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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 651-659
NEOPLASIA
Distribution and pattern of BCL-6 mutations throughout
the spectrum of B-cell neoplasia
Daniela Capello,
Umberto Vitolo,
Laura Pasqualucci,
Silvia Quattrone,
Giuseppe Migliaretti,
Lucia Fassone,
Cristiano Ariatti,
Daniela Vivenza,
Annunziata Gloghini,
Cristina Pastore,
Carlo Lanza,
Josep Nomdedeu,
Barbara Botto,
Roberto Freilone,
Daniela Buonaiuto,
Vittorina Zagonel,
Eugenio Gallo,
Giorgio Palestro,
Giuseppe Saglio,
Riccardo Dalla-Favera,
Antonino Carbone, and
Gianluca Gaidano
From the Divisions of Internal Medicine and Epidemiology, Department
of Medical Sciences, Amedeo Avogadro University of Eastern Piedmont,
Novara, Italy; Division of Hematology, A. O. San Giovanni Battista
della Citta' di Torino, Torino, Italy; Division of Oncology,
Department of Pathology, College of Physicians & Surgeons, Columbia
University, New York, NY; Center for Oncologic Prevention, CPO
Piedmont, Torino; Divisions of Pathology and of Medical Oncology B,
Centro di Riferimento Oncologico, Istituto Nazionale Tumori, Aviano,
Italy; Departments of Pediatric Sciences and Adolescence and of
Biomedical Sciences and Human Oncology, University of Torino, Torino,
Italy; Department of Hematology, Hospital de la Santa Creu i Sant Pau,
Barcelona, Spain; Division of Internal Medicine and Hematology,
Department of Clinical and Biological Sciences, University of Torino,
Orbassano, Italy.
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Abstract |
BCL-6 mutations are accumulated during B-cell transit
through the germinal center (GC) and provide a histogenetic marker for B-cell tumors. On the basis of a comprehensive analysis of 308 B-cell
neoplasms, we (1) expand the spectrum of tumors associated with
BCL-6 mutations; (2) corroborate the notion that mutations cluster with GC and post-GC B-cell neoplasms; and (3) identify heterogeneous mutation frequency among B-lineage diffuse
large cell lymphoma (B-DLCL) subsets. Mutations are virtually
absent in acute lymphoblastic leukemia (P < .001) and
mantle cell lymphoma (P < .05), whereas they occur
frequently in GC or post-GC neoplasms, including lymphoplasmacytoid
lymphoma, follicular lymphoma, MALT lymphomas, B-DLCL and Burkitt
lymphoma. Among B-DLCL, mutations occur frequently in systemic nodal
B-DLCL, primary extranodal B-DLCL, CD5+ B-DLCL,
CD30+ B-DLCL, and primary splenic B-DLCL,
suggesting a similar histogenesis of these B-DLCL subsets. Conversely,
mutations are rare in primary mediastinal B-DLCL with
sclerosis (10.0%; P < .01), supporting a distinct
histogenesis for this lymphoma. Longitudinal follow-up of
B-DLCL transformed from follicular lymphoma shows that they BCL-6 mutations may accumulate during histologic progression. Mutations also occur in some B-cell chronic lymphocytic leukemias, small lymphocytic lymphomas, and hairy cell leukemias, consistent with
the hypothesis that a fraction of these lymphoproliferations are
related to GC-like cells. Finally, the molecular pattern of 193 mutational events reinforces the hypothesis that mutations of
BCL-6 and immunoglobulin genes are caused by similar mechanisms.
(Blood. 2000;95:651-659)
© 2000 by The American Society of Hematology.
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Introduction |
The BCL-6 proto-oncogene has been originally
identified because of its involvement in chromosomal translocations
affecting band 3q27 in B-lineage diffuse large cell lymphoma
(B-DLCL).1-3 The BCL-6 protein is a POZ/zinc finger
trascriptional repressor which, in the B-cell lineage, is expressed
selectively by germinal center (GC) B cells, but not by immature B-cell
precursors or differentiated plasma cells.4 Experimental
animal models have demonstrated that expression of BCL-6 is an
absolute requirement for GC formation and function.5,6
The BCL-6 gene may be affected by 2 types of molecular
alterations. The first type of BCL-6 alteration is represented
by chromosomal translocations that lead to substitution of the gene
promoter with heterologous sequences derived from the partner
chromosome.1-3,7 Gross rearrangements of BCL-6 are
virtually restricted to 30% B-DLCL.8-11 The second type of
genetic alteration affecting BCL-6 is represented by point
mutations of the 5' noncoding region of the gene.12
These mutations are somatic in nature, are often multiple in the same
tumor, may be biallelic, and occur independent of cytogenetic
alterations of band 3q27. The sequences affected by these mutations lie
in the proximity of the BCL-6 promoter and overlap with the
major cluster of chromosomal breakpoints. Mutations of BCL-6
are regarded as a marker of B-cell transit through the GC because, in
normal lymphoid tissues, they occur in approximately 30% to 50% of GC
and memory B cells, whereas are absent in pre-GC, virgin B
cells.13-15 On this basis, BCL-6 mutations have
been proposed as a genetic marker for defining the histogenesis of
B-cell lymphoproliferations.
