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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1308-1316
Transcription Factor B-Cell-Specific Activator Protein
(BSAP) Is Differentially Expressed in B Cells and in Subsets of B-Cell
Lymphomas
By
Laszlo Krenacs,
Andreas W. Himmelmann,
Leticia Quintanilla-Martinez,
Thierry Fest,
Agostino Riva,
Axel Wellmann,
Eniko Bagdi,
John H. Kehrl,
Elaine S. Jaffe, and
Mark Raffeld
From the Hematopathology Section, Laboratory of Pathology, National
Cancer Institute and the B cell Molecular Immunology Section,
Laboratory of Immunoregulation, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, MD; and
the Department of Pathology, Albert Szent-Gyorgyi Medical University,
Szeged, Hungary.
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ABSTRACT |
The paired box containing gene PAX-5 encodes the
transcription factor BSAP (B-cell-specific activator protein), which
plays a key role in B-lymphocyte development. Despite its known
involvement in a rare subtype of non-Hodgkin's lymphoma (NHL), a
detailed examination of BSAP expression in NHL has not been previously reported. In this study, we analyzed normal and malignant lymphoid tissues and cell lines, including 102 cases of B-cell NHL, 23 cases of
T- and null-cell NHL, and 18 cases of Hodgkin's disease. Normal
lymphoid tissues showed strong nuclear BSAP expression in mantle zone B
cells, less intense reactivity in follicular center B cells, and no
expression in cells of the T-cell-rich zones. Monocytoid B cells
showed weak expression, whereas plasma cells and extrafollicular large
transformed B cells were negative. Of the 102 B-cell NHLs, 83 (81%)
demonstrated BSAP expression. All of the 13 (100%) B-cell chronic
lymphocytic leukemias (B-CLLs), 21 of (100%) mantle cells (MCLs), and
20 of 21 (95%) follicular lymphomas (FLs) were positive. Moderate
staining intensities were found in most B-CLL and FL cases, whereas
most MCLs showed strong reactions, paralleling the strong reactivity of
nonmalignant mantle cells. Eight of 12 (67%) marginal zone lymphoma
cases showed negative or low BSAP levels, and 17 of 24 (71%) large
B-cell lymphomas displayed moderate to strong expression. None of the
23 T- and null-cell lymphomas reacted with the BSAP antisera, whereas
in Hodgkin's disease, 2 of 4 (50%) nodular lymphocytic predominance and 5 of 14 (36%) classical cases showed weak nuclear or nucleolar BSAP reactions in a fraction of the tumor cells. Western blot analysis
showed a 52-kD BSAP band in B-cell lines, but not in non-B-cell or
plasma cell lines. We conclude that BSAP expression is largely
restricted to lymphomas of B-cell lineage and that BSAP expression
varies in B-cell subsets and subtypes of B-cell NHL. The high levels of
BSAP, especially those found in large-cell lymphomas and in some
follicular lymphomas, may be a consequence of deregulated gene
expression and suggest a possible involvement of PAX-5 in
certain B-cell malignancies.
This is a US government work. There are no restrictions on its use.
| |
INTRODUCTION |
B-CELL-SPECIFIC activator protein (BSAP)
is a 52-kD transcription factor originally identified as a mammalian
homologue of the sea urchin tissue-specific activator protein
(TSAP).1 BSAP is encoded by the Pax-5 gene, a
member of the highly conserved paired box (Pax) gene family of
transcription factors,2 and is equivalent to the
NF-HB,3 S -BP,4 NFSµ-B1,5
LR1,6 and EBB-17 B-cell specific nuclear
factors identified by different investigators.8 Among
hematopoietic cells, Pax-5 gene expression is restricted to the
B-cell lineage.1 Pax-5 gene transcription is
initiated in pro-B cells and is abundant at the pre-B- and mature
B-cell differentiation stages, but absent in terminally differentiated
plasma cells.1,2 Pax-5 gene expression also occurs
in the mesencephalon and spinal cord during embryogenesis and in the
adult testis.2
BSAP has been implicated in the regulation of several B-cell-specific
genes, in controlling B-cell development, and in mediating the balance
between B-cell proliferation and Ig secretion.8,9 BSAP is
believed to be a key protein for transcriptional regulation of the CD19
gene2,10 and is identical to EBB-1,8 a factor reported to regulate transcription of the surrogate light chain genes
VpreB and  5.7 BSAP binding sites are found upstream of
and within Ig switch regions, suggesting a role for BSAP in regulating
class switching.4-6,9,11 The functional significance of a
BSAP binding site located in the promoter of the CD20 gene remains to
be clarified.12 Targeted disruption of the Pax-5 gene in mice blocks B-cell development at the pro-B-cell stage, implicating a role for BSAP in the control of B-cell
development.13 Recently, BSAP has been shown to regulate
mature B-cell proliferation.9,11 The addition of
Pax-5 antisense oligonucleotides to splenic B-cell cultures
reduced BSAP levels and impaired the proliferative response to
lipopolysaccharide (LPS) stimulation. Conversely,
transient overexpression of BSAP enhanced the LPS
response.8,9,11,14 BSAP may also have an important
repressor function for Ig heavy chain expression by suppressing the
activity of the 3 heavy chain enhancer.3,8,14,15
Thus, the downregulation of BSAP that occurs in plasma cells may be
essential for the high rate of Ig production found in these cells.
