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NEOPLASIA
From the Department of Pathology, Weill Medical College
of Cornell University, New York, NY.
The organization and expression of the BCL-6 gene in normal and
neoplastic thymic T cells has not been fully determined. We examined 8 precursor T-cell lymphoblastic lymphomas (T-LBLs) and found significant
BCL-6 expression in 4 cases. Three of the BCL-6+ cases
expressed a common thymocyte phenotype (CD4+,
CD8+), and one expressed a precursor thymocyte phenotype
(CD4 BCL-6 is a transcriptional repressor belonging to
the POZ/zinc finger family of transcription factors that is implicated
in normal lymphoid development and lymphomagenesis.1
Recent in vitro studies suggest that BCL-6 may function as an
anti-apoptotic molecule.2,3 The BCL-6 gene was identified
because of its involvement in chromosomal translocations affecting band
3q27, which is a frequent break-point site in diffuse large B-cell
lymphomas (DLBCLs).4-8 Rearrangements of this gene can be
identified by Southern blot analysis in 30% to 45% of DLBCLs, in 6%
to 10% of follicular lymphomas, and in 20% of acquired
immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphomas
(NHLs).9-12 Most rearrangement break points cluster within
a 4-kilobase (kb) region spanning the BCL-6 promoter and first
noncoding exon, resulting in the fusion of BCL-6 coding sequences
(exons 2-10) to heterologous promoters from other chromosomes, leading
to deregulated expression by the mechanism of promoter
substitution.13
Subsequent analysis of BCL-6 has revealed the presence of point
mutations and/or small deletions in 70% of DLBCLs, 45% of follicular
lymphomas, and 58% of AIDS-associated NHLs.14,15 BCL-6
point mutations have also been identified in 43% of polymorphic post-transplantation lymphoproliferative disorders and 90% of post-transplantation lymphoproliferative disorders classified as NHL or
multiple myeloma.16 These mutations occur within a 4-kb
region spanning the first exon but tend to cluster in the 5' region of
the first intron and overlap with the major cluster of chromosomal
break points. Analyses of both BCL-6 gene rearrangements and mutations
have shown that structural alterations in the 5' noncoding region of
BCL-6 are present in virtually all cases of DLBCL and in most cases of
follicular and AIDS-related NHLs.14,15 These findings
imply that BCL-6 may contribute to B-cell lymphomagenesis, presumably
by altering BCL-6 gene expression. BCL-6 expression was also reported
in NHLs in the absence of any genetic alterations, suggesting the
existence of other as yet unknown regulatory mechanisms resulting
in its abnormal expression.17
BCL-6 protein expression has also been reported in CD30+
T-cell anaplastic large cell lymphomas and in some neoplastic cells in
angioimmunoblastic T-cell lymphomas,18,19 but structural alterations in the BCL-6 gene have not been reported in T-cell malignancies.
In normal lymphoid tissue, BCL-6 is preferentially expressed by
germinal center B cells but not by immature B-cell precursors or
differentiated plasma cells.17,20 Within the T-cell
compartment, BCL-6 expression is found in CD4+ T cells
within germinal centers and in scattered T cells in the perifollicular
areas.17,21,22 BCL-6 also has been reported to be
detectable in cortical thymocytes.17,21,23 However, no
detailed immunohistochemical studies relating BCL-6 expression to
discrete stages of thymocyte development have been undertaken. Similarly, the pattern of expression and genetic integrity of the BCL-6
gene in neoplastic thymic cells have not yet been fully characterized.
Therefore, the goal of these studies was to assess the presence of
structural alterations of the BCL-6 gene in precursor T-cell
lymphoblastic lymphomas (T-LBLs) and analyze BCL-6 protein expression
in these lymphomas as well as prenatal and postnatal human thymus to
better understand its role in normal thymic ontogeny and possible
implications for T-LBL lymphomagenesis. In addition, to gain
additional insight into the possible role of other anti-apoptotic molecules in thymic lymphomagenesis, we analyzed the expression of
BCL-2 and BCL-XL in T-LBLs and selected thymuses and
determined their coexpression with BCL-6. Finally, we compared the
expression of BCL-6, BCL-2, and BCL-XL in normal thymus and
T-LBL to that of follicle centers of reactive lymph nodes and
follicular lymphoma to determine whether T-LBL and follicular lymphoma
share similarities in the abnormal pattern of their coexpression.
