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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2220-2228
Expression Status of BCL-6 and Syndecan-1 Identifies Distinct
Histogenetic Subtypes of Hodgkin's Disease
By
Antonino Carbone,
Annunziata Gloghini,
Gianluca Gaidano,
Silvia Franceschi,
Daniela Capello,
Hans G. Drexler,
Brunangelo Falini, and
Riccardo Dalla-Favera
From the Division of Pathology, Centro di Riferimento Oncologico,
IRCCS, Istituto Nazionale Tumori, Aviano, Italy; the Division of
Internal Medicine, the Department of Medical Sciences, University of
Torino at Novara, Novara, Italy; the Division of Epidemiology, Centro
di Riferimento Oncologico, IRCCS, Istituto Nazionale Tumori, Aviano,
Italy; the German Collection of Microorganisms & Cell Cultures, Human
and Animal Cell Culture Collection, Braunschweig, Germany; the
Institute of Hematology, University of Perugia, Italy; and the Division
of Oncology, the Department of Pathology, College of Physicians and
Surgeons, Columbia University, New York, NY.
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ABSTRACT |
The tumor cells in most cases of Hodgkin's disease (HD) have been
recently recognized to originate from the B-cell lineage, but their
precise differentiation stage is not fully clarified. Recently, we have
reported that the histogenesis of B-cell lymphomas may be assessed by
monitoring the expression pattern of BCL-6, a transcription factor
expressed in germinal center (GC) B cells, and CD138/syndecan-1
(syn-1), a proteoglycan associated with post-GC, terminal B-cell
differentiation. In this study, we have applied these two markers to
the study of HD histogenesis. We have found that in nodular lymphocyte
predominance HD (NLPHD) tumor cells consistently display the
BCL-6+/syn-1 phenotype, indicating their
derivation from GC B cells. Conversely, classic HD (CHD) is
heterogeneous because the tumor cells of a fraction of CHD display the
BCL-6/syn-1+ phenotype of post-GC B-cells,
whereas another fraction of CHD is constituted by a mixture of tumor
cells reflecting the GC (BCL-6+/syn-1) or
post-GC (BCL-6/syn-1+) phenotypes.
BCL-6/syn-1+ tumor cells of CHD are mostly
found surrounded by T cells expressing CD40L, consistent with the
observation that CD40 signaling downregulates BCL-6 expression. These
data indicate that tumor cells of NLPHD uniformly display a GC B-cell
phenotype, whereas the phenotype of tumor cells of CHD appears to be
modulated by the surrounding cellular background, particularly
CD40L+ reactive T cells.
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INTRODUCTION |
HODGKIN'S DISEASE (HD) is characterized
histologically by scanty neoplastic cells interspersed in the context
of a reactive cellular background.1-3 Based on the
characteristics of neoplastic cells and of the reactive background, HD
is distinguished into two major categories termed nodular lymphocyte
predominance HD (NLPHD) and classic HD (CHD). Whereas NLPHD generally
follows an indolent course, CHD is fatal without
therapy.3,4
The detailed characterization of the HD neoplastic population has been
a matter of debate for many years.3 Recently, single-cell analysis of Ig genes has shown that tumor cells of NLPHD and of most
CHD of B-cell lineage derive from germinal center (GC) B cells that
have been stimulated and selected by antigen.5-9 Despite their common origin, however, neoplastic cells of CHD, known as Reed-Sternberg (RS) cells, and neoplastic cells of NLPHD, known as
lymphocytic and histiocytic (L&H) cells, differ markedly in terms of
morphology, phenotype, and infection pattern by Epstein-Barr virus
(EBV).1-3 Whereas the features of L&H cells are relatively homogeneous, RS cells of CHD display a high degree of polymorphism which remains unexplained.1,3
Progression of normal GC B cells to later stages of B-cell
differentiation may be monitored by following the expression of biologic markers associated specifically with distinct subsets of
mature B cells. We have recently shown that expression of BCL-6 and
CD138/syndecan-1 (syn-1) can reliably discriminate between GC and
post-GC B cells.10 The BCL-6 protein is a zinc finger transcriptional repressor encoded by the BCL-6 proto-oncogene and
implicated in the pathogenesis of B-cell diffuse large cell lymphoma
(B-DLCL).11 The BCL-6 protein is expressed by GC B cells
and is required for GC formation and function.12-14
Conversely, expression of BCL-6 is negative in all other stages of
B-cell differentiation, including virgin and memory B cells as well as plasma cells.12 Challenging of GC B cells either with
antigen or through the CD40/CD40L pathway causes downregulation of
BCL-6.15-17 Similarly, induction of the EBV-encoded latent
membrane protein-1 (LMP-1) downregulates expression of BCL-6 in B cells
reflecting the GC phenotype.17 Syn-1 is a proteoglycan
belonging to the syndecan family, which mediates cell-to-extracellular
matrix interactions.18,19 Among mature B cells, syn-1 is
expressed in post-GC B cells, including immunoblasts and plasma cells,
whereas it is absent in GC B cells.19-21
Here we report data suggesting a histogenetic model for HD development.