There are 9 paired box genes known in human (PAX-1-9) and mouse
(Pax-1-9). Each encodes a transcription factor that uses the paired box domain to recognize DNA elements in their target
genes.16-19 Mammalian PAX genes are transiently
expressed in various organs during embryogenesis and play an important
role in regulating organogenesis.20-22 Loss-of-function
mutations of some PAX genes are important in some rare
congenital human diseases,21,22 and by several criteria,
paired box genes are nuclear proto-oncogenes. First, the overexpression
of different Pax genes in mouse fibroblasts causes their
malignant transformation as these cells can form tumors in nude
mice.23 Second, alterations in PAX genes,
particularly gain-of-function mutations and overexpression, have been
reported in a variety of unrelated human malignancies, possibly
contributing to tumorigenesis in these cancers.24-30
Deregulated PAX-5 gene expression has been observed in human
medulloblastomas31 and in adult astrocytic
neoplasms.32 In the latter tumors, PAX-5 expression
levels correlated with the degree of malignancy.32 In
addition, BSAP (PAX-5), PAX-2, and PAX-8 proteins are capable of
inhibiting the function of p53 in vitro, suggesting another mechanism
by which these genes may contribute to the development of
neoplasia.33 Most recently, the PAX-5 gene has been
implicated in a rare subset of lymphoma with plasmacytic
differentiation through translocation with the Ig heavy chain gene
locus.34 The above-mentioned data and the proposed key role
of BSAP in B-cell differentiation and proliferation suggest that
dysregulation of PAX-5 gene function may contribute to
tumorigenesis in lymphoid malignancies.
We report here results from an immunohistochemical study of BSAP
expression in reactive lymphoid tissues and neoplastic lymphoid tumors,
using a specific polyclonal antisera to the human protein. Immunoblot
analysis of hematolymphoid cell lines, including those representing all
stages of B-cell maturation, was also performed. We found that BSAP
expression is restricted to B cells in reactive tissues and
non-Hodgkin's lymphomas (NHLs) of B-cell lineage. Furthermore, the
data are consistent with a role for BSAP in the pathogenesis of some
forms of human NHLs.
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MATERIALS AND METHODS |
Tissue samples.
Routinely fixed and processed malignant lymphoma and reactive lymphoid
tissue samples were selected from the histopathology files of the
Hematopathology Section, Laboratory of Pathology, National Cancer
Institute, National Institutes of Health (Bethesda, MD) and the
Department of Pathology, Albert Szent-Gyorgyi Medical University
(Szeged, Hungary). All cases included in the study were classified
according to the Revised European-American Classification of Lymphoid
Neoplasms (REAL).35 All cases had been previously immunophenotyped in either paraffin or frozen section
immunohistochemistry. Polyclonal CD3 (DAKO Corp, Carpinteria, CA),
CD15/LeuM1 (Becton Dickinson [BD], Mountain View, CA), CD20/L26
(DAKO), CD30/BerH2 (DAKO), CD43/Leu22 (BD), CD45R0/A6 (Zymed, South San
Francisco, CA), CD56/123C3 (Monosan/Caltag Laboratories, Burlingame,
CA), CD68/KP1 (DAKO), and CD79a/JCB117 (DAKO) were used in paraffin sections, whereas CD2/Leu5b (BD), CD3/Leu4 (BD), CD4/Leu3a (BD), CD5/Leu1(BD), CD7/Leu9 (BD), CD8/Leu2a (BD), CD11b/Leu15 (BD), CD19/Leu12 (BD), CD22/Leu14 (BD), and CD56/Leu19 (BD) were used for
frozen sections.
BSAP antibodies.
