetics and Department of Pathology, Columbia University, New York, New York 10032 [L. P., R. D-F., A. M.], and Servizio di Ematologia, Istituto di Scienze Mediche, Università di Milano, Ospedale Maggiore IRCCS, 20122 Milano, Italy [A. N., L. B.]
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Until recently, the process of somatic hypermutation was believed to be
restricted to the immunoglobulin loci, including heavy (H) and light
chain V region genes. However, it has been shown that another locus,
BCL-6, can be targeted by somatic mutations in the GC
(9, 10, 11)
. BCL-6 is a proto-oncogene encoding for
a POZ/Zinc finger transcriptional repressor expressed at high levels in
GC B-lymphocytes and required for GC development (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
.
Mutations of its 5' regulatory region (
3.5 kb spanning the first
noncoding exon and the first intron) are frequently found in normal GC
and memory B cells, as well as in lymphoid malignancies displaying
GC/post-GC phenotype and harboring mutated IgVH
genes, but not in naive B cells or in other tumor types (9, 10, 11
, 23
, 24)
. BCL-6 mutations are multiple, often
biallelic and heterogeneous, and display features of the immunoglobulin
somatic hypermutation process, which suggests a common mechanism
(9, 10, 11
, 23)
. On the basis of these findings, BCL-6 protein
expression has been considered a marker of GC phenotype in B cells and
BCL-6 gene mutations have been proposed as a molecular
marker of transit through the GC.
As a further approach to determining the cellular origin of B-CLL, this study was aimed at investigating the presence and distribution of BCL-6 mutations in this disease. In addition, we assessed the relationship between the presence of BCL-6 and IgVH mutations, and the expression of the BCL-6 protein.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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40% in two
cases. Genomic DNA was prepared from Ficoll-separated PBMCs by the
"salting-out" procedure as described previously (25)
.
PCR Amplification of the BCL-6 Sequence.
A 781-bp genomic fragment, located within the first intron of the
BCL-6 gene and previously reported to represent the major
cluster for mutations (23)
, was amplified using 100 ng
high molecular weight DNA and the following primers: sense,
5'-CGCTCTTGCCAAATGCTTTG; and antisense, 5'-CTCTCGTTAGGAAGATCACG. The
reaction was carried out in a 50-µl volume containing Expand High
Fidelity buffer (BMB), 1.75 mM
MgCl2, 200 µM each dNTP,
10 pmol of each primer, and 2.5 units Taq DNA polymerase (Life
Technologies, Inc.). Amplification conditions consisted of an initial
denaturation step at 95°C for 5 min, followed by 30 cycles at 95°C
for 30 s, 57°C for 30 s, 72°C for 45 s, and a final step at
72°C for 7 min.
PCR Amplification of IgVH Sequences.
The protocol for amplification of the IgVH genes
has been reported previously (26)
. Briefly, a set of six
VH family-specific primers annealing to
sequences in the framework region I was used in separate reactions,
along with a JH primer mix. PCR was
performed for 34 cycles, and a 5-µl aliquot of the reaction mixture
was analyzed on ethidium bromide-stained 2% agarose gel. In case of
amplification failure, the sense primers were replaced with
oligonucleotides complementary to the Leader sequences of the
VH genes (27)
.
DNA Sequencing Analysis.
PCR products were purified directly or by gel excision using the
QIAquick PCR purification kit (QIAGEN) and were directly sequenced from
both strands using the same primers as in the amplification reaction.
The procedure was accomplished by the dideoxy chain termination method
on an ABI377 sequencer (Perkin-Elmer, Applied Biosystem Division,
Norwalk, CT). Sequencing analysis and alignments were performed using
the GCG software (Genetics Computer Group, Madison, WI) and the GenBank
data library as well as
DNAPLOT4
for comparison of the rearranged IgVH genes with
the most homologous germ line sequences. In addition,
IgVH sequences were compared with each other and
with our own database to rule out contamination with rearrangements
previously amplified in the laboratory. Sequences of somatically
mutated V region genes were submitted to the National Center for
Biotechnology Information data library under accession numbers
(GenBank accession nos. AF304488–AF304517). The first
nucleotide of the BCL-6 cDNA (GenBank accession no. U00115) was defined
as position +1.
