Virchows Arch (2003) 442:284–293 DOI 10.1007/s00428-002-0735-5
ORIGINAL ARTICLE
Susanne Annette Steimle-Grauer · Marianne Tinguely · Laila Seada · Christian Fellbaum · Martin-Leo Hansmann
Expression patterns of transcription factors in progressively transformed germinal centers and Hodgkin lymphoma Received: 5 July 2002 / Accepted: 18 September 2002 / Published online: 5 February 2003 Springer-Verlag 2003
Abstract The World Health Organization (WHO) classification of Hodgkin lymphoma (HL) distinguishes two types: Classical Hodgkin lymphoma (CHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). Both groups have in common that they mostly derive from B cells with rare classical cases originating from T cells. They differ in their histomorphology, immunophenotype, and clinical behavior. One of the subtypes of CHL, designated as lymphocyte-rich classical Hodgkin lymphoma (LRCHL), shares some morphological features with NLPHL. The transcription factors BSAP, BOB.1, Oct2 and MUM1 are sequentially expressed in normal Bcell development. In order to investigate the relationship between the CHL subgroups and NLPHL, we examined the protein expression of these transcription factors using immunohistochemistry in 15 reactive processes and 4 different subtypes of 58 HL cases. Our findings underline the B-cell origin of HL, without evidence, that reactive processes like progressively transformed germinal centers (PTGCs) are precursor lesions of HL. Furthermore, they demonstrate that LRCHL is distinct from NLPHL and that it is closely related to the mixed cellularity CHL (MCHL) in respect of BSAP, BOB.1, and Oct2 expression. It therefore occupies an intermediate position between MCHL and NLPHL. Based on MUM1 staining, LRCHL exhibits a more mature phenotype than NLPHL. Keywords Hodgkin lymphoma · Transcription factors · BSAP/Pax5 · Oct2 · BOB.1/OBF1 · MUM1/IRF4
Both authors, S.A. Steimle-Grauer and M. Tinguely, contributed equally to this work. S.A. Steimle-Grauer ()) · M. Tinguely · L. Seada · C. Fellbaum · M.-L. Hansmann Senckenbergisches Institute of Pathology, Johann Wolfgang Goethe-University of Frankfurt, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany e-mail:
[email protected] Tel.: +49-69-63015761 Fax: +49-69-63015241
Introduction Hodgkin lymphoma (HL) is one of the most common lymphomas in the Western world. Its cellular origin has been a controversial issue for years. Single-cell analyses of Hodgkin and Reed Sternberg (HRS) cells revealed a majority of about 98% of classical Hodgkin lymphoma (CHL) as being derived from B cells and only about 2% from T cells [6, 12, 29, 43]. In recent years, intensive research has confirmed the division into two main groups, the CHL and the rare nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). They are distinguished by their different morphology as well as by their immunophenotype. The malignant lymphocytic and histiocytic (L&H) cells in NLPHL express B-cell associated molecules as CD20, CD79a, J-chain and immunoglobulins, but lack CD30 and CD15. In contrast, the HRS cells in CHL typically express CD30 and often CD15 but, in most instances, are devoid of CD20, CD79a, J-chain and immunoglobulins [41, 52]. Genotypically, all HLs contain rearranged immunoglobulin genes; however, both types have different patterns [5, 19, 22, 23, 26, 27]. In 1995, four cases of a variant of classical Hodgkin’s disease were described as follicular Hodgkin’s disease [2]. Recently, this variant of CHL, which morphologically resembled NLPHL in terms of nodular growth and lymphocyte richness, and CHL in terms of the immunophenotype, was designated as the common nodular variant of lymphocyte-rich classical Hodgkin lymphoma (LRCHL). The nodules in LRCHL, predominantly consisting of B cells, differ from those in NLPHL in that they represent expanded mantle zones with atrophic germinal centers (GCs) [1, 13]. Single-cell analyses for immunoglobulin gene rearrangements of LRCHL revealed that its tumor cells are derived from antigen experienced B cells with a mutation pattern similar to that of CHL (Bruninger et al. unpublished observations). To gain new insights into the cellular origin and the maturation stage of HL, we first examined the localization of the tumor cells in HL, focusing on LRCHL and NLPHL. Thereafter, B-cell specific activator protein
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(BSAP/Pax5) [32, 35, 36], B-cell Oct binding factor1 (OBF-1/BOB.1) [11, 16, 20, 25, 42], octamer binding protein2 (Oct2) [15, 44, 45] and multiple myeloma-1 protein (MUM1/IRF4) [9, 49, 50] were selected for immunohistochemistry, because of their sequential expression at different stages of B-cell development and their importance for the regulation of gene expression during B-cell development. Their expression pattern in HL was evaluated by considering staining intensity and localization of the tumor cells with respect to the lymphnode architecture. Reactive lymphoid tissue and progressively transformed germinal centers (PTGCs) were taken for comparison.