The distribution of BCL-6 mutations in B-cell neoplasia has
been investigated to a certain extent and, on the basis of available data, it has been suggested that these genetic lesions predominate in
tumors displaying a GC or a post-GC phenotype.12,13,15 However, a number of issues remain to be defined. For example, several
histologic types of B-cell lymphomas recognized by the Revised
European-American Lymphoma (REAL) classification have not been tested
for BCL-6 mutations.12,13,15,16 Also, although BCL-6 mutations occur frequently in B-DLCL,12,13,15
knowledge of their exact distribution among different subsets of the
disease is lacking and may provide a clue to clarify the marked
heterogeneity of this lymphoma.16
The aim of this study was a comprehensive analysis of BCL-6
mutations throughout the spectrum of B-cell neoplasia recognized by the
REAL classification and, in particular, throughout distinct subsets of
B-DLCL. Overall, our results (1) expand the spectrum of B-cell
disorders associated with BCL-6 mutations; (2) corroborate the
notion that mutations cluster with neoplasms of GC and post-GC B cells;
and (3) identify heterogeneity of mutation frequency in different
subsets of B-DLCL, suggesting heterogeneity in the histogenesis of this
lymphoma. In addition, extensive analysis of the BCL-6 mutation
pattern in B-cell neoplasia reinforces the hypothesis that
BCL-6 mutations may be caused by a mechanism similar to somatic
hypermutation of immunoglobulin (Ig) genes.
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Materials and methods |
Tumor samples and DNA extraction
This study was based on 308 tumor samples representative of the
spectrum of B-cell neoplasia recognized by the REAL
classification.16 Tumor samples were derived from lymph
nodes, bone marrow, or other involved organs obtained during routine
diagnostic procedures. In all instances, with the exception of B-DLCL
transformed from a follicular phase, the specimens were collected at
diagnosis before specific therapy. Diagnosis was based on morphology
and immunophenotypic analysis of cell surface markers and was
complemented by immunogenotypic analysis of antigen receptor gene
rearrangement.11 In most cases, the fraction of malignant
cells was  70% and in all cases 30%.
On the basis of the REAL classification,16 samples were
classified as precursor B-cell acute lymphoblastic leukemia (precursor B-cell ALL; n = 46), B-cell chronic lymphocytic leukemia (B-CLL; n = 30), small lymphocytic lymphoma (SLL; n = 9),
lymphoplasmacytoid lymphoma (LPL; n = 5), mantle cell lymphoma (MCL;
n = 20), follicular lymphoma (FL; n = 15), mucosa associated
lymphoid tissue (MALT) lymphoma (n = 15), hairy cell leukemia (HCL;
n = 8), B-DLCL (n = 125), and Burkitt lymphoma (BL; n = 35).
Precursor B-cell ALL was representative of different molecular variants
of the disease and included cases associated with hyperdiploidy
(n = 5), rearrangement of BCR/ABL (n = 13), rearrangement
of MLL (n = 9), rearrangement of TEL/AML-1 (n = 6),
or no known genetic lesion (n = 13). MALT lymphomas originated in the
gastrointestinal tract (n = 13) or in the thyroid (n = 2). B-DLCL
samples were further subdivided into distinct subsets of the disease,
which were either representative of specific B-DLCL categories formally
recognized by the REAL classification16 or were arbitrarily
identified on the basis of the phenotypic, clinical, or biologic
peculiarities of the lymphoma. On these grounds, B-DLCL were subdivided
into systemic B-DLCL arising de novo without clinical evidence of
previous lymphoma (systemic de novo B-DLCL; n = 66), systemic B-DLCL
transformed from a previous follicular lymphoma (transformed B-DLCL;
n = 5), primary mediastinal B-DLCL with sclerosis (n = 10),
CD5+ B-DLCL (n = 9), primary splenic B-DLCL (n = 15),
primary extranodal B-DLCL (n = 12), CD30+ anaplastic
B-DLCL (n = 5), and primary central nervous system B-DLCL
(n = 3). Primary extranodal B-DLCL originated in the gastrointestinal tract (n = 8), thyroid (n = 2), testis (n = 1), or lung (n = 1) and included only cases without evidence of accompanying nodal involvement at diagnosis. A fraction of B-DLCL (n = 72),
predominantly represented by systemic de novo B-DLCL, primary
splenic B-DLCL, and primary extranodal B-DLCL, had been classified also
according to the Working Formulation.17 Genomic DNA was
purified by cell lysis followed by digestion with proteinase K,
"salting out" extraction, and precipitation by
ethanol.11
Oligonucleotides
All the oligonucleotides used in this study were synthesized by the
solid phase triester method. The sequence of oligonucleotides used as
primers for the mutational analysis of BCL-6 was as follows: E1.21B, 5'-CTCTTGCCAAATGCTTTG-3' and E1.24,
5'-TAATTCCCCTCCTTCCTC-3' (for fragment 1.10); E1.23,