Western Blot Analysis.
Cell lysates were obtained from exponentially growing cell lines and
from the PBMCs of 10 B-CLL patients, as described previously
(28)
. Total protein lysates (50 µg) were fractionated by
8% SDS-PAGE, transferred to nitrocellulose membrane (Schleicher &
Schuell), and immunostained according to standard methods. Briefly, the
membrane was blocked in Tris-buffered saline (TBS)-0.5% Tween with 5%
milk for 1 h and was incubated overnight at 4°C with a 1:1000
dilution of the anti-BCL-6 polyclonal antibody N3 (Santa Cruz) in
TBS-0.2% Tween with 3% BSA. Membranes were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated antirabbit
IgG antibody (1:5000) in TBS-0.2% Tween with 5% milk. Reactive bands
were detected using an ECL system (Amersham Pharmacia Biotech;
no. RPN2106). Filters were then stripped according to the protocol
described by the manufacturer and reprobed for ß-actin as a control
for protein integrity and amounts.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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and previously shown to be altered in >95% of the
lymphoid tumors and tonsillar GC lymphocytes displaying
BCL-6 mutations (9
, 23) . Sequence analysis was
performed by direct sequencing of PCR products, thus eliminating the
detection of nucleotide changes generated by Taq polymerase errors.
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and Table 1
. Three previously described population polymorphisms were detected: a T
insertion at position +876 (50% of the cases), a G to C substitution
in position +753 (26% of the cases) and a G to A substitution in
position +858 (2 cases; not shown; 9
, 23
). Case 1231
harbored an insertion T within a poly-T stretch in position 1104–1111,
that could also be ascribed to a genetic polymorphism because it was
previously seen in all IgD-
CD27- naive B cells individually sorted from the
peripheral blood of a healthy
individual.5
These changes were found alone or in combination and were not
considered in the calculation of the mutation frequency.
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0.12 x 10-2/bp; Ref. 10
), which suggests
that there was no apparent selection for BCL-6 mutations during
tumorigenesis. Sequencing analysis of cloned fragments from 2 of the 4
cases carrying more than one nucleotide change indicated that mutations
were biallelically distributed (data not shown), as previously observed
in normal B cells and B-cell-derived tumors (9
, 23)
. None
of the mutations were seen in more than one B-CLL patient. However,
some nucleotide changes had been previously observed either in normal
GC lymphocytes (positions +451; +626; +1002; +1090) or in tumor cells
of various lymphoma subtypes (+490, +499, +953, and +1090), which
suggests that some mutations may be recurrent.
Analysis of IgVH Genes.
To assess the relationship between the occurrence of BCL-6 mutations
and the immunoglobulin somatic hypermutation process, we characterized
the rearranged IgVH genes of the B-CLL cases by
PCR amplification and sequencing of genomic DNA. Clonal rearrangements
were obtained from 33 samples; one case (case 1330, Table 1
) did not
yield a positive amplicon with either framework region I or
leader-specific primers and, therefore, was not included in the
comparative analysis. In seven cases (21.2%), we detected more than
one rearranged IgVH gene (Table 1)
. Sequencing
analysis showed that in five of these seven cases, the two isolated
VH sequences corresponded to the productively and
nonproductively rearranged allele; in the remaining two cases (6%),
both of the VDJ rearrangements amplified—three in sample
1287—were productive and displayed distinct VH
genes, which was consistent with a lack of allelic exclusion
(29)
, or amplification of V genes from bystander
normal B cells, or oligoclonality of the leukemic population.