Tumor cells of NLPHD consistently express the
BCL-6+/syn-1 phenotype and thus closely
reflect GC B cells. Conversely, the majority of CHD display only or
predominantly BCL-6/syn-1+ RS cells. The
HD variants identified by BCL-6 and syn-1 differ in terms of amount and
distribution of CD40L+ reactive T lymphocytes, suggesting
that interactions between CD40 (on neoplastic cells) and CD40L (on
reactive T cells) is a crucial event in modulating the differentiation
stage and phenotype of HD.
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MATERIALS AND METHODS |
Samples
The study was based on 53 HD cases for which frozen tissue samples were
available. The HD panel included 10 NLPHD (all B-cell phenotype) and 43 CHD (31 nodular sclerosis and 12 mixed cellularity) with B-cell (n = 10) or undetermined (n = 33) phenotype
(Table 1). NLPHD was diagnosed according to
morphologic and immunophenotypic criteria.1,22 The
CD30+, CD45, CD15+,
EMA (epithelial membrane antigen)
diagnostic profile was required for the diagnosis of
CHD.1 The Rye modification of the Lukes and Butler
classification was used to classify the histologic subtypes of
CHD.23 Frozen and/or paraffin-embedded tissues from 12 clinical samples with nonneoplastic lymphoid proliferations were
also included in the study.
Immunohistochemistry (IHC)
IHC was performed with the alkaline phosphatase anti-alkaline
phosphatase (APAAP) method as described.24 The
protocol used for each antigen tested is described below. Negative
control experiments, which were invariably negative, consisted of
omission of the primary antibody, substitution with phosphate-buffered
saline (PBS), or staining with irrelevant isotype-matched mouse Ig.
BCL-6 protein.
The BCL-6 protein was detected by the PG-B6 monoclonal antibody (MoAb)
that has been recently generated in the laboratory of one of the
investigators (B.F.) by immunizing BALB/c mice with a glutathione
S-transferase-BCL-6 fusion protein.25
Immunostaining for BCL-6 was performed on frozen or formalin-fixed,
paraffin-embedded sections by the APAAP method.24
Paraffin-embedded tissue sections were pretreated in a microwave oven
(Jet 900 W; Philips, Eindhoven, The Netherlands) for
30 minutes at 250 W in EDTA solution (0.05 mmol; pH 8).
Syn-1 antigen.
Anti-B-B4 MoAb (Serotec, Oxford, UK), which specifically recognizes
the syn-1 antigen,21 was applied to frozen tissues from all
HD cases. For morphologic control purposes, the MoAb was also applied
to paraffin-embedded tissues from a representative subset of HD cases
(NLPHD, 4 cases; nodular sclerosis CHD, 15 cases; mixed cellularity
CHD, 6 cases). Immunohistochemistry for syn-1 was performed as
previously described.10
CD40 and CD40L.