Polyclonal antibodies reactive with human BSAP were generated by
immunizing rabbits either with an N-terminal (MDLEKNYPTPRTSRC; antibody
7077) or a C-terminal peptide (CPPAAATAYDRH; antibody 7083) coupled to
Keyhole limpet hemocyanin (KLH). These antibodies have previously been
shown to be specific for BSAP protein by the following criteria. (1) In
electromobility shift assay, both antibodies supershifted a complex
formed with a B-cell nuclear extract prepared from HS-Sultan cells and
a DNA probe containing a high-affinity BSAP binding site. (2) Both
antisera reacted with a major product of the appropriate molecular
weight (52 kD) by Western blot analysis. (3) Both antisera react with B
cells and not T cells after immunohistochemical staining of lymphoid
tissue (A. Riva, manuscript in preparation).
Immunohistochemical detection of BSAP.
For immunohistochemical detection of BSAP, antibody 7077 was used in
all cases, whereas antibody 7083 was used for only a proportion of the
samples. All immunoreactions were performed after microwave antigen
retrieval, according to a protocol modified from methods previously
described.36,37 Briefly, the antigen retrieval was
performed for 40 minutes in a microwave pressure cooker (Nordic Ware,
Minneapolis, MN) using either DAKO Target Retrieval Solution or 10 mmol/L citrate buffer (pH 6.0) containing 0.1% Tween-20 (Sigma, St
Louis, MO). The sections were incubated overnight with 7077 antiserum
(1/1,000) at room temperature. Immunoreactions were detected with
biotinylated swine F(ab)2 antirabbit immunoglobulins (DAKO; 1/400) and streptavidin-peroxidase conjugate (DAKO; 1/600). The
peroxidase reaction was developed with 3,3 diaminobenzidine tetrahydrochloride (DAB; Sigma). All BSAP immunostains were compared with consecutively stained conventional hematoxylin and eosin sections
as well as with sections stained with CD20 and CD3 to determine the
tissue and immunolocalization of BSAP.
Double immunostaining was also performed on representative reactive
lymph nodes and cases to more precisely evaluate coexpression of BSAP
with B- (CD20) and T- (CD3) cell markers. For these reactions, BSAP was
detected as described above, except that the reaction was developed
using a Peroxidase Vectastain Elite ABC kit (Vector Laboratories,
Burlingame, CA) and DAB (Sigma) to generate a brown precipitate. In
sequential reactions, either CD20 (L26) or CD3 was detected after 1 hour of incubation with the primary antibody and 30 minutes of incubation with the secondary antibody. These reactions were developed using the Elite ABC kit and DAB with nickle
enhancement to generate a contrasting black precipitate or with VIP
substrate (Vector Laboratories) to develop a contrasting dark purple
precipitate.
Western analysis of BSAP.
Human hematolymphoid cell lines were cultured in RPMI 1640 supplemented
with 10% (vol/vol) fetal calf serum, 1% (wt/vol)
penicillin/streptomycin, and 2 mmol/L L-glutamine. Exponentially
growing cells (about 106 cells/mL) were subjected to
protein extraction in modified RIPA lysis buffer (50 mmol/L Tris-HCl,
pH 7.6, 150 mmol/L NaCl, 0.5% [wt/vol] Na-deoxycholate, 1%
[vol/vol] Nonidet P-40, and 1% [wt/vol] sodium dodecyl sulfate
[SDS]) supplemented with proteinase inhibitors (25 mg/mL
leupeptin, 50 mg/mL aprotinin, and 1 mmol/L phenylmethylsulphonyl fluoride). The cells were lysed on ice, and the lysates were vortexed for 10 seconds in 10-minute intervals for a total time of 60 minutes. The protein content of centrifuged lysates was determined using a
Bio-Rad Protein Assay kit (Bio-Rad, Richmond, CA), according to the
manufacturer's instructions. Identical amounts (75 mg of total
extracted protein) were boiled in Laemmli's sample buffer and
separated by electrophoresis on 10% (wt/vol) SDS-polyacrylamide gels.
After transfer to a nitrocellulose membrane (Schleicher & Schuell,
Keene, NH), immunoblotting was performed as follows. The membrane was
blocked for 1 hour at room temperature in TTBS (50 mmol/L Tris-buffered
saline, pH 7.6, with 0.05% Tween-20) containing 5% nonfat dry milk,
10% normal goat serum (GIBCO Laboratories, Grand Island, NY), and
0.1% NaN3. The nitrocellulose membranes were probed either
with 7077 or 7083 immunosera (both diluted in 1/1,000) and developed
using biotinylated swine F(ab)2 antirabbit Igs
(1/10,000; Dako), streptavidin-peroxidase conjugate (1/5,000; Dako),
and ECL (Amersham Life Science, Arlington Heights, IL) detection
reagents.
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RESULTS |
Use of BSAP antisera for immunocytochemistry.