As shown in Table 1
, 9 (27%) of 33 cases displayed unmutated
VH genes, whereas significant levels of somatic
mutations were found in the remaining 24 cases (73%). Among the
mutated sequences, 19 (63%) differed by 5% or more from the closest
germ line immunoglobulin gene. The average frequency of mutations in
the IgVH genes, including both of the alleles in
cases with more than one rearrangement, was 6.5 x 10-2/bp. The VH3,
VH4, and VH1 families were
the most commonly used in our B-CLL panel, being observed in 56, 27,
and 12%, respectively, of the rearranged sequences.
BCL-6 Mutations Are Associated with IgVH
Mutations.
On the basis of the occurrence of BCL-6 and
IgVH mutations, the B-CLL panel can be divided
into three groups (Table 2)
. The first, represented by 8 (24%) of 33 samples, displays mutations
in both BCL-6 and IgVH sequences; the
second, accounting for 49% of the cases, is characterized by
mutated IgVH genes and germ line BCL-6
sequences; in the third group, which includes 9 cases (27%), both of
the genes were in germ line configuration. None of the cases
investigated displayed BCL-6 mutations in the absence of
IgVH mutations (Table 2)
. In the mutated cases,
the average mutation frequency corresponded to 0.14 and 6.5% for the
BCL-6 and the IgVH genes,
respectively, consistent with previous reports of a 10- to 100-fold
difference between the frequency of nucleotide changes in the two loci
(9, 10, 11)
. The total number of mutational events in
the BCL-6 gene (18)
was too limited to define
the features of mutations. Taken together, these observations indicate
that, analogous to normal GC cells and to GC/post-GC-derived B-cell
malignancies, BCL-6 mutations are restricted to B-CLLs displaying
IgVH mutations.
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were
examined for BCL-6 protein steady-state levels by Western blot analysis
(n = 10) and for BCL-6
RNA levels by Northern blot analysis (n = 20). The results indicated that all of the B-CLL cases express BCL-6
RNA (not shown) and protein (see Fig. 2
for representative results) at very low levels. These amounts
corresponded to one-tenth to one-twentieth of those expressed by normal
or transformed GC B cells (Fig. 2)
but are higher than in memory
B-cells, which lack BCL-6 protein expression.5
Although
some variability in the levels of BCL-6 mRNA and protein was observed
among the cases studied, no difference was detectable between the
various subgroups identified by the presence of BCL-6 and
IgVH mutations. These results suggest that B-CLL cells
express BCL-6 at significantly lower levels than do GC B cells or
GC-derived tumors and that these levels are not apparently influenced
by the presence of mutations.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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BCL-6 Mutations in B-CLL.
The percentage of B-CLLs carrying BCL-6 mutations reported
here (24%) is not significantly different from that reported in a
previous study (30%) as part of a survey involving major subtypes of
B-cell malignancies (24)
. These percentages are also not
significantly different from those observed in other categories of
B-cell malignancies including BL (37%), mucosa-associated lymphoid
tissue lymphoma (40%), and multiple myeloma (33%), whereas higher
frequencies may be present in follicular lymphoma and
DLCL (50–73%; Refs. 9
, 11
, 24
). These values are
also comparable with the percentage (30–40%) of normal GC and memory
B cells carrying BCL-6 mutations (9, 10, 11)
. Thus,
the number of B-CLL cases displaying BCL-6 mutations may
reflect the fraction of normal B cells undergoing BCL-6
hypermutation. In terms of average frequency of mutations per mutated
case, B-CLLs appear to have a lower load of mutations (0.14%) than do
DLCLs (0.5%; Ref. 23
), which suggests a higher
exposure of DLCL cells to the hypermutation process.
Association between BCL-6 Mutations and
IgVH Mutations.
When examining the relationship between IgVH and
BCL-6 mutations, the analysis of B-CLL is particularly
informative because, differently from other B cell tumor types, which
display IgVH mutations in almost 100% of cases,
B-CLL are heterogeneous, with a fraction of cases carrying unmutated
IgVH sequences. Thus, assessing the distribution
of the two types of mutations can support or disprove the hypothesis of
their derivation from the same mechanism. Our results suggest that
BCL-6 mutations are restricted to B-CLL cases displaying
IgVH mutations, whereas in no instance, could
they be detected in the subset of B-CLL that lacks
IgVH mutations. This observation strongly
supports the notion that BCL-6 mutations are introduced by
the same mechanism that generates IgV hypermutation.