Anti-CD40 MoAb 89 (kindly provided by Dr J. Bancherau, Centre de
Recherche, Schering-Plough, Dardilly, France) was applied to
paraffin-embedded tissue sections from all HD cases. Anti-CD40L MoAb
M90 (Genzyme Diagnostic, Cambridge, MA) was applied to frozen sections
from all cases included in the study because of its lack of reactivity
in paraffin embedded tissue sections.
Lineage assignment.
Further immunophenotyping and lineage assignment of HD cases was
performed with antibodies against conventional B- and
T-cell-associated antigens, as reported in detail
previously.26,27
Assessment of BCL-6 and syn-1 Staining in HD Samples
At least 100 neoplastic cells per section, as defined by histologic and
immunohistologic criteria (CD30 positivity), were independently counted
by two of us (A.C., A.G.). The percentage of BCL-6+ or
syn-1+ neoplastic cells was assigned into one of the
following categories: 0, <10%, 10% to 25%, 25% to 50%, 50% to
75%, and >75%. Only definite and unambiguous staining on
unequivocally malignant cells was accepted as positive.
Two-Color Staining
Multiple immunocytochemical staining was performed to detect BCL-6 plus
syn-1 and BCL-6 plus LMP-1 in selected HD samples as previously
described,10 with minor modifications. Briefly, frozen
section were cut and fixed in acetone-chloroform (1:1) solution for 5 minutes and stored at  80°C until use. Slides were then
brought to room temperature (RT), fixed in acetone for 5 minutes at RT,
air dried, fixed in buffered 10% formalin for 10 minutes at RT, rinsed
in PBS pH 7.4, preincubated with normal rabbit serum (Dakopatts A/S,
Glostrup, Denmark) for 5 minutes at RT, and incubated with MoAb BCL-6
(undiluted, with the addition of 3% normal human serum)
for 1 hour at RT. After washing in 0.05 mol/L Tris-buffered saline
(TBS) pH 7.5, they were fixed in cold methanol at 20°C for
10 minutes and then immunostained by the APAAP method24
using naphthol AS-MX phosphate along with fast blue BB salt (Sigma
Chemical Co, St Louis, MO) for the development of alkaline phosphatase.
Subsequently, sections were treated twice for 5 minutes in citrate
buffer (pH 6) in a microwave oven to denature bound antibody molecules
and to inactivate alkaline phosphatase present in the APAAP complex.
Finally, sections were incubated overnight at 4°C with anti-syn-1
MoAb or anti-LMP-1 MoAb and immunostained by the APAAP method using
naphthol AS-MX phosphate along with fast red TR salt (Sigma) for the
development of alkaline phosphatase.
Multiple immunocytochemical staining was also performed to detect CD40L
plus BCL-6 and CD40L plus syn-1 in selected HD samples. Double-immunostaining assays were performed on frozen sections using
CD40L as first antibody. These experiments were performed following the
above-described method with the exception that the CD40L MoAb was
applied after the fixation in methanol.
To define the relative abundance of CD40L+ lymphocytes in
the reactive background of HD, a semi-quantitative analysis was
performed. In each case analyzed, the number of CD40L+
lymphocytes directly surrounding (ie, rosetting) each RS cell was
calculated on a total of 100 to 200 RS cells counted.
Analysis of Viral Infection
All HD samples included in this study were subjected to determination
of tumor infection by EBV. EBER in situ hybridization (ISH) studies
were performed on HD samples to identify the nature and distribution of
EBV-infected cells.28 In all the samples, immunostaining
for LMP-1 was performed with an LMP-1 specific antibody (Dakopatts A/S)
on Bouin or formalin-fixed paraffin-embedded tissue sections, as
described above. The percentage of LMP-1+ neoplastic cells
was assigned to one of the following categories: 0, <10%, 10% to
25%, 25% to 50%, 50% to 75%, and >75%.
Cell Lines
The characteristics of the human CHD cell lines L-428, KM-H2, SUP-HD1,
SBH-1, CO, HD-MY-Z, HDLM-2, and L-540 were described in detail
previously.29 CHD-derived cell lines were obtained through
the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).