Abnormal coexpression of BCL-6, BCL-2, and BCL-XL in T-LBLs
as well as follicular lymphoma might imply a similar contribution of
these anti-apoptotic genes in the pathogenesis of these 2 types of lymphoma.
Samples
Immunohistochemistry
Immunohistochemistry for BCL-6 Formalin-fixed, paraffin-embedded tissue sections of all T-LBL cases, as well as sections of prenatal and postnatal thymuses, were evaluated for BCL-6 expression using monoclonal antibody PG-B6 (Dako) and the Cap-Plus Peroxidase Detection System (Zymed Laboratory) according to the modified MIP protocol. Antigen retrieval was performed in a pressure cooker using 10 mM citrate buffer, pH 6.0. T-LBL cases were considered positive when more than 10% of the neoplastic cells showed nuclear staining, as previously described.18 In addition, staining intensity and distribution of BCL-6-expressing neoplastic cells and thymocytes was also evaluated.Two-color immunohistochemistry To define the phenotypic characteristics of BCL-6-expressing tumor cells and thymocytes, all T-LBL cases expressing BCL-6 and selected thymuses were double stained for BCL-6 and CD1a, CD2, CD3, CD4, CD5, CD7, or CD8. In addition, selected cases of T-LBL expressing BCL-6 and selected thymuses, as well as reactive lymph nodes and cases of follicular lymphoma, were double stained for BCL-6 and BCL-2 or BCL-XL.Paraffin tissue sections were stained for BCL-6 as described above, after which antigen retrieval was again performed in a pressure cooker with a 10 mM citrate buffer at pH 6. The expression of the second antigen was determined with the ChemMate Alkaline Phosphatase Detection System (Ventana Medical Systems) using the modified MIP protocol. The alkaline phosphatase reaction was developed by BT Red Reagent Substrate (Ventana Medical Systems) or Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories). Three-color immunohistochemistry To evaluate the expression of BCL-6 by CD4+ and CD8+ cortical thymocytes, paraffin sections of selected thymuses were stained by 3-color immunohistochemistry for BCL-6, CD4, and CD8 using modified MIP protocol (Ventana Medical Systems). Before each round of staining, sections were retrieved in a pressure cooker using 10 mM citrate buffer, pH 6.0. BCL-6 protein was detected using LSAB2 Alkaline Phosphatase Kit (Dako). The alkaline phosphatase reaction was developed employing BT Red Alkaline Phosphatase Substrate (Ventana Medical Systems). CD4 antigen was detected using EnVision Peroxidase Mouse Detection System (Dako). Peroxidase reaction was developed employing Liquid DAB Substrate Chromogen System (Dako). CD8 antigen was detected with alkaline phosphatase antialkaline phosphatase (APAAP) method25 using APAAP mouse monoclonal and goat antimouse immunoglobulins (Dako). Alkaline phosphatase reaction was developed employing Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories). Incubation times of the paraffin sections with reagents of each individual detection system were adjusted according to the manufacturers' protocols.Southern blot hybridization analysis for BCL-6 gene rearrangements Genomic DNA was extracted from frozen tissue blocks using a salting-out procedure.26 Five-microgram aliquots of genomic DNA were digested with BamHI and XbaI, respectively (Boehringer-Manheim, Indianapolis, IN), electrophoresed in 0.8% agarose gels, denatured with alkali, neutralized, and transferred to nitrocellulose filters according to Southern. The filters were hybridized to a 32P-labeled BCL-6 probe (Sac4.0) as previously described.4Single-strand conformation polymorphism analysis Five sets of primers, spanning a 741-base pair region that is altered in close to 70% of DLBCLs, were used for single-strand conformation polymorphism analysis. These sets have been designated E1.9 through E1.13 as follows: E1.9: 5'-GGGTTCTTAGAAGTGGTG-3' and 5'-CAAAGCATTTGGCAAGAG-3'; E1.10: 5'-CTCTTGCCAAATGCTTTG-3' and 5'-TAATTCCCCTCCTTCCTC-3'; E1.11: 5'-AGGAAGGAGGGGAATTAG-3' and 5'-AAGCAGTTTGCAAGCGAG-3'; E1.12: 5'-TTCTCGCTT- GCAAACTGC-3' and 5'-CACGATACTTCATCTCATC-3'; E1.13: 5'-GATGAGATGAAGTATCGTG-3' and 5'-ACACTGAAAGGCATCGCA-3'. Polymerase chain reactions were performed with 100 ng genomic DNA, in the presence of 10 pmol of each primer, 25 µM deoxyribonucleoside triphosphate, 1 µCi [ -32P]deoxycytidine triphosphate (NEN; specific
activity, III TBq/mmol), and 1.5 mM MgCl2. Thirty
cycles of denaturation (94°C), annealing (56°C for E1.10, 58°C
for E1.11, and 54°C for E1.10), and extension (72°C) were
performed. The reaction mixture (2 µL) was diluted 1:25 in 0.1%
sodium dodecyl sulfate, 10 mM EDTA, and further mixed 1:1 with a
sequencing stop solution (95% formamide, 20 mM EDTA, 0.05%
bromophenol blue, and 0.05% xylene cyanol). Samples were heat-denatured and electrophoresed in a 6% acrylamide-TBE
(Tris-borate-EDTA) gel containing 10% glycerol.
Expression of BCL-6 in T-LBL The immunophenotypic profiles of the 8 T-LBLs corresponded to the common or precursor thymocyte phenotype (Table 1). Three T-LBLs were positive for both CD4 and CD8 (double-positive), 4 T-LBLs were CD4 ,
CD8 (double-negative), and one case was positive only for
CD4 (immature single-positive; CD4+, CD8,
TCR , TCR  ). The BCL-6 protein
was expressed in 4 of 8 (50%) T-LBLs analyzed. Three of these cases
were double-positive T-LBL, corresponding to a common thymocyte
phenotype, and one case was double-negative T-LBL, corresponding to a
precursor thymocyte phenotype. BCL-6 protein was expressed by more than
90%, 80%, and 50% of malignant T lymphoblasts in 3 double-positive
T-LBL cases, respectively (Figure 1A), and by 50% of malignant T
lymphoblasts in the one double-negative T-LBL case (Figure
1B). In all positive cases, anti-BCL-6
monoclonal antibody diffusely stained the nuclei of malignant T
lymphoblasts with moderate to strong intensity. Two-color staining
demonstrated that the malignant T lymphoblasts of all BCL-6+ T-LBLs expressed both BCL-6 and CD1a, CD2, CD3, CD5,
CD7 (shown for CD3 in Figure 1B) and, in addition, in 3 double-positive
T-LBLs, CD4 and CD8. All BCL-6+ T-LBLs were confirmed to
have T-cell receptor (TCR) or chain rearrangements by
Southern blot, polymerase chain reaction analysis, or TCR protein
expression by immunohistochemistry, except for the single
BCL-6+, double-negative (CD4,
CD8) T-LBL, which showed a germline configuration of the
TCR and chain genes (data not shown). Three of 4 BCL-6 T-LBLs displayed a double-negative phenotype
(CD4, CD8) of precursor thymocytes; one
BCL-6 T-LBL displayed a phenotype of immature
single-positive (CD4+, CD8)
precursor thymocytes.