To test the reactivity of the two BSAP antisera, 7083 and 7077, in
paraffin-embedded formalin-fixed tissues, we examined the ability of
the antisera to appropriately mark reactive lymphoid tissues.
Consistent with the known nuclear location of BSAP, a specific
immunoreaction was observed only in nuclei within B-cell zones and in
lymph node, tonsil, and spleen. Furthermore, these initial studies
established that mantle zone B cells have a consistently high level of
BSAP expression, allowing these or residual mantle cells in the
subsequent stained tumor sections to serve as endogenous controls in
all BSAP immunoreactions (Fig 1A). The
strong positivity of normal mantle zone cells and scattered (residual)
small B lymphocytes was also used to evaluate the relative staining
intensity of other BSAP-positive cells as follows: strong BSAP
expression, consistent with the staining of mantle zone cells; moderate
expression, weaker but significant reaction intensity; and weak
expression, detectable nuclear staining above the background level.
Double-staining with antibodies directed against B- or
T-cell-associated antigens clearly identified the BSAP-positive cells
as B cells (Fig 2A and B). Identical results were obtained using either the
amino-terminus-specific 7077 or the carboxy-terminus-specific 7083 antiserum. However, because of its clearer reaction pattern, antibody
7077 was systematically used in the study.
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| Fig 1.
(A) Hyperplastic lymph node showing BSAP expression
primarily restricted to follicular regions, with strong nuclear
staining present in mantle zone cells and less intense staining in
follicle center cells. (B through D) Lymphomas with strong BSAP
expression: (B) mantle cell lymphoma, (C) FL, and (D) mediastinal large
B-cell lymphoma. (E and F) Lymphomas with no BSAP expression. (E)
Primary monocytoid B-cell (MALT-) lymphoma of parotid gland and (F)
angioimmunoblastic T-cell lymphoma. Note the positive control staining
of residual mantle zone cells and normal B cells in the negative cases.
(Streptavidin-biotin-peroxidase method; original magnification [A]
×200, [B] ×400, [C] ×200, [D] ×600, [E] ×200, and
[F] ×400.)
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| Fig 2.
(A and B) Parallel sections of a hyperplastic
lymph node stained for BSAP and CD20 in (A) and for BSAP and CD3 in
(B). In (A), BSAP expression (brown nuclear staining) is shown
restricted to the CD20 positive B cells (purple-black) that are
localized mainly in the germinal center and mantle zone (lower half of
both photomicrographs), whereas the CD3+ (purple-black)
interfollicular T cells in (B) are essentially negative for BSAP. (C
and D) Mantle cell lymphoma stained for BSAP (brown) and CD20
(purple-black) in (C) and BSAP (brown) and CD3 (purple-black) in (D).
The CD20+ tumor cells are strongly positive for BSAP,
whereas the CD3+ T cells are BSAP negative. (E and F)
Mediastinal large-cell lymphoma stained for BSAP and CD20 in (E) and
BSAP and CD3 in (F). The CD20+ tumor cells stain strongly
for BSAP, whereas the nonneoplastic infiltrating CD3+ T
cells are negative for BSAP. (G) FL, grade 1 stained for BSAP (brown)
and CD3 (purple-black). Note the occasional CD3+,
BSAP-negative T cells, and rare CD3 BSAP-negative
cells that most likely represent dendritic cells. (H) AILD-like T-cell
lymphoma stained for BSAP (brown) and CD20 (purple-black). The tumor
cells are negative for BSAP, whereas 2 residual B cells show nuclear
BSAP staining and membrane CD20. (Original magnifications [A] ×400,
[B] ×400, [C] ×400, [D] ×400, [E] ×1,000, [F]
×1,000, [G] ×1,000, and [H] ×1,000.)
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Two hundred twenty-eight cases were collected in the initial group.
Seventy-three of these cases showed poor or no BSAP immunoreaction, despite the presence of residual small normal B cells, and therefore were excluded from further evaluation. These technically negative samples included either cases fixed in B5 or those for which only unstained paraffin slides stored at room temperature for years were
available for study. The loss of immunoreactivity over time in paraffin
sections (especially in B5-fixed tissues) is well documented with some
nuclear and nonnuclear antigens.38
Among the 155 cases judged to be appropriate for evaluation of BSAP
expression, there were 12 hyperplastic lymphatic tissues (including
nonspecific follicular hyperplasias of 3 tonsils and 7 lymph nodes, and
2 cases of Toxoplasma lymphadenitis), 102 B-cell lymphomas, 23 non-B-cell lymphomas including 12 peripheral T-cell lymphomas (PTCLs),
3 precursor T-cell leukemia/lymphomas, 8 anaplastic large-cell
lymphomas of T- and null-cell types (ALCL), and 18 cases of Hodgkin's
disease (HD).