5'-AGGAAGGAGGGGAATTAG-3' and IP1.6, 5'-AAGCAGTTTGCAAGCGAG-3' (for fragment 1.11); IP1.7,
5'-TTCTCGCTTGCAAACTGC-3' and E1.26,
5'-CACGATACTTCATCTCATC-3' (for fragment
1.12).12 Overall, these oligonucleotides amplify 3 partially overlapping PCR products spanning a 739 base pair (bp) region
located downstream the first noncoding exon of BCL-6. The
oligonucleotides used as primers for the mutational analysis of
p53 exons 5 through 8 have been previously
reported.11 Primers used for analysis of Ig heavy chain
variable (IgVH) genes included sense VH
family-specific and 3' antisense JH primers and have
been reported previously.18,19
Polymerase chain reaction-single strand conformation polymorphism
(PCR-SSCP)
PCR-SSCP was performed as previously reported.11,12
Briefly, 100 ng of genomic DNA, 10 pmol of each primer, 2.5 µmol
dNTPs, 1 µCi  -32P]dCTP (Amersham Life Sciences,
Amersham, UK; specific activity, 3.000 Ci/mmol; 1 Ci = 37 Gbq), 10 mmol/L Tris-HCl (pH 8.8), 50 mmol/L KCl, 1 mmol/L MgCl2,
0.01% gelatin, and 0.5 U AmpliTaq polymerase (Taq gold, Perkin-Elmer,
Norwalk, CT) were mixed in a final volume of 10 µL. Thirty-five
cycles of denaturation (94°C), annealing (annealing temperatures
were optimized for each pair of primers), and extension (72°C) were
performed in a temperature controller (DNA Thermal Cycler Cetus;
Perkin-Elmer, Norwalk, CT). Samples were heated at 95°C for 5 minutes, chilled on ice, and immediately loaded (3 µL) onto a 6%
acrylamide/Tris-borate-EDTA (TBE) gel containing 10% glycerol. Gels
were run at 8 W for 16 to 18 hours at room temperature, air dried, and
analyzed by autoradiography using an intensifying screen (Quanta III;
Dupont-NEN, Boston, MA) for 6 to 48 hours. By using the conditions
described previously, reconstruction experiments have shown that the
sensitivity of the PCR-SSCP method allows the detection of mutations
present in 10% of the cell populations tested (sensitivity
limit = 10%).
Sequencing procedures of BCL-6 gene
For DNA sequencing of BCL-6 5' noncoding regions, a
unique PCR product encompassing fragments E1.10, E1.11, and E1.12
(nucleotides +404 to +1142) was amplified by primers E1.21B and E1.26.
For all samples subjected to BCL-6 DNA sequencing, the
amplified PCR fragment was directly sequenced with appropriate primers
using a commercially available kit (ThermoSequenase, Amersham Life
Sciences). -33P]-labeled terminator dideoxynucleotides
(Amersham Life Science) were included in the sequencing mixture. For
each DNA fragment analyzed, sequencing of both strands was performed on
independent PCR reactions. In selected cases, PCR products showing
mutations by direct sequencing were also sequenced after subcloning
into the TA plasmid vector (Invitrogen, Leek, The Netherlands). DNA sequencing analysis of p53 exons 5-8 was performed as
previously reported.11
Southern blot analysis and DNA probes
For Southern Blot analysis,11 6 to 10 µg of genomic
DNA were digested with the appropriate restriction endonuclease,
electrophoresed in a 0.8% agarose gel, denaturated, neutralized,
transferred to Hybond C+ filters (Amersham Life Science),
and hybridized to probes that had been -32P]-labeled by
the random priming extension method using a commercially available kit
(Amersham Life Sciences). Filters were washed in 0.2 × NaCl-Na
Citrate (SSC)/0.5% sodium dodecyl sulfate (SDS) for 1 hour at 60°C
and then autoradiographed using intensifying screens. Ig gene
rearrangement analysis was performed using a JH probe on
BamHI, EcoRI, and HindIII digests and a J
probe on BamHI digests.11 The organization of the
BCL-6, c-MYC, and BCL-2 loci was investigated
as previously reported.11
Statistical analysis of the distribution and pattern of
BCL-6 mutations
Mutation data were handled in a spreadsheet format using Excel
(Microsoft Corp, Redmond, WA). The SPSS software (version 6.0 for
Windows) was used for statistical elaboration. Statistical analysis was
based on nonparametric methods; significant differences were considered
when P-values < .05. The Fisher Exact test was used to
compare the number of cases harboring BCL-6 mutations among the
different categories of B-cell neoplasia. The relation between
BCL-6 rearrangements and BCL-6 mutations was evaluated with  2 test. Differences among all groups were evaluated
with the Kruskal Wallis test (nonparametric 1-way ANOVA); differences
between pairs of groups were evaluated with the Mann-Whitney test with
Bonferroni adjustment for multiple comparison (significative level
P < .05). Mutation frequencies were normalized on the basis
of the base composition of the sequence analyzed. The normalized
mutation frequencies of each individual nucleotide were calculated by
multiplying the mutation frequency of each single nucleotide by
0.25/f, where f is the frequency of occurrence of the
specific nucleotide in the region sequenced.