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Materials and methods Seventy-three paraffin-embedded tissue samples were retrieved from the files of the Senckenbergisches Institute of Pathology, reference center for lymph-node pathology, Johann Wolfgang Goethe-University, Frankfurt am Main, and reviewed according to the World Health Organization (WHO) classification. For immunohistochemistry of transcription factors, 11 cases of NLPHL and 47 cases of CHL were investigated. The latter group included 12 cases of the common nodular variant of LRCHL, 12 cases of mixed cellularity CHL (MCHL) and 23 cases of nodular sclerosis CHL (NSHL). In addition, reactive lesions were included, comprising five lymph nodes with signs of a lymphadenitis (LA) and ten lymph nodes with PTGCs. Immunohistochemistry Immunohistochemistry was performed on paraffin-embedded, formalin-fixed lymph-node tissue. Nonmalignant lymph nodes with LA and no signs of progressive GC transformation were used as positive controls. Sections 4–5 m thin were cut, dried, deparaffinized in xylene and rehydrated in graded alcohol. Before the staining procedure, antigen retrieval was carried out by heating in citrate buffer (pH 6.0, 10 mmol) for BSAP and BOB.1 and EDTA buffer (pH 8.0, 1 mmol) for Oct2 and MUM1. The primary antibodies (all from Santa Cruz Biotechnology, CA) for staining were as follows: BSAP staining – Pax-5 antibody (C-20, polyclonal goat), Oct2 staining – Oct2 antibody (C-20, polyclonal rabbit), BOB.1 staining – BOB.1 antibody (C-20, polyclonal rabbit) and for MUM1 staining – IRF-4/ICSAT antibody (M-17, polyclonal goat). Staining procedure was carried out with a GENESIS RSP 200 (Fa. Tecan, Deutschland GmbH, Germany) according to standard protocols. Streptavidin-biotin-coupled alkaline phosphatase (StrAviGen Multi Link Kits, Biogenex) and Fast Red as chromogen (Biogenex) were used to detect the bound antibodies. Counterstaining was performed with hemalaun (Sigma).
Transcription factors in PTGCs Morphological findings in PTGCs Ten lymph nodes showing between one to six PTGCs were investigated. In contrast to the alterations in LA, GCs seem to be broken up and consist of sheets of blasts as well as centrocyte clusters. The mantle zones seem to invade and dissect the GC structures, leading to an irregular border between mantle zone and GC. Immunohistochemical findings of transcription factors in PTGCs l
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Results Transcription factors in LA Immunohistochemical findings of transcription factors in GCs of LA l
BSAP. In the GCs of reactive LA, an intense nuclear staining of small lymphocytes in the follicular mantle and a weak to moderate positivity of GC cells (some dark-zone centroblasts and some light-zone centro-
cytes) was seen. A moderate staining of some interfollicular blasts was also detectable (Fig. 1A). Oct2. A strong nuclear positivity of GC cells was found. The small lymphocytes in the follicular mantle zone were characterized by a less intense nuclear staining. A few lymphoid cells in the marginal zone and in the interfollicular areas showed an intense nuclear staining as well (Fig. 1B). GC cells, a few dispersed mantle zone cells, and a few interfollicular lymphocytes exhibited a strong nuclear positivity. The small mantle zone lymphocytes showed a weak nuclear staining (Fig. 1C). MUM1. A strong MUM1 expression was preferentially localized in GC cells of the light zone and in plasma cells. Centrocytes showed a strong nuclear and less intense cytoplasmic staining whereas plasma cells often presented with an intense nuclear and cytoplasmic immunostaining. Lymphocytes of the mantle zone were usually negative for MUM1. Furthermore, a positive reaction of some lymphocytes in the T-cell area was detected (Fig. 1D).
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BSAP. Small lymphocytes with an intense nuclear staining were concentrated in the enlarged and irregularly shaped mantle zone. A few strongly positive cells were interspersed in the interfollicular area. Centroblast-like cells showed weak to moderate immunostaining in their nuclei (Fig. 1a). Oct2 and BOB.1. Cells showing an intense nuclear positivity for Oct2 and BOB.1 were not clearly defined to GC structures anymore, rather scattered throughout the follicular mantle and along the outer border of the mantle zone. Less intensely stained small lymphocytes of the mantle zone were also detected (Fig. 1b, c). MUM1. Intense nuclear and less intense cytoplasmic stained medium sized cells were irregularly dispersed over the PTGC. The mantle zones often remained free from strongly positive cells whereas the interfollicular areas were full of them (Fig. 1d).