Reactive lymphoid tissues.
In addition to the homogenous strong BSAP staining found in mantle zone
cells, follicular center (FC) B cells displayed a variable but usually
less intense, weak to moderate, BSAP reaction (Fig 1A). Weak to
moderate nuclear staining was found in the dark zone centroblasts and
in some light zone centrocytes, whereas weak reactions occurred in most
light zone FC B cells, reflecting the characteristic polarization of
reactive follicles. The majority of normal monocytoid B cells
characteristically seen in cases of toxoplasma lymphadenitis were
negative, but rare cells demonstrated weak reactions. No staining
occurred in plasma cells, extrafollicular large B cells, histiocytes,
and T cells.
B-cell NHLs.
Overall, 81% (83/102) of the B-cell lymphomas demonstrated BSAP
positivity (Table 1). With rare exceptions,
all tumor cells in positive cases showed BSAP expression. Weak
expression of BSAP was found in tumor cells in 1 of the 2 precursor
B-cell leukemia/lymphoma cases tested. All 13 cases (100%) of B-cell
chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL)
expressed BSAP and most stained with moderate intensity. Cells of
proliferation centers usually displayed less intense, negative to weak,
BSAP expression, although in 1 case, small cells were negative, whereas
prolymphocytes and paraimmunoblasts showed moderate BSAP reactivity.
BSAP expression was demonstrated in 21 of 21 (100%) mantle cell
lymphomas (MCLs), with strong reactions occurring in the majority of
cases (Fig 1B). Double-staining experiments demonstrated that BSAP was
present exclusively in CD20+ tumor cells (Fig 2C and D).
Only 1 of the 21 FLs (5%) was BSAP-negative. Of the 20 positive cases,
2 (both grade III cases [10%]) showed weak BSAP expression, 11 (1 grade I, 8 grade II, and 2 grade III cases [55%]) displayed moderate
expression and 7 (1 grade I, 2 grade II, and 4 grade III cases
[35%]) were strongly BSAP-positive (Fig 1C). Loss of polarization of
neoplastic follicles was clearly seen with BSAP staining (Fig 1C).
Double-staining studies showed that only the CD20+ B cells
expressed BSAP, whereas the occasional intratumoral CD3+
cell and dendritic cell were negative for expression of the
transcription factor (Fig 2G). Of the 12 marginal zone B-cell lymphomas
tested, 5 (42%) were negative and 3 (25%) were weakly, 3 (25%) were
moderately, and 1 (8%) was strongly BSAP-positive. Four of the 5 monocytoid B-cell lymphomas of the parotid were either negative or
weakly focally positive (Fig 1E). Nuclear staining was not demonstrated in 5 plasmacytomas tested, including 2 cases of myeloma and 3 extramedullary plasmacytomas. Of the 2 lymphoplasmacytoid lymphoma (immunocytoma) cases, 1 showed a positive BSAP reaction. Nineteen (79%) of 24 diffuse large B-cell lymphomas (LBCL) displayed BSAP expression of moderate to strong intensity, including 3 of 3 (100%) cases of mediastinal LBCL with strong positivity (Fig 1D). Again, double-staining studies indicated that only the CD20+ tumor
cells contained detectable BSAP (Fig 2E and F).
BSAP levels in T- and null-cell NHLs and HD.
BSAP immunoreactivity was not seen in 8 anaplastic large-cell lymphomas
of T- and null-cell types, in 12 nonanaplastic peripheral T-cell
lymphomas (Figs 1E and 2H), or in the 3 cases of T-cell lymphoblastic
lymphoma. Of the 18 cases of HD, 7 (39%) showed weak nuclear or
nucleolar positivity in a fraction of tumor cells, including 2 of 4 (50%) nodular lymphocytic predominance (LP) and 5 of 14 (36%)
classical cases (4 nodular sclerosis and 1 lymphocyte-depleted subtype).
Western blot analysis for BSAP.