To estimate the occurrence of mutations within specific nucleotide
triplets, the number of mutations in each triplet was normalized for
the frequency of the triplet in the sequence analyzed and compared with
the expected mutation frequency by the goodness-of-fit 2
test. The expected mutation frequency for any triplet was obtained by
multiplying the overall mononucleotide mutation frequency by 3. The
expected frequency of mutations falling within RGYW (A/G G C/T A/T)
motifs or the inverse sequence WRCY was obtained by multiplying the
overall mononucleotide mutation frequency by 4, assuming no target preferences.
Sequencing analysis of IgVH genes
IgVH gene rearrangements were amplified with a set of 6 VH gene family-specific primers and a JH primer
mix in separate reactions for each VH primer, as
described.18 The VH primers used in this study
hybridize to sequences in the framework region I of the respective
VH families. PCR was performed for 38 cycles as described previously. The PCR buffer was represented by the Expand HF buffer (Boehringer-Mannheim, Monza, Italy). Annealing temperatures were 61°C for VH1, VH2, VH5, and
VH6 reactions and 65°C for VH3 and VH4 reactions. PCR products were directly sequenced
according to the sequencing procedures described previously. Because of the lack of an amplification product in some cases, the VH
framework region I primers were replaced by VH leader
specific primers, as previously reported.19 Sequences were
compared with the V BASE sequence directory (MRC Centre for Protein
Engineering, Cambridge, UK) using MacVector 6.0.1 software (Oxford
Molecular Group PLC, Oxford, UK) for comparison of the rearranged IgV
genes to the most homologous germline sequences.
Analysis of overall survival in B-DLCL with and without
BCL-6 mutations
Analysis of overall survival (OS) in B-DLCL with and without
BCL-6 mutations was performed in 72 patients with previously untreated de novo B-DLCL (either systemic de novo B-DLCL or primary extranodal B-DLCL), for whom complete clinical data were available. Patients had been diagnosed and treated at 3 Italian institutions from
1990 to 1995 and monitored through March 1999 or until death. The
median follow-up duration from initiation of treatment for censored
patients was 68 months. All patients were treated with an
antracycline-containing regimen. Ten patients with localized stage of
disease without adverse prognostic features were treated with 3 courses
of either ACOPB (adriamycin, cyclophosphamide, vincristine, prednisone,
bleomycin) or CHOP (cyclophosphamide, adriamycin, vincristine,
prednisone), followed by locoregional radiotherapy. Twenty-one patients
with localized stage and adverse prognostic features or advanced stage
disease were treated with CHOP or a third-generation chemotherapy
scheme such as MACOPB (methotrexate, adriamycin, cyclophosphamide,
vincristine, prednisone, bleomycin) or VACOPB (etoposide,
adriamycin, cyclophosphamide, vincristine, prednisone, bleomycin).
Twenty-six elderly patients, over 65 years of age, received PVEBEC
(prednisone, vinblastine, epirubicin, bleomycin, etoposide,
cyclophosphamide) or VMP (etoposide, mitoxantrone, prednimustine)
chemotherapy. Fifteen patients with advanced stage and adverse
prognostic features were treated with MACOPB per 8 weeks plus MAD
(mitoxantrone, high dose ara-C, dexamethasone) plus autologous stem
cell transplantation with myeloablative chemotherapy BEAM (carmustine,
etoposide, ara-C, melphalan) as preparative regimen. Assessment of
response was performed as reported previously.20 All the
patients who began treatment were considered assessable. OS includes
all patients and was measured from the beginning of treatment to the
date of death or last follow-up visit. OS curves were plotted according
to the method of Kaplan-Meier and differences between curves were
evaluated by the log-rank test.21 All calculations were
made by applying the BMDP program (1985) developed at the Health
Science Computing facility, UCLA (NIH) Special Research Resources.
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Results |
Characterization of the tumor panel
All cases of B-cell neoplasia displayed a major monoclonal B-cell
population based on immunophenotypic and/or immunogenotypic analysis.
Distribution of BCL-6 mutations throughout the
clinico-pathologic spectrum of B-cell neoplasia
All 308 samples of B-cell neoplasia were subjected to PCR-SSCP
analysis of 3 partially overlapping fragments encompassing a 739 bp
region within BCL-6 intron 1 (fragments E1.10, E1.11, E1.12).
The selection of these PCR fragments was based on the observation that
these sequences represent mutational hotspots of the gene, harboring > 90% mutations reported in B-cell neoplasia and that these
sequences are consistently mutated in all cases carrying BCL-6
mutations.12 Cases of B-cell neoplasia were scored positive
for mutation when 1 or more PCR-SSCP fragments displayed a variant
pattern that could not be attributed to a population polymorphism.
Results are summarized in Table 1 and Table
2 and representatively shown in Figure
1 and Figure 2.
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| Fig 1.
PCR-SSCP analysis of BCL-6 mutations in
representative B-cell neoplasms.