Cytospin preparations of HD-derived cell lines were fixed in
acetone-chloroform at room temperature for 10 minutes and immunostained with BCL-6 and anti-B-B4 MoAbs by the APAAP method.24
Genetic Studies of BCL-6
The presence of mutations of BCL-6 5 noncoding regions
was tested by two independent methods, including polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA
direct sequencing. PCR-SSCP analysis of BCL-6 5
noncoding regions was performed on three partially overlapping PCR
fragments (E1.10, E1.11, E1.12) spanning 739 bp. This 739-bp DNA
fragment is located downstream of the first BCL-6 noncoding
exon and has been shown to harbor greater than 95% of BCL-6
5 mutations detected in B-cell non-Hodgkin's
lymphomas.30,31 DNA direct sequencing was performed on a
unique PCR product encompassing fragments E1.10, E1.11, and E1.12 using
a commercially available kit (Thermosequenase; Amersham Life Sciences,
Amersham, UK). Mutations were investigated in CHD cell
lines and, for comparative purposes, in B-cell lymphomas known to
derive from the GC, including 102 cases of B-DLCL and 20 cases of
follicular lymphoma (FL). The gross configuration of BCL-6
alleles was explored by Southern blot hybridization using probes that
recognize the cluster of BCL-6 rearrangements detected in
B-cell lymphoma.32 Quantitative analysis of the
hybridization signal was performed using a Molecular Imager System
(Biorad, Hercules, CA).
Statistical Methods
Difference in rosetting of CD40L+ T cells by BCL-6/syn-1
profile was assessed by means of Wilcoxon rank-sum test for unpaired data and analyses of variance.33
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RESULTS |
Expression Profile of BCL-6, syn-1, and CD40L in Nonneoplastic Lymph
Nodes
We first assessed the expression pattern of BCL-6, syn-1, and CD40L in
nonneoplastic lymphoid tissues. The GC B cells of 12/12 (100%)
nonneoplastic lymph nodes displayed a strong and specific reactivity
for BCL-6. The B cells of mantle and paracortical zones stained
negative for BCL-6, with the exception of a subset of large B cells,
which were localized around the follicles. These BCL-6+ large B cells were also seen at the margins of the
GC. A strong staining for syn-1 was found on plasma cells, but not
other cell populations. Overall, these data show that BCL-6 and syn-1
map to lymph node areas that are populated by B cells at different stages of differentiation. In particular, B cells within the GC are
BCL-6+/syn-1, whereas plasma cells,
which predominate in interfollicular areas, are
BCL-6/syn-1+.
Expression of CD40L in nonneoplastic lymph nodes was restricted to
small lymphocytes. These cells usually displayed a prominent dotlike or
punctate paranuclear staining and occasionally exhibited a membrane
staining pattern. In the mantle and germinal center light zone of
secondary follicles, CD40L+ cells paralleled the
distribution of CD4+ T lymphocytes.
Expression Profile of BCL-6, syn-1, and CD40/CD40L in NLPHD
In 10/10 (100%) cases of NLPHD, the majority of neoplastic (L&H) cells
(range, 75% to 100%) expressed BCL-6 (Table 1 and Fig 1A). Syn-1 expression was
consistently negative in L&H cells of all NLPHD (Fig 1B), which thus
could be ascribed to the BCL-6+/syn-1
phenotype (Fig 1A and B).
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| Fig 1.
(A and B) Frozen sections from nodular
lymphocyte predominance HD stained with the BCL-6 MoAb (A) and the
syn-1 MoAb (B). L&H cells show a strong nuclear positivity for BCL-6
(A), whereas no syn-1 expression is detectable (arrows). Residual
plasma cells show cytoplasmic staining for syn-1 MoAb (B). (C and D)
Frozen sections from classic (nodular sclerosis) HD stained with the
BCL-6 MoAb (C) and the syn-1 MoAb (D). No BCL-6 expression is
detectable; a BCL-6 RS cell is shown (arrow) (C).