Expression of BCL-6 in normal thymus The BCL-6 protein was expressed by 60% to 90% of cortical thymocytes and by 10% to 20% of the medullary thymocytes in the 6 normal fetal thymuses analyzed (Figure 2A). BCL-6 was expressed as early as 14 weeks of gestational age. The pattern of expression was similar in the 6 postnatal thymuses. Most cortical thymocytes expressed both BCL-6 and CD1a, CD2, CD3, CD4, CD5, CD7, or CD8 (shown for CD3 in Figure 2B). However, a small fraction of BCL-6-expressing cortical thymocytes were negative for these antigens. The rare BCL-6+ cells in the medulla also expressed CD2, CD3, CD4, CD5, CD7, or CD8 (not shown). Three-color staining for BCL-6, CD4, and CD8 showed that BCL-6 was expressed by most double-positive (CD4+, CD8+) cortical thymocytes (Figure 3).
Expression of BCL-2 and BCL-XL in normal thymus and T-LBL and coexpression with BCL-6 In the normal fetal and postnatal thymuses, BCL-2 was uniformly expressed by the mature medullary thymocytes; however, only scattered cortical thymocytes were BCL-2+ (Figure 4A). The BCL-2+ cortical thymocytes did not coexpress BCL-6 (Figure 4B), except for the very rare cell. Conversely, BCL-XL was expressed by most cortical thymocytes but only by scattered medullary thymocytes. BCL-XL+ cortical thymocytes also coexpressed BCL-6 (not shown). All 4 BCL-6+ T-LBLs, as well as all 4 BCL-6 T-LBLs, coexpressed BCL-2 (Figure 4C) and
BCL-XL (not shown). The BCL-2 expression was usually
strong, while BCL-XL expression was variable.
Comparison between the pattern of BCL-6, BCL-2, and BCL-XL expression in normal thymus and BCL-6+ T-LBL to that in follicle centers of reactive lymph nodes and follicular lymphoma The pattern of BCL-6 and BCL-2 expression in thymic cortex and BCL-6+ T-LBL paralleled that seen in follicle centers of reactive lymph nodes and follicular lymphoma, respectively. Follicle centers of reactive lymph nodes expressed BCL-6, while BCL-2+ cells were seen primarily outside follicles (Figure 5A). BCL-XL was expressed by most follicle center cells (not shown). Only scattered cells in follicle centers were BCL-2+ and did not express BCL-6 (Figure 5B), except for the very infrequent cell. Follicular lymphoma, similarly to BCL-6+ T-LBL, showed abnormal coexpression of BCL-6 and BCL-2 (Figure 5C) and also coexpressed BCL-XL (not shown).
Analysis of BCL-6 gene rearrangements and mutations Southern blot analysis was performed to assess the presence of rearrangement in the 5' region of the BCL-6 gene. All 6 T-LBLs examined showed a distinct band in the germline configuration, and no rearrangements were identified using this method (results not shown). None of the 6 T-LBLs examined showed evidence of mutations or deletions in the 5' noncoding region of the BCL-6 gene, as evaluated by single-strand conformation polymorphism analysis.