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Fig. 1A–D Transcription factors in normal germinal centers (GCs) (A–D) and progressively transformed germinal centers (PTGCs) (a–d) of lymphadenitis, 100. Aa Intense nuclear BSAP staining in mantle zone cells, weak to moderate positivity in GC cells. Bb Strong nuclear Oct2 positivity in GC cells, less intense staining in
mantle zone cells. Cc Strong nuclear BOB.l positivity in GC cells, weaker staining in some mantle zone cells. Dd Strong nuclear MUM1 expression, preferentially localized in GC cells of the light zone and in plasma cells. Negative mantle zone cells
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Fig. 2 Transcription factors in nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). A Strong nuclear BOB.1 staining of lymphocytic and histiocytic (L&H) cells, 400. B Strong nuclear Oct2 staining of L&H cells; in addition, a faint spill over to the
cytoplasm, 1000. Strong nuclear staining of L&H cells (inset, 400). C L&H cells, weaker BSAP stained than mantle zone cells, 400
Table 1 Staining pattern of transcription factors in tumor cells of Hodgkin lymphoma subgroups. +++ strong, ++ moderate, + weak, – no staining; n analyzed cases per subgroup: for each HL subgroup 0–4 cases could not be evaluated for technical reasons for some stains (BSAP, Oct2, BOB.1). Therefore, the number of actually evaluated cases can be different from n in the text. Bold print prevailing staining pattern in the majority of analyzed cases, referring to more than 75% of the tumor cells. Brackets with numbers staining pattern in the remaining cases
Transcription factors in HL subtypes
BSAP NLPHL (n=11) LRCHL (n=12) MCHL (n=12) NSHL (n=23) 1 2 3 4 5 6 7
++ – (++)2 – (++)2 – (++)4
Oct2 +++ +++ (-)3 – (++)4 – (++)6
BOB.1
MUM1 1
+++ (-) – (++)4 – (++)5 – (++)7
++ (–) ++ +++ +++
One case with no stained tumor cells Three cases with more than 75% of tumor cells positive Five cases with only a minority of or no tumor cells positive Four cases with more than 75% of tumor cells positive Two cases with more than 75% of tumor cells positive Seven cases with more than 75% of tumor cells positive One case with more than 75% of tumor cells positive
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Table 1 shows the staining pattern of transcription factors in tumor cells of HL subgroups. Morphological findings in NLPHL The lymph-node architecture was partially or totally replaced by a nodular or combined nodular and diffuse infiltrate. In particular, the nodular infiltrate consisted predominantly of small lymphocytes, histiocytes, epitheloid cells and intermingled L&H cells. Immunophenotypically, L&H cells were positive for B-cell markers, but negative for CD30 and CD15. The background was mixed up with numerous CD57 positive T cells.
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Fig. 3 Transcription factors in lymphocyte-rich classical Hodgkin lymphoma (LRCHL). A Strong nuclear BOB.1 stained Hodgkin and Reed Sternberg (HRS) cells laying next to weaker stained mantle zone cells, 400. B Intense nuclear Oct2 staining and
Immunohistochemical findings of transcription factors in NLPHL l
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BSAP. The malignant L&H cells showed a moderate nuclear positivity, clearly weaker than the staining of normal mantle zone cells. There was no difference in the staining intensity according to the localization of the L&H cells. In addition, a weak cytoplasmic staining was observed in some cells of a few cases. BSAP was expressed in more than 75% of the malignant cells in all cases examined (Fig. 2C). Oct2 and BOB.1. The prevailing staining pattern was a strong nuclear one with occasional cells showing a faint cytoplasmic expression of Oct2 or BOB.1. Oct2 was highly expressed in all cases, BOB.1 in 10 of the 11 cases in the majority of tumor cells. For BOB.1 one case showed no tumor cell staining (Fig. 2A, B). MUM1. MUM1 was expressed inconsistently in NLPHL. The L&H cells showed a moderate nuclear staining in addition to a weaker cytoplasmic one. In
negativity (inset) in HRS cells, two different cases, 400. C Nuclear MUM1 positivity in HRS cells with a spill over to the cytoplasm, 400
eight cases, more than 75% of the malignant cells were positive. In the remaining three cases, two had less than 50% of the malignant cells positive and one case was negative. In addition to the malignant cells, plasma cells and mature lymphocytes in the nodules as well as in T zones were strongly positive. Morphological findings in LRCHL LRCHL presents with a nodular background mixed with histiocytes and few neutrophils and eosinophils. Besides the presence of a varying proportion of neoplastic cells, exhibiting morphologic features of L&H cells, many tumor cells have the cytomorphological appearance of classical HRS cells. The neoplastic cells are often scattered within the expanded mantle zones, thereby rarely forming aggregates. These cells express CD30 and/ or CD15. A few cells are positive for CD20, but negative for the J-chain. Furthermore, small GCs can be identified
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Fig. 4 Transcription factors in mixed cellularity classical Hodgkin lymphoma (MCHL) and nodular sclerosis classical Hodgkin lymphoma (NSHL). A Nuclear Oct2 expression in a majority of Hodgkin and Reed Sternberg (HRS) cells of MCHL, laying next to
a negative HRS cell in the same case (*), 400. B No Oct2 expression in lacunar cells of NSHL, 400. C Consistent nuclear MUM1 expression in HRS cells of NSHL and by-stander cells, 400
in the periphery of the nodular structures, distinguishing these nodules from those of NLPHL, which do not contain GCs. Immunostaining with CD21 confirms the presence of eccentrically placed small GCs with a sharply depicted meshwork of follicular dendritic cells (FDCs). The mantle zone is expanded and contains an ill-defined loose meshwork of FDCs. The neoplastic cells are trapped in this meshwork and often surrounded by T-cell rosettes (CD3+).