Conversely, several RS cells show cytoplasmic and membrane staining for
syn-1 (D). (E and F) Serial frozen sections from a case of classic
(nodular sclerosis) HD stained with the BCL-6 MoAb (E) and the syn-1
MoAb (F). Few RS cells show a nuclear positivity for BCL-6 (E), whereas
the majority of RS cells express syn-1 (F); the intensity of staining
of RS cells for syn-1 (arrows) is lower than that of reactive plasma
cells in the background (asterisk). (G and H) Frozen sections from
cases of classic (nodular sclerosis) HD tested by two-color staining
with BCL-6 MoAb and syn-1 MoAb (see Materials and Methods). In both
cases BCL-6+ (nuclear, blue) RS cells and
syn-1+ (cytoplasmic and membranous, red) RS cells
(arrows) are present. Note positive staining of bystander plasma cells
(asterisks). No coexpression of BCL-6 protein is detectable in the
syn-1+ RS cells. APAAP immunostaining, hematoxylin
counterstain (A through F). Two-color staining, no counterstain (G and
H). Original magnification × 180 (A and B), × 250 (C through G), × 400 (H).
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CD40 was strongly expressed by L&H cells of 100% NLPHD. Reactive
CD40L+ T cells in the tumor nodules of NLPHD were rare and
distributed in a scattered fashion. Rosetting of L&H cells by
CD40L+ T cells was absent in all fields analyzed,
suggesting that CD40/CD40L interactions between L&H cells and reactive
T lymphocytes is not a feature of NLPHD.
L&H cells of all NLPHD scored negative for LMP-1, consistent with
absence of infection by EBV.
Expression Profile of BCL-6, syn-1, and LMP-1 in CHD
The expression pattern of BCL-6 in CHD was characterized by a certain
degree of heterogeneity. In the majority of CHD cases (25/43; 58%), RS
cells did not express BCL-6 (Table 1 and Fig 1C). A fraction of CHD
(18/43; 42%) displayed a low proportion of BCL-6+ RS cells
(Fig 1E) (<10% in 14/18). Only 4/43 (9%) CHD contained >10%
BCL-6+ RS cells.
A positive staining for syn-1 was detected in all CHD (43/43; 100%),
although the proportion of syn-1+ RS cells and the
intensity of staining was variable among the cases (Table 1 and Fig 1D
and F). In cases containing both syn-1+ and
BCL-6+ RS cells (n = 18; Table 1 and Fig 1E and F),
double-staining experiments showed that expression of these two
antigens was mutually exclusive in the same RS cell (Fig 1G and H).
Comparison of BCL-6 and syn-1 expression in each individual CHD led to
the identification of two major phenotypic profiles of the disease
(Tables 1 and 2). The first phenotypic
profile associated with 25/43 (58%) CHD and was characterized by
BCL-6/syn-1+ RS cells (Fig 1C and D) in
the absence of BCL-6+/syn-1 RS cells.
The second phenotypic profile associated with 18/43 (42%) CHD and was
characterized by the coexistence of
BCL-6/syn-1+ and
BCL-6+/syn-1 RS cells (Fig 1E through H)
in the same biopsy.
Because CHD may be subclassified in terms of morphology (nodular
sclerosis v mixed cellularity) and expression of conventional lymphoid markers (B-cell phenotype v undetermined phenotype), we proceded to compare the expression of BCL-6 and syn-1 with the
morphologic and phenotypic variant of CHD. This analysis showed that
BCL-6+/syn-1 and
BCL-6/syn-1+ RS cells were found in both
nodular sclerosis and mixed cellularity subtypes and in all
conventional phenotypes (Table 1).
Nine of 43 CHD were infected by EBV. In these cases, variable
proportions (5% to 75%) of RS cells expressed LMP-1. In cases displaying both LMP-1+ and BCL-6+ RS cells (n = 4), double-staining experiments ruled out the coexpression of BCL-6 and
LMP-1 by the same RS cell.