Representative results obtained for PCR-SSCP fragments E1.10 and E1.12
are shown. Samples of B-cell neoplasms are indicated at the top of each
lane by a numbered code. A positive control (POS), represented by a
tumor sample known to harbor BCL-6 mutations, as well as a
normal (N) sample, represented by a lymphoblastoid cell line, are also
included for each PCR-SSCP fragment shown. Samples were scored positive
when their migration pattern differed from the normal control (N) and
the migration abnormalities could not be ascribed to population
polymorphisms. Among the samples shown in the figure, cases scored as
positive included cases 1063 (primary splenic B-DLCL), 166 (primary
extranodal B-DLCL), 1955 (primary central nervous system B-DLCL) for
PCR product E1.10, and cases 998 (systemic de novo B-DLCL), 374 (systemic de novo B-DLCL), 1053 (primary splenic B-DLCL), 1054 (primary
splenic B-DLCL), and 187 (primary extranodal B-DLCL) for PCR product
E1.12.
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| Fig 2.
Nucleotide sequencing analyses of BCL-6 mutations
in representative B-cell neoplasms.
The sequence of each case shown in the figure is matched to the
sequence of a normal control (N) displaying germline BCL-6
alleles or to the sequence of a tumor sample harboring mutations at a
different site. The position of mutations is indicated by the
nucleotide number of the corresponding BCL-6 germline sequence
(the first nucleotide of the BCL-6 cDNA was arbitrarily chosen
as position +1). Cases included in the figure include systemic de novo
B-DLCL (1176, 1198, 998), primary central nervous system B-DLCL (1955),
and primary splenic B-DLCL (1063, 1569).
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On these bases, mutations of 5' noncoding regions of
BCL-6 were consistently absent among precursor B-cell neoplasms
(n = 46; P < .001), independent of the molecular variant
of the disease (Table 1). Among mature B-cell neoplasia, mutations were
detected in 105/262 (40.0%) cases. In particular, mutations occurred
in LPL (2/5; 40.0%), FL (9/15; 60.0%), B-DLCL (61/125; 48.8%), BL (13/35; 37.1%), MALT lymphomas (5/15; 33.3%) as well as in a subset of B-CLL (9/30; 30.0%), SLL (2/9; 22.2%), and HCL (2/8; 25.0%) (Table 1). Conversely, mutations in MCL (2/20; 10.0%) were
significantly less frequent than in other mature B-cell neoplasms
(P < .05) (Table 1).
In the case of B-DLCL, BCL-6 mutations occurred at different
frequencies in different subsets of the disease (Table 2). In particular, BCL-6 mutations were virtually absent in primary
mediastinal B-DLCL with sclerosis (1/10; 10%; P < .01),
although occurred at a substantially similar frequency in all other
B-DLCL variants, including systemic de novo B-DLCL (33/66; 50.0%),
transformed B-DLCL (3/5; 60.0%), CD5+ B-DLCL (4/9;
44.4%), primary splenic B-DLCL (9/15; 60.0%), primary extranodal
B-DLCL (7/12; 58.3%), CD30+ anaplastic B-DLCL
(2/5; 40.0%), and primary central nervous system B-DLCL (2/3; 66.6%).
In B-DLCL cases that had been also classified according to the Working
Formulation (n = 72; predominantly represented by systemic de novo
B-DLCL, primary splenic B-DLCL, and primary extranodal B-DLCL),
mutations of BCL-6 distributed equally between Working
Formulation category G (19/38; 50.0%), and category H (17/34; 50.0%).
Longitudinal follow-up of BCL-6 mutations
In an attempt to clarify the timing of BCL-6 mutation
acquisition in transformed B-DLCL, we studied 5 B-DLCL transformed from a previous follicular phase before and after histologic progression. Mutations occurred in 1/5 follicular phases and in 3/5 transformed samples. In particular, 2 patients (cases 1931 and 1935 in Figure 3) displayed BCL-6 mutations in the
transformed, but not in the follicular phase, suggesting that mutations
had been accumulated at the time of histologic progression. One patient
displayed identical BCL-6 mutations in both phases of the
disease.
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| Fig 3.
Characterization of BCL-6 mutations in 46 representative B-cell neoplasms.
(A) Schematic representation of the BCL-6 gene. Coding and
non-coding exons are indicated by filled and empty boxes,
respectively. The PCR fragment amplified for mutational analysis is
approximately positioned below the BCL-6 gene map and expanded
in panel B to show the distribution of mutations. (B) Each line of
rectangles represent the BCL-6 sequence of a different B-cell
neoplasm. Each case is indicated by a numbered code, together with the
corresponding diagnosis (on the left; B-CLL, B-cell chronic lymphocytic
leukemia; LPL, lymphoplasmacytoid lymphoma; HCL, hairy cell leukemia;
MCL, mantle cell lymphoma; FL, follicular lymphoma; B-DLCL, B-lineage
diffuse large cell lymphoma). Each rectangle represents a 20 bp
interval of the BCL-6 sequence; nucleotide positions are
indicated in the top line. The first nucleotide of the BCL-6
cDNA was arbitrarily chosen as +1. Filled rectangles indicate the
presence of mutation(s) in the corresponding 20 bp of the BCL-6
sequence. The characteristics of individual mutations are detailed on
the right (eg, G549C, G  C at nucleotide position 549).