In this study we show that BCL-6 is expressed by a significant proportion of precursor T-cell lymphoblastic lymphomas. Four of 8 cases of T-LBL that we investigated expressed high levels of BCL-6. None of these cases had rearrangements or mutations in the regulatory region of the BCL-6 gene. This result indicates that, while structural alterations of the BCL-6 gene are uncommon in T-LBL, BCL-6 protein expression can be identified in a significant proportion of cases. Of the 4 T-LBLs in our series expressing the BCL-6 protein, 3 were
double-positive (CD4+, CD8+), displaying a
common thymocyte phenotype. The fourth T-LBL had a double-negative
phenotype (CD4 Staining for BCL-6 on normal prenatal and postnatal thymus showed
that BCL-6 is expressed by most cortical thymocytes and is
down-regulated in the medulla, where it is expressed only by a small
proportion of thymocytes. Two-color immunohistochemistry of normal
thymus showed that BCL-6 is expressed by double-negative (CD4 Recent in vitro studies have suggested that BCL-6 may function as
an anti-apoptotic molecule. These studies have shown that cross-linking
of B-cell antigen receptor (BCR) with anti-IgM on a BCL-6+,
Epstein-Barr virus In contrast to our understanding of the surface receptors that
regulate progression through thymocyte development, downstream signal
transduction pathways are not completely understood.31 Nevertheless, considerable progress has been made in unraveling the
nature of signaling pathways involved in T-cell ontogeny, largely
resulting from studies on gene-manipulated animals in which the
activity of some of those molecules was either knocked out or enhanced,
as well as by the availability of more specific inhibitors of signal
transduction pathways.32 For To further address a possible role of apoptosis in T-LBL lymphomagenesis, we analyzed the expression of 2 other anti-apoptotic genes, BCL-2 and BCL-XL, in T-LBLs and selected thymuses and determined their coexpression with BCL-6. Within the thymus, BCL-2 and BCL-XL showed a characteristic reciprocal pattern of expression, as previously reported.36-39 Flow cytometry has shown that BCL-2 is expressed in nearly all CD4+ and CD8+ single-positive cells but in only a few CD4+, CD8+ (double-positive) immature thymocytes. Conversely, BCL-XL is highly expressed in immature double-positive cells but absent from mature single-positive thymocytes.36-38 Moreover, there is evidence to suggest that expression of BCL-2 and BCL-XL may be coupled to TCR-mediated signals. BCL-2 expression is up-regulated during positive selection and persists in mature T cells in the periphery,40,41 while BCL-XL appears to play an essential role in the survival of double-positive thymocytes prior to selection signals. Interestingly, all 4 of our BCL-6+ T-LBL cases expressed BCL-2 and showed variable levels of BCL-XL. The abnormal coexpression of BCL-6, BCL-2, and BCL-XL may be useful in the differential diagnosis of small biopsies from anterior mediastinal masses in distinguishing T-LBL from residual normal thymus. Whether coexpression of BCL-6, BCL-2, and BCL-XL represents a marker of malignancy or may occur also as a result of microenvironmental alterations in thymocyte maturation and development, such as observed in thymomas,42 has yet to be determined. The abnormal coexpression of BCL-2, BCL-XL, and BCL-6 in T-LBL is also highly reminiscent of coexpression in follicular lymphomas 43 and suggests that, in conjunction, expression of these genes may contribute to T-LBL evolution. In addition, the pattern of BCL-2, BCL-6, and BCL-XL expression in thymus and normal germinal centers shows striking similarities, because most BCL-6+ cortical thymocytes lack BCL-2 expression and express BCL-XL, analogous to normal germinal center B cells.20,23,37,39,44 Our results show that, in our series, BCL-6 protein is expressed by cortical thymocytes and a subset of T-LBLs, in particular all of those with a double-positive phenotype. These results suggest that T-LBLs expressing BCL-6 may arise from normal cortical thymocytes, perhaps through inability to down-regulate BCL-6 during positive selection. The dysregulated BCL-6 expression does not appear to be caused by structural alterations in the regulatory region of this gene, and the mechanism causing this dysregulation remains to be elucidated. Nevertheless, the pattern of BCL-6, BCL-2, and BCL-XL expression in normal thymuses is remarkably similar to that identified in germinal centers, and the abnormal coexpression of BCL-2, BCL-XL, and BCL-6 is seen in both T-LBL and follicular lymphoma.17,43,44 Expression of BCL-6 may be regulated by signals important for T-cell development, differentiation, and survival, and dysregulation of those signals may contribute to thymic lymphomagenesis.
Submitted June 9, 2000; accepted September 7, 2000.
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.
Reprints: Daniel M. Knowles, Department of Pathology, Weill Medical College of Cornell University, 1300 York Ave, New York, NY 10021; e-mail: dknowles{at}med.cornell.edu.
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© 2001 by The American Society of Hematology.
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Top
Abstract
Introduction
70°C from 7 T-LBLs and formalin-fixed, paraffin-embedded tissue from all cases were
used in this study. The diagnosis of T-LBL was based on correlative
morphologic, immunophenotypic, and genotypic analysis.