Relationship Between RS Cell Phenotype and CD40/CD40L Interactions in
CHD
In all CHD, RS cells strongly expressed CD40 but were consistently
negative for CD40L. Conversely, CD40L was expressed by numerous T
lymphocytes present in the reactive background. Single-color IHC showed
that CD40L+ T cells localized preferentially in close
proximity of RS cells, a phenomenon known as rosetting. Therefore, by
means of serial section two-color IHC, we explored the relationship
between CD40L+ T cells and the BCL-6/syn-1 profile of RS
cells in six different CHD cases (see Materials and Methods). These
data showed that in most instances
BCL-6+/syn-1 RS cells did not display
rosetting by CD40L+ T cells (65% of the
BCL-6+/syn-1 RS cells examined)
(Fig 2). Conversely, 93% of
BCL-6/syn-1+ RS cells were rosetted by
 1 CD40L+ T cells (Fig 2). CD40L+ T cells were
of a significantly higher number around
BCL-6/syn-1+ RS cells (mean = 1.39;
median = 1) than around
BCL-6+/syn-1 RS cells (mean = 0.39;
median = 0) (Wilcoxon 2-sample test, P <.001). Analyses of variance showed that the most
important source of difference was the BCL-6/syn-1 profile (F value = 345.5; P < .001), and not specific examined cases (F value = 0.69; P = .63).
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| Fig 2.
(A through E) Frozen sections from a case of classic
(nodular sclerosis) HD tested by two-color staining with CD40L and
BCL-6 MoAbs (A, C, and D) and CD40L and syn-1 MoAbs (B and E) (see
Materials and Methods). Only scattered CD40L+
(cytoplasmic, blue) lymphoid cells are detectable in the proximity of
BCL-6+ (nuclear, red) RS cells (A), whereas expression of
CD40L (cytoplasmic, blue) can be observed on several reactive
lymphocytes that surround syn-1+ (cytoplasmic and
membranous, red) RS cells (B). (A, inset) A higher magnification of
BCL-6+ RS cells with two CD40L+ lymphoid
cells (arrows). (C through E) RS cells of classic HD displaying the
BCL-6+ (C), BCL-6 (D), and
syn-1+ (E) phenotype, respectively. The
BCL-6+ RS cell does not display close association with
CD40L+ T cells (C). Conversely, both BCL-6
(D) and syn-1+ (E) RS cells are accompanied by several
CD40L+ T cells. Two-color staining, no counterstain.
Original magnification × 180 (A), × 250 (inset A), × 400 (B), × 630 (C, D, and E).
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Analysis of Mutations of the 5 Noncoding Regions of BCL-6
in CHD Cell Lines
To define the histogenesis of
BCL-6/syn-1+ RS cells, we have
investigated CHD for the occurrence of mutations of BCL-6
5 noncoding sequences. These mutations are regarded as a genetic
marker of GC derivation that is achieved during B-cell transition
through the GC and is subsequently retained during further
differentiation.30,31 Because of the paucity of RS cells in
CHD biopsies, we have used CHD cell lines representative of
B-cell-derived CHD (KM-H2, SUP-HD1, SBH-1, L-428) expressing the
BCL-6/syn-1+ phenotype. Mutational
analysis by DNA direct sequencing showed that 3 out of 4 of these cell
lines (KM-H2, SUP-HD1, and L-428) harbored BCL-6 5
mutations clustering in the proximity of the BCL-6 promoter
(Table 3 and
Fig 3). The frequency of BCL-6
mutations in CHD cell lines (Table 3) was within the range of mutation rates detected in other lymphomas known to derive from the GC, as shown
by our analysis of 102 cases of B-DLCL and 20 cases of FL (G.G., D.C.,
A.C., unpublished observation, May 1998). Also, the
spectrum of BCL-6 mutations observed in CHD cell lines was similar to that of B-DLCL, as shown by a predominance of
single-nucleotide substitutions over point insertions and deletions, as
well as a relative excess of transitions versus transversions.
Mutations of BCL-6 5 noncoding regions appeared to be
specific for CHD cell lines of B-cell origin, because the CHD cell
lines L-540, HDLM-2, CO, and HD-MY-Z, which derive from lineages other
than B cells, were devoid of mutations.