The finding that not all cases carrying mutated
IgVH genes have BCL-6 mutations is
entirely consistent with the above hypothesis, based on the observation
that only 1 of 3 normal B cells displaying IgVH
mutations also harbor BCL-6 mutations. Thus, the three
groups shown in Table 2
may in fact represent two types of B-CLL cells:
those that have been exposed to the somatic hypermutation mechanism and
acquired IgVH mutations and, in a fraction of
cases, BCL-6 mutations; and those that have not been exposed
to the process and, therefore, lack both types of mutations.
Two studies addressing the same issues were reported while this article was in preparation/review. In complete agreement with our findings, Capello et al. (30) showed a concordant distribution of BCL-6 and IgVH mutations in B-CLL. Conversely, Sahota et al. (31) reported the presence of subclonally distributed BCL-6 mutations in 4 of 10 B-CLL patients with unmutated IgVH sequences and suggested that a diverse mechanism targets the two genes. Because, in the latter study, sequences were obtained after the cloning of PCR products (as opposed to direct sequencing; Ref. 30 and our analysis) the two sets of results are difficult to compare. However, the detection of a few subclonal BCL-6 mutations in the absence of IgV mutations does not necessarily imply a distinct molecular mechanism, because a low activity of the same somatic hypermutation process may stochastically target one of the two loci, leading to a discordant pattern of mutations.
Role of BCL-6 Mutations in B-CLL.
The role of BCL-6 mutations in normal GC B cells as well as
in their various transformed counterparts is presently unknown. Initial
results indicate that some specific BCL-6 mutations may be
responsible for BCL-6 overexpression in DLCL.5
However, no
differences were detectable between wild type and mutated alleles in
five B-CLL cases tested;5
consistently, our analysis on
BCL-6 protein expression showed no difference in the levels of BCL-6,
irrespective of the BCL-6 mutation status. Thus, it is
possible that BCL-6 mutations are not functionally
significant in B-CLL or, alternatively, that they confer subtle
regulatory disturbances to gene expression.
Implications for the Cellular Origin of B-CLL.
Previous studies have proposed that B-CLL cases displaying
IgVH mutations originate from antigen-selected
memory B cells, whereas those carrying germ line
IgVH sequences may derive from naive B cells or
from memory B cells that were selected by antigens unable to induce
IgVH hypermutation (5)
. The
concordant distribution of BCL-6 and
IgVH mutations shown here confirms the existence
of two distinct subgroups of B-CLL. However, both groups express the
BCL-6 protein, a marker of GC phenotype, at levels significantly lower
than those detectable in normal GC B cells or in their transformed
counterparts (follicular lymphoma, BL, DLCL; Ref. 17
). A
cell population with such BCL-6 expression levels and variable presence
of IgVH and BCL-6 mutations has not
been identified in normal B cells. This may be attributable to either
the incomplete knowledge of the functional program of CD5+ B cells or
to phenotypic differences associated with the transformed status of
B-CLL cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH Grant CA75553. L. P.
is a recipient of a Fellowship from the American Italian Cancer
Foundation. 
2 To whom requests for reprints should be
addressed, at Institute of Cancer Genetics, Columbia University, 1150
St. Nicholas Avenue, Room 303, New York, NY 10032. Phone: (212)
304-7380; Fax: (212) 304-5537; Email: rd10{at}columbia.edu
3 The abbreviations used are: B-CLL,
B-cell chronic lymphocytic leukemia; GC, germinal center; IgV,
immunoglobulin variable (region); IgVH, IgV heavy chain;
PBMC, peripheral blood mononuclear cell; BL, Burkitt lymphoma; DLCL,
diffuse large cell lymphoma.
4 Internet address:
www.genetik.uni-köln.de.
Received 5/22/00. Accepted 8/22/00.
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REFERENCES |
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