Western blot analysis with the 7077 antibody demonstrated a specific
protein band of the expected size (52 kD) in cell lines corresponding
to precursor and mature B cells, but not in terminally differentiated
plasma cells, T cells, and other hematopoietic cell lines
(Table 2 and
Fig 3). Identical results were obtained with the carboxy terminus-specific 7083 antibody in the cell lines tested. In addition, the pre-B-cell line PB-697 also displayed an
intense band of 46 kD (Fig 3) that was only detected with the 7077 antisera. The pre-B-cell line Nalm-6 had a similar 46-kD band,
although at a much lower intensity. However, gel shift analysis of
PB-697 nuclear extracts did not show an additional BSAP gel shift (data
not shown), and immunoblotting of nuclear and cytoplasmic extracts
indicated that the 46-kD protein resides in the cytoplasm rather than
in the nucleus. Furthermore, reverse transcription polymerase chain
reaction (PCR) and sequencing of BSAP from PB697 cells
failed to identify a novel mRNA transcript or evidence for a frame
shift mutation (A. Rivas, unpublished observation). Thus, this band is unlikely to represent a c-terminally truncated form of
BSAP, as recently described in the human pro-B-cell line
REH.39
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| Fig 3.
Western blot analysis for BSAP in hematolymphoid
tumor cell lines. (A) YT, (B) Jurkat, (C) K562, (D) PB-697, (E)
Nalm-6, (F) Ramos, (G) Granta 519, (H) SU-DHL-5, (I)
SU-DHL-6, and (J) NCEB 1806. A 52-kD band of expected size is
demonstrated only in the B-cell lines (D through J). PB-697 and Nalm-6
also display a second band of 46 kD, which has a much lower intensity
in Nalm-6. This band originates from a cytoplasmic cross-reactive
protein that does not appear to be related to BSAP (see text).
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DISCUSSION |
Using two polyclonal BSAP antisera, we evaluated the expression of BSAP
in reactive and neoplastic lymphoid tissues by immunocytochemistry and
in representative B-cell and non-B-cell lines by immunoblotting. We
found that the majority of tissue B cells and greater than 80% of the
B-cell lymphomas reacted with the BSAP antisera. Double-staining studies demonstrated that the vast majority of BSAP-positive cells coexpressed CD20 and that few, if any, expressed CD3. The levels of
BSAP expression varied among different B-cell subsets and among B-cell
NHL subtypes. In addition, 40% of the HD cases tested had weak BSAP
expression in occasional tumor cells. In comparison, normal or
neoplastic T cells and other hematolymphoid tissues consistently lacked
BSAP. To the best of our knowledge, this is the first study of in vivo
BSAP expression in primary tumor samples of malignant lymphomas.
In reactive lymphoid tissues, BSAP expression was found only in the
nucleus of B lymphocytes. Interestingly, the different B-cell
compartments displayed significant differences in the intensities of
their BSAP immunoreactivity. Cells of the follicular mantle contained
the highest levels of BSAP, whereas germinal center B cells had
considerably lower levels. Extrafollicular transformed B cells
(immunoblasts), along with plasma cells, lacked detectable immunoreactivity. The high BSAP levels found in mantle zone B cells
relative to the germinal center B cells is curious because of the
purported role of BSAP in B-cell proliferation and promoting Ig class
switching.6,9,11 Because switch recombination apparently requires cell division40 and is believed to occur in the
germinal center large cells (centroblasts),41 one might
have expected high BSAP levels in these cells. The importance of the
decline of BSAP levels in the germinal center B cells remains to be
determined. Our observation that mantle zone B cells express high
levels of BSAP suggests the presence of important BSAP target genes in
these IgM- and IgD-expressing B cells. Finally, the absence of BSAP in
immunoblasts and plasma cells is consistent with its known downregulation in Ig-secreting cells.1,2
The neoplastic B cells also displayed varying levels of BSAP that to
some extent paralleled the levels found in the corresponding normal
cells. Strong BSAP expression was observed in mantle cell lymphomas.
This may reflect the derivation of this lymphoma from the strongly
positive mantle zone B cells. Whether the high level of BSAP in these
tumors contributes to their unfavorable clinical course in comparison
with other low grade lymphomas remains to be determined. All cases of
B-cell CLL/SLL expressed BSAP, but the staining was usually less
intense than in the mantle cell lymphoma cases. Weaker immunoreactivity
of prolymphocytes and paraimmunoblasts than of small lymphoma cells was
a characteristic finding. In some cases, BSAP immunoreactivity even
spared the proliferation centers. Because prolymphocytes and
paraimmunoblasts have a higher proliferative activity compared with the
surrounding tumor cells,42 their lower BSAP levels
contrasts with the positive correlation between BSAP levels and B-cell
proliferation.9,11 The lowered BSAP expression in these
cells might be related to an abortive differentiation toward cells with
increasing Ig heavy chain transcription.43
All but 1 (95%) of the 21 follicular lymphomas were moderately to
strongly BSAP positive. In many cases, these levels approximated the
high levels seen in normal mantle cells, in contrast to the low to
moderate levels seen in their corresponding nonneoplastic counterpart,
reactive follicle center B cells. This observation suggests that BSAP
may be expressed at inappropriately high levels in some FLs. No
correlation was found between the cytological grade of the FL, as
proposed in the REAL Classification, and the intensity of BSAP
positivity. In contrast to follicular lymphoma, the majority of
monocytoid B cells in toxoplasma lymphadenitis, as well as in tumor
cells of marginal zone lymphomas (composed of monocytoid cells),
displayed no or low levels of BSAP expression. Among lymphomas, this
phenomenon was most apparent in primary monocytoid B-cell (MALT-type)
lymphomas of the parotid gland. Monocytoid and marginal zone B cells
are considered to be memory B cells.44-46 This finding may
indicate that modulation of BSAP expression may be involved in the
generation of memory B cells.