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| Fig 3.
Mutational analysis of the 5 noncoding regions of
BCL-6 in HD cell lines. (A) Analysis by PCR-SSCP.
Representative results obtained for PCR products E1.11 and E1.12 are
shown. HD cell lines are indicated at the top of each lane by their
conventional denomination. A positive control (POS), represented by a
tumor sample known to harbor BCL-6 5' 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, cell lines scored as positive
included L-428 for PCR products E1.11 and 1.12 and KM-H2 for PCR
product E1.12. (B) Analysis by DNA direct sequencing. The nucleotide
sequence of each case shown in the figure is matched to the sequence of
a normal control (N) displaying germline BCL-6 alleles. The
position of mutations is indicated (arrow) 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).
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| |
DISCUSSION |
The aim of this study was to investigate the histogenesis of the
pathologic spectrum of HD. The implications suggested by our data are
twofold. First, different categories of HD with B-cell or undetermined
phenotype correspond to different stages of B-cell maturation. Second,
the maturation stage of the HD neoplastic clone is associated with a
different composition of the reactive background of HD.
The expression profile of BCL-6 and syn-1 in the neoplastic cells of HD
segregates two major phenotypic categories of the disease, ie,
BCL-6+/syn-1 and
BCL-6/syn-1+. In normal lymphoid
tissues, these phenotypic profiles correspond to GC and post-GC B
cells, respectively.10,12 The
BCL-6+/syn-1 profile associates with
100% NLPHD, thus corroborating the notion that NLPHD is a relatively
homogenous disorder closely reflecting the GC phenotype
(Fig 4).22,34 The
BCL-6/syn-1+ profile associates with the
majority of CHD, indicating that RS cells are frequently represented by
post-GC B cells that have undergone preterminal differentiation (Fig
4). Although BCL-6/syn-1+ RS cells no
longer express GC-restricted phenotypic markers, their histogenetic
derivation from GC cells is indicated by the association with molecular
hallmarks of GC transition. In fact, this and previous studies have
shown that RS cells of CHD harbor mutations of BCL-6 and Ig
genes, which are regarded as markers of B-cell transition through the
GC.5-7
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| Fig 4.
A model of HD histogenesis. The proposed model is based
on the expression pattern of BCL-6 and syn-1 throughout physiologic
B-cell differentiation. B cells within the GC display the
BCL-6+/syn-1 phenotype, whereas B cells
that have exited the GC and have undergone further maturation toward
the plasma cell stage exhibit the
BCL-6/syn-1+ phenotype. On this basis,
neoplastic cells of NLPHD consistently express the
BCL-6+/syn-1 phenotype and thus closely
reflect the GC phenotype. Conversely, RS cells of CHD may express
either the BCL-6+/syn-1 phenotype or the
BCL-6/syn-1+ phenotype, consistent with
further maturation toward the late stages of B-cell differentiation.
Most CHD cases display only BCL-6/syn-1+
RS cells, whereas a fraction of CHD displays a mixture of
BCL-6+/syn-1 and
BCL-6/syn-1+ RS cells, suggesting
heterogeneity in the differentiation stage of the neoplastic clone. The
CD40 molecule is expressed on neoplastic cells of both NLPHD and HD,
whereas expression of CD40L by reactive T cells is restricted to the
case of CHD and is consistently absent in NLPHD. In CHD containing both
BCL-6+/syn-1 and
BCL-6/syn-1+ RS cells,
CD40L+ T cells preferentially cluster around
BCL-6/syn-1+ RS cells. This model suggests
that CD40/CD40L-mediated interactions between tumor and reactive cells
modulate the differentiation of the neoplastic clone and is consistent
with the in vitro observation that CD40/CD40L interactions downregulate
BCL-6 in B cells with a GC phenotype.