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Characterization of BCL-6 mutations
The detailed characterization of the BCL-6 mutations in
B-cell neoplasia was investigated by studying 193 mutational events observed in 46 samples representative of the clinico-pathologic spectrum of these disorders. Representative sequences are shown in
Figure 2, whereas the characteristics of BCL-6 mutations are graphically represented in Figure 3. The overwhelming majority of
mutations included single base-pair substitutions (n = 185), whereas
single point deletions (n = 3), single point insertions (n = 1),
and deletions/insertions of a short DNA stretch (n = 4) were observed
only rarely. The frequency of mutation ranged from 0.0675 to
1.9 × 10-2/bp. Among cases harboring mutations, the
mean frequency of mutation was overall similar in patients belonging to
different categories of B-cell neoplasia.
Of a total of 185 single base-pair substitutions observed, 83 were
transitions and 102 were transversions. The observed
transition/transversion ratio was 0.81 (expected 0.5;
P < .005). The frequency of each type of nucleotide
substitution is shown in Table 3. The
distribution of base substitutions was calculated using the coding
strand and data were normalized for the nucleotide representation in
the BCL-6 sequence analyzed. Analysis of the nature of the
nucleotide substitutions indicated that T was mutated most frequently
(29.4% of all mutations), followed by C (28.7%) and G (27.5%) (Table 3). Conversely, A was mutated less frequently than expected by chance
alone (14.4%; P < .01) (Table 3). Pyrimidine transitions were more frequent than expected (23.5% of all mutations;
P < .02), whereas purine transversions were less frequent
than expected (20.8%; P < .001). Preference for G A
transitions (P < .001), T C transitions
(P < .05), and C G transversions (P < .01) was also present (Table 3). Conversely, nucleotide transversions C A (P < .05), A T (P < .05),AC
(P < .05), and GT (P < .005) occurred less
frequently than expected (Table 3).
Clustering of BCL-6 mutations within specific DNA motifs
To determine whether the pattern of BCL-6 mutations
exhibited sequence-specific preferences, we determined the mutation
frequency in each of the 64 combinations of nucleotide triplets (Table
4). The observed frequency of mutations in
specific triplets was found to differ significantly from that expected
based on chance alone (2 goodness-of-fit test). In
particular, triplets AGC, GCT, TAC, CTA, GTT, CTG, TTA, and TGC were
mutated at a frequency higher than expected (Table 4), whereas triplets
CCC, CTC, GGG, TCC, CCT, GGA, GCC, AAA, ATG, and AAG were mutated at a
frequency lower than expected (Table 4).
A variety of studies have suggested that the quadruplet motif RGYW (A/G
G C/T T/A) and the inverse repeat WRCY are a target for increased
mutational activity in Ig genes.22-24 To test whether such
motifs are also preferentially targeted by the BCL-6 mutation mechanism, we analyzed the presence of mutations occurring in the RGYW
quadruplet and in the inverse repeat WRCY. Together, RGYW/WRCY motifs
represented the 17.7% of the total sequence. Mutations of RGYW/WRCY
accounted for 31.0% of all nucleotide substitutions and occurred at a
frequency higher than expected (P < .05). In particular,
BCL-6 mutations preferentially targeted specific RGYW/WRCY motifs, including the AGCT, AGCA and TGCT nucleotide quadruplets (Table
4).
Comparative analysis of mutations of BCL-6 and
IgVH genes
The occurrence of mutations of BCL-6 and IgVH
genes was compared in a selected panel of samples, including FL, MALT
lymphomas, and HCL. Results are summarized in Table
5. Mutations of IgVH genes were
detected in 15/15 (100%) FL, 15/15 (100%) MALT lymphomas, and 6/8
(75.0%) HCL. Comparison of BCL-6 and IgVH
mutations in these cases revealed that all BCL-6 mutated
samples harbored mutations of IgVH genes (Table 5).
Conversely, only a fraction of IgVH mutated cases harbored
mutations of BCL-6, including 9/15 FL (60.0%), 5/15 (33.3%)
MALT lymphomas, and 2/8 (25.0%) HCL (Table 5). In all samples
investigated, the frequency of IgVH mutations was approximately 10-fold higher than the frequency of BCL-6
mutations (Table 5).