BSAP/PAX-5 is thought to repress Ig heavy chain transcription in
immature and mature B cells by inhibiting the activity of the Ig heavy
chain 3 chain enhancer in these cells.3,14,15 In
plasma cells, downregulation of BSAP may remove this repressive activity on the Ig heavy chain 3 enhancer, facilitating high levels of Ig heavy chain transcription. Because the BSAP antibodies failed to react in decalcified bone marrow core biopsies, we could study only a limited number of plasmacytoma/myeloma cases. We did not
find nuclear BSAP expression in either normal or neoplastic plasma
cells. Consistent with our results, a recent study on PAX-5 mRNA expression in human multiple myeloma cell lines and primary myeloma cells also reported the failure to detect PAX-5
expression in these cells.47
A substantial fraction (5/24 [21%]) of the LBCL cases was negative
for BSAP. Most LBCLs are thought to arise from peripheral antigen
stimulated B cells; however, the clinical, pathological, and molecular
heterogeneity of these lymphomas suggests their derivation from B cells
of various stages of differentiation, and this heterogeneity was
reflected in their BSAP expression. The BSAP-negative LBCL cases might
be derived from postfollicular transformed B cells representing the
transition from mature B cells to plasma cells. Almost half (10/24
[42%]) of the LBCL cases, including all 3 mediastinal LBCL cases
tested, displayed strong BSAP positivity. The strong BSAP expression
may be part of their malignant phenotype, representing overexpression
of the protein. Furthermore, the low surface Ig expression
characteristic of mediastinal LBCLs48,49 may be related to
their high-level expression of BSAP as a result of the Ig repressor
function of BSAP. Whether the variable expression level of BSAP in
LBCLs defines clinically relevant subgroups remains to be tested.
In addition to LPHD, which is generally accepted to be a B-cell
process,35 weak BSAP expression was also found in 38%
(6/16) classical HD cases. Our result is contrary to the findings in the HRS cell lines, L428, HDLM-1, and KM-H2, in which BSAP was not
detected by gel mobility shift assay.50 The weak BSAP
expression is consistent with the developing consensus of a B-cell
derivation for classical HD in a high proportion of
cases.51,52
The nonrandom chromosomal translocation t(9;14)(p13;q32) has been
reported in cases of the lymphoplasmacytoid lymphoma subtype of
NHL.34,53 This translocation was recently cloned and shown to result in the juxtaposition of the Ig heavy chain Sµ region to the
9p13 locus,34,54 in which the PAX-5 gene has been
mapped,18,55 leading to overexpression of the PAX-5
gene product.34 This is further evidence that BSAP/PAX-5
may be involved in the pathogenesis of some B-cell lymphomas.
Lymphoplasmacytoid lymphomas are relatively rare; in the current study,
none was included.
B-cell-restricted expression of BSAP was also confirmed by Western
blot analysis. BSAP was detected in cell lines corresponding to
precursor and mature B-cell stages, but not in terminally
differentiated plasma cell, T-cell, or other hematopoietic cell lines
(Table 2 and Fig 3). In addition to the 52-kD band known to be specific for BSAP, a 46-kD band was also found in the lysate of the PB-697 cell
line, a cell line derived from a patient with pre-B ALL. A similar,
although less prominent band was also found in lysates from the
pre-B-cell line Nalm-6. Because we detected the 46-kD band only with
the N-terminal BSAP-specific antiserum, it is likely to differ from the
52-kD form of BSAP at the carboxy-terminus. Distinct Pax-5 isoforms
generated by alternative splicing have been described in murine neural,
B-lymphoid, and testicular tissues.8 Although we initially
though that the 46-kD band represented a distinct BSAP isoform, further
analysis has suggested that it represents a cross-reactive band present
in the two pre-B-cell lines.