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The differences observed in BCL-6 and syn-1 expression throughout the
pathologic spectrum of HD may be caused by differences in the
composition of the reactive background. It is well established that
rosetting of RS cells by activated CD4+ T lymphocytes is
mediated in part by the CD40/CD40L adhesion pathway and that signaling
between RS cells and rosetting T lymphocytes actively involves the
CD40/CD40L system.35-37 Whereas CD40 is consistently expressed by both L&H and RS cells, the abundance and distribution of
CD40L+ T lymphocytes varies markedly in different HD
categories34,38 (and this study). Our data indicate that
CD40/CD40L signaling between neoplastic and reactive cells is a
prominent feature of CHD, but not of NLPHD, and that it preferentially
clusters with RS cells displaying the
BCL-6/syn-1+ phenotype.
The phenotypic profiles identified by BCL-6, syn-1, and LMP-1 in RS
cells, as well as the preferential distribution of CD40L+ T
cells around BCL-6/syn-1+ RS cells, are
consistent with in vivo observations derived from other lymphoma types
and with experimental evidence gained from in vitro cellular models. In
vivo, it has been reported that BCL-6 expression is mutually exclusive
with syn-1 and LMP-1 also in the context of systemic acquired
immunodeficiency syndrome (AIDS)-related lymphomas and primary central
nervous system lymphomas (PCNSL).10,28,39 In these two
settings, the
BCL-6+/syn-1/LMP-1
profile associates with cases displaying a large noncleaved cell morphology and reflecting a GC phenotype, whereas the
BCL-6/syn-1+/LMP-1+ profile
associates with cases displaying an immunoblastic morphology and
reflecting a post-GC phenotype. In vitro, it has been shown that
CD40/CD40L signaling is able to downregulate BCL-6 in B cells with a GC
phenotype.15,17 A similar effect is exerted in vitro also
by LMP-1, which, in fact, is functionally homologous to
CD40.17,40
On this basis, it may be postulated that CD40 ligation may be the major
determinant of the phenotype of RS cells. In EBV-infected HD, the
effect mediated by CD40 ligation may be substituted by RS expression of
LMP-1. According to this model, NLPHD retains BCL-6 expression and,
consequently, the GC phenotype, because L&H cells are not exposed to
CD40L signaling. Conversely, CD40/CD40L interactions and/or
LMP-1 expression induce RS cells of CHD to downregulate BCL-6, thus
allowing further maturation of the tumor clone to assume a post-GC
phenotypic profile. Presumably, cases of CHD containing a mixture of
BCL-6/syn-1+ and
BCL-6+/syn-1 RS cells represent tumors
in which, for unknown reasons, the differentiation process of RS cells
is not complete in a fraction of cells and/or is still ongoing
at the time of observation.
| |
FOOTNOTES |
Submitted May 11, 1998;
accepted July 7, 1998.
Supported in part by the Associazione Italiana per la Ricerca sul
Cancro, Milan, Italy; by "Fondazione CRT," Torino, Italy; and by
National Institutes of Health Grant No. CA-37295. D.C. is being
supported by a fellowship from Fondazione "Piera Pietro e Giovanni
Ferrero," Alba, Italy.
Address reprint requests to Antonino Carbone, MD, Division of
Pathology, Centro di Riferimento Oncologico, IRCCS, via Pedemontana Occidentale, Aviano I-33081, Italy; e-mail acarbone{at}ets.it.
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.
| |
ACKNOWLEDGMENT |
The authors thank Ivana Zanette, Paola Ceolin, and Barbara Canal for
excellent technical assistance in immunohistochemistry experiments,
multiple immunocytochemical staining, and EBER in situ hybridization
studies.
| |
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May 1, 1999;
11(5):
739 - 744.
[Abstract]
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A. Carbone, A. Gloghini, L. M. Larocca, A. Antinori, B. Falini, U. Tirelli, R. Dalla-Favera, and G. Gaidano
Human Immunodeficiency Virus-Associated Hodgkin's Disease Derives From Post-Germinal Center B Cells
Blood,
April 1, 1999;
93(7):
2319 - 2326.
[Abstract]
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Abstract
Introduction
Abstract