Relationship between BCL-6 mutations, BCL-6
rearrangements and other genetic lesions occurring in B-DLCL
Because B-DLCL is molecularly heterogeneous,10,11,16 we
compared the distribution of BCL-6 mutations with that of
several other genetic lesions of this lymphoma, including
rearrangements of BCL-6, BCL-2, and c-MYC, as
well as mutations of p53. Rearragements of BCL-6 were
detected in 29/115 (25.2%) B-DLCL, including 20/66 (30.3%) systemic
de novo B-DLCL, 2/15 (13.3%) primary splenic B-DLCL, 6/12 (50.0%)
primary extranodal B-DLCL, and 1/4 (25.0%) CD30+
anaplastic B-DLCL (Table 6). Notably,
rearrangements of BCL-6 were absent in primary mediastinal
B-DLCL with sclerosis (n = 8), transformed B-DLCL (n = 5), and
primary central nervous system B-DLCL (n = 3) (Table 6). Comparison
of the distribution of BCL-6 rearrangements and mutations
confirmed that mutations can occur independent of the concomitant
presence of rearrangements in all tested categories of B-DLCL (Table
6). Comparison of the distribution of BCL-6 mutations with that
of alterations of c-MYC, BCL-2, and p53 showed
that BCL-6 mutations occurred in B-DLCL cases both positive and
negative for these genetic alterations (Table 6).
View this table:
[in this window]
[in a new window]
|
Table 6.
Distribution of genetic lesions in different subsets of
B-lineage diffuse large cell lymphoma (B-DLCL) with and without BCL-6
mutations
|
|
Overall survival in de novo B-DLCL with and without
BCL-6 mutations
The OS according to BCL-6 mutations was assessed in 72 de
novo B-DLCL for whom complete clinical data were available. At a median
follow-up of 68 months, OS rate was 58% in cases harboring BCL-6 mutations and 44% in cases devoid of BCL-6
mutations (Figure 4), with no significant
difference between the 2 groups.
View larger version (7K):
[in this window]
[in a new window]
| Fig 4.
Overall survival (OS) according to the presence or
absence of BCL-6 mutation.
OS rates at 68 months 58% (BCL-6 mutated, solid line) versus
44% (BCL-6 nonmutated, dotted line).
|
|
| |
Discussion |
The aim of this study was a comprehensive investigation of the
distribution and pattern of BCL-6 mutations throughout the spectrum of B-cell neoplasia. On the basis of the analysis of 300 B-cell tumors, we report that BCL-6 mutations cluster with B-cell neoplasms thought to originate from GC and post-GC B cells. In
the case of B-DLCL, BCL-6 mutations associate with most, though not all, subsets of the disease, suggesting heterogeneity in the histogenesis of this lymphoma. Mutational analysis performed on almost
200 events indicates that BCL-6 mutations may be due to a
molecular mechanism similar to somatic hypermutation of Ig genes.
Analysis of BCL-6 mutations throughout the spectrum of B-cell
neoplasia confirms that mutations are virtually absent in B-cell tumors
deriving from precursor and virgin B-cells, namely, precursor B-cell
ALL and MCL. This observation is consistent with the fact that
BCL-6 mutations arise at the time of B-cell transit through the
GC.13-15 Conversely, mutations occur at sustained frequency in LPL, FL, MALT lymphomas, B-DLCL, and BL for which a derivation from
GC-related B cells has been either proven or
postulated.18,25-30 Also, mutations are found in a fraction
of HCL, which, in most cases, are thought to derive from post-GC B
cells.31,32 The occurrence of BCL-6 mutations in a
fraction of B-CLL and SLL is noteworthy, because these disorders have
been traditionally viewed as proliferations of virgin B
cells.16 In addition to BCL-6 mutations, however,
other features of GC-related B cells have been recently reported in a
subset of B-CLL,33,34 suggesting that the histogenesis of
the disease may be more heterogeneous than previously thought.
According to the REAL classification,16 the term
B-DLCL is thought to include more than 1 disease entity. The
heterogeneity of B-DLCL reflects heterogeneity in the phenotype,
clinical history, and primary site of the disease.16 With
the exception of primary mediastinal B-DLCL with sclerosis,
BCL-6 mutations distribute uniformly throughout most B-DLCL
variants, including systemic de novo B-DLCL, systemic B-DLCL
transformed from FL, CD5+ B-DLCL, primary splenic B-DLCL,
primary extranodal B-DLCL, and primary central nervous system B-DLCL.
Because BCL-6 mutations are a histogenetic marker of B-cell
transit through the GC,13-15 it is conceivable that all
B-DLCL subsets harboring BCL-6 mutations share a common origin
from GC-related B cells. This notion is substantiated by data of
somatic Ig gene mutation analysis.18,30,35 Remarkably, the
association of CD5+ B-DLCL with both BCL-6 and
somatic Ig gene mutations (this report and ref. 35) indicates that the
histogenesis of this lymphoma differs from that of other malignancies
of the CD5+ B-cell lineage, namely, MCL, B-CLL, and SLL
which, in the majority of cases, display features of virgin B
cells.16 In contrast to other B-DLCL subsets, the absence
of BCL-6 mutations in primary mediastinal B-DLCL with sclerosis
points to a different histogenetic pathway for this lymphoma, possibly
related to a noncirculating B cell normally residing in the thymic
medulla.16,36,37 The molecular and histogenetic
peculiarities of primary mediastinal B-DLCL with sclerosis are also
reinforced by the virtual absence of BCL-6 rearrangements (ref.
38 and this report) and by the observation that these lymphomas fail to
express the BCL-6 protein (A.Carbone et al, unpublished
observation, 1999), a marker of GC-like phenotype that is commonly
expressed by other B-DLCL variants.4
The finding of BCL-6 mutations in transformed B-DLCL, but not
in the FL biopsy, which precedes histologic progression, is noteworthy.