In nonhematopoietic tumors, PAX gene products acquire their oncogenic
activity through inappropriate expression and/or
gain-of-function alterations. The strong expression of PAX-5
found in B-cell lymphomas may possibly indicate a role for this paired
box protein in some lymphomas. On the other hand, because BSAP is
strongly expressed normally during some stages of normal B-cell
differentiation (eg, mantle cells), we can only infer involvement of
BSAP in those lymphomas for which the intensity of expression exceeded
the expression level of the putative normal cellular counterpart
(deregulated expression). In this respect, the strong BSAP expression
in LBCL (especially the mediastinal large B-cell lymphomas) and some
FLs can be considered to be particularly interesting for further
analysis.
We conclude that BSAP expression is unique to the B-cell lineage among
hematopoietic cells and tumors. Double-staining studies demonstrated
that the vast majority of BSAP-positive cells coexpress CD20 and that
few, if any, express CD3. BSAP levels vary among B-cell subsets and
among subtypes of B-cell lymphoma, with the highest levels detected in
normal and neoplastic mantle cells, in some FLs, and in a proportion of
LBCL, including all mediastinal large B-cell lymphomas studied. In
addition, weak BSAP expression was also detected in about one third of
all classical HD cases. We speculate that the high levels of BSAP,
particularly those found in LBCL, and in some cases of FLs, may be a
result of deregulated BSAP/PAX-5 expression, suggesting a possible role
for PAX-5 in the pathogenesis of some B-cell malignancies.
| |
FOOTNOTES |
Submitted October 20, 1997;
accepted April 8, 1998.
L.K. and A.W.H. contributed equally to this study.
Supported in part by a grant from the Swiss National Foundation to
A.W.H.
Address reprint requests to Mark Raffeld, MD, Hematopathology Section,
Laboratory of Pathology, Bldg 10, Room 2N110, National Cancer
Institute, National Institutes of Health, 9000 Rockville Pike,
Bethesda, MD 20892.
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 are grateful to Alan Epstein for providing SU-DHL-1, -4, -5, and -10 cell lines; to Michael Kuehl for Jim3, KMS11, and OPM2; to
Ian Magrath for CA-46 and BL41; to Thomas Tedder for PB-697 and Nalm-6;
to A. Karpas for Karpas 299; to Junji Yodoi for YT; to Takemi Otsuki
for NCEB 1806; and to Martin Dyer for the Granta 519 cell line. Cell
lines not mentioned above were obtained from the American Type Cell
Culture (ATCC, Rockville, MD). We also thank Cynthia A. Harris and
Sarah E. Delay-Brown for their expert technical assistance and Ralph L. Isenberg for his photographic assistance.
| |
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409 - 426.
[Abstract]
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H. Husson, E. G. Carideo, D. Neuberg, J. Schultze, O. Munoz, P. W. Marks, J. W. Donovan, A. C. Chillemi, P. O'Connell, and A. S. Freedman
Gene expression profiling of follicular lymphoma and normal germinal center B cells using cDNA arrays
Blood,
January 1, 2002;
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[Abstract]
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E. Torlakovic, A. Tierens, H. D. Dang, and J. Delabie
The Transcription Factor PU.1, Necessary for B-Cell Development Is Expressed in Lymphocyte Predominance, But Not Classical Hodgkin's Disease
Am. J. Pathol.,
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[Abstract]
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C. Tunyaplin, M. A. Shapiro, and K. L. Calame
Characterization of the B lymphocyte-induced maturation protein-1 (Blimp-1) gene, mRNA isoforms and basal promoter
Nucleic Acids Res.,
December 15, 2000;
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[Abstract]
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H. Stein, H.-D. Foss, H. Durkop, T. Marafioti, G. Delsol, K. Pulford, S. Pileri, and B. Falini
CD30+ anaplastic large cell lymphoma: a review of its histopathologic, genetic, and clinical features
Blood,
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[Abstract]
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B. Falini, M. Fizzotti, A. Pucciarini, B. Bigerna, T. Marafioti, M. Gambacorta, R. Pacini, C. Alunni, L. Natali-Tanci, B. Ugolini, et al.
A monoclonal antibody (MUM1p) detects expression of the MUM1/IRF4 protein in a subset of germinal center B cells, plasma cells, and activated T cells
Blood,
March 15, 2000;
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H.-D. Foss, R. Reusch, G. Demel, G. Lenz, I. Anagnostopoulos, M. Hummel, and H. Stein
Frequent Expression of the B-Cell-Specific Activator Protein in Reed-Sternberg Cells of Classical Hodgkin's Disease Provides Further Evidence for Its B-Cell Origin
Blood,
November 1, 1999;
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Abstract
Introduction
Abstract