Despite the limited number of cases investigated, these results suggest
that accumulation of BCL-6 mutations might be involved in the
molecular mechanisms of FL transformation and mandate functional
studies aimed at defining the precise biologic relevance of
BCL-6 mutations in FL histologic progression.
Our analysis of 193 mutational events corroborates and expands the
characteristics of the mutational pattern of BCL-6. First, single nucleotide substitutions predominate, whereas
insertions/deletions are rare. Second, nucleotide transitions occur
more frequently than expected. Third, mutations cluster at high
frequency in specific nucleotide triplets and RGYW/WRCY motifs.
Finally, the pyrimidine T is more frequently mutated than the purine A,
suggesting a mutational mechanism exhibiting strand polarity.
Collectively, these features reinforce the hypothesis that
BCL-6 mutations may result from a mechanism similar to that
causing somatic hypermutation of Ig genes.13-15,39-41
Infact, both BCL-6 mutations and somatic Ig gene hypermutation
display a net preference for single nucleotide substitutions, an excess
of nucleotide transitions over transversions and a high degree of
clustering in similar hotspots, including specific RGYW/WRCY motifs and
selected nucleotide triplets (13-15,39-41 and this report). The hypothesis that the BCL-6 locus may be recognized by the
somatic Ig gene hypermutation mechanism is consistent with results
obtained in animal models showing that non-Ig sequences may be targeted by somatic Ig gene hypermutation.42,43
Although previous studies yielded controversial results regarding the
strand polarity of BCL-6 mutations,13,14 the
asimmetry of A and T mutations observed in the present report suggests
that the BCL-6 mutation mechanism preferentially targets 1 DNA
strand. At variance with strand polarity associated with somatic Ig
hypermutation, which preferentially targets A over T,39,44
the mutation mechanism of BCL-6 appears to preferentially
target T over A. This discrepancy in strand polarity between Ig and
BCL-6 mutations may be due to specific features of the gene
sequences, as also suggested by experiments performed on other genes
that have been artificially exposed to the somatic Ig hypermutation
mechanism.42
Comparative analysis of BCL-6 and IgVH mutations in
several B-cell malignancies deriving from GC or post-GC B cells has
revealed that the proportion of cases harboring IgVH
mutations is higher than the proportion of cases harboring
BCL-6 mutations (this study and ref. 13). Also, the frequency
of BCL-6 mutations in a given tumor sample is generally lower
than that of IgVH mutations (this study and ref. 13).
Notably, mutations of BCL-6 never occurred in the absence of
IgVH mutations in B-cell malignancies analyzed to date for
both mutations, including B-DLCL),13 FL, MALT lymphoma, HCL, and B-CLL. Absolute coincidence of BCL-6 and
IgVH mutations should not be expected in view of the lower
frequency of BCL-6 mutations, compared with IgVH
mutations, in normal GC and post-GC B cells.13-15 In fact,
BCL-6 mutations are found in approximately 30% to 50% normal
GC and post-GC B cells, whereas IgVH mutations occur in
virtually all such cells. Also, the mutation frequency of BCL-6
in normal GC and post-GC B cells is approximately 10-fold lower than
that of IgVH genes. The precise reasons for the different incidence and frequency of BCL-6 versus IgVH
mutations are not currently known. It is possible that the mutational
mechanism targets BCL-6 with a lower efficiency compared with
IgVH genes or that BCL-6 mutations occur relatively
later than IgVH mutations in the progression of the cell
transfer through the germinal center.
Currently, detection of BCL-6 mutations provides information on
the B-cell compartment from which a given B-cell tumor originates and
therefore bears implications for the disease histogenesis. Although a
pathogenetic role for BCL-6 mutations has not been formally
established, indirect observations suggest that mutations may carry
functional consequences. In particular, it is remarkable that
BCL-6 mutations cluster in highly conserved genomic regions, suggesting that some mutations may affect regulatory domains of BCL-6 and thus deregulate the physiologic expression of the
gene.12 To define the precise pathogenicity of these
genetic alterations, however, further studies are needed to test the
ability of tumor-derived BCL-6 alleles to deregulate the
expression of BCL-6.
| |
Footnotes |
Submitted May 12, 1999; accepted September 23, 1999.
Supported by ISS, II Programma nazionale di ricerca sull'AIDS
1998-Progetto Patologia clinica e terapia dell'AIDS, Rome, Italy; "Fondazione Piera, Pietro e Giovanni Ferrero," Alba, Italy; by "Fondazione CRT," Torino, Italy; and by Associazione Italiana per
la Ricerca sul Cancro (A.I.R.C.), Milan, Italy. D.C. is being supported
by a fellowship from A.I.R.C., Milan, Italy.
Reprints: Gianluca Gaidano, Division of Internal
Medicine, Department of Medical Sciences, "Amedeo Avogadro"
University of Eastern Piedmont, Via Solaroli 17, 28100 Novara, Italy.
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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Top
Abstract
Introduction