cells. The staining intensity was comparable to that of NLPHL. Oct2 and BOB.1. The staining intensity was similar to that in NLPHL. In contrast to NLPHL, in LRCHL more cases were negative for Oct2 and BOB.1. For Oct2, seven cases expressed Oct2 either in all neoplastic cells or in at least the majority (75%). Three cases remained completely negative, whereas in two cases only a minority of malignant cells was positive. For BOB.1, six cases remained negative, two presented with less than 50% of their tumor cells positive and four with at least the majority positive. Only three cases were positive for Oct2 and BOB.1 (Fig. 3A, B). MUM1. The neoplastic cells showed a strong nuclear staining pattern in addition to a less intense cytoplasmic expression of MUM1. In all cases, at least 75% of the cells were positive. Furthermore, a prominent moderate to strong nuclear positivity of rosetting cells
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Immunohistochemical findings of transcription factors in LRCHL l
BSAP. In eight cases, the majority of neoplastic cells were completely negative or expressed BSAP only in less than 50% of their malignant cells. Only three cases expressed BSAP in more than 75% of their tumor
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was observed, corresponding presumably to activated T cells (Fig. 3C). Morphological findings in NSHL and MCHL The morphology and immunophenotype of both subtypes presented with the known, classical features. Immunohistochemical findings of transcription factors in NSHL and MCHL l
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BSAP. As expected from the classical immunophenotype, the majority of NSHL and MCHL cases were negative for BSAP. In detail, 18 of the NSHL and seven of the MCHL cases remained negative in the overwhelming majority of tumor cells. In four cases of NSHL nearly all of the tumor cells expressed BSAP. In MCHL, three cases presented with their majority of tumor cells positive. The nuclear staining intensity was moderately strong. Oct2 and BOB.1. The staining intensity for both markers was moderate in most cases. In both subtypes, Oct2 was more frequently expressed than BOB.1, which was negative in most cases. Four cases of MCHL and only seven cases of NSHL showed a moderate to strong nuclear positivity for Oct2 in more than 75% of their tumor cells. In the majority of tumor cells, six cases of MCHL and 14 of NSHL remained negative; 18 cases of NSHL and seven cases of MCHL were negative for BOB.1. Only in one case of NSHL and in two cases of MCHL was BOB.1 expressed in more than 75% of the malignant cells (Fig. 4A, B). MUM1. In contrast to NLPHL and LRCHL, in all cases 100% of the malignant HRS cells showed a strong nuclear positivity with a weak to moderately strong spill over to the cytoplasm. In addition, some of the HRS cells were surrounded by positive small lymphocytes (Fig. 4C).
Discussion NLPHL was first described by Jackson in 1937 as an “early HD” [18]. In the past decade, evidence has accumulated that NLPHL exhibits features of a B-cell lymphoma with its own characteristics. Differences between the CHLs have been shown regarding the antigen profile, the immunoglobulin gene rearrangement pattern as well as the clinical behavior and prognosis [1, 5, 8, 26]. The common nodular variant of LRCHL, on the one hand, shares morphological, genotypical and clinical features with NLPHL; on the other hand, it presents with the same immunophenotype as CHL [8]. PTGCs were reported in association with 15% of patients with NLPHL and about 2% of cases with CHL [12]. Therefore, the
Fig. 5 Predominant, nuclear expression of transcription factors in B cells during the germinal center pathway. Blue BSAP, purple Oct2, pink BOB.l, green MUM1. Intense/hatched colors represent the strong/weaker expression of transcription factors; bars represent correlation between intensity of expression and maturation stage of B cells. MZ mantle zone, GC germinal center
issue was raised whether these disorders play a role as precursor lesions of HL. B lymphopoiesis, like other developmental processes, entails the ordered, sequential expression of structural and regulatory genes. Different transcription factors are differentially expressed during B-cell maturation and are important for the regulation of gene expression during Bcell development. Whereas BSAP, regulated by the Pax5 gene, usually starts occurring in early developmental stages, Oct2 and its cofactor BOB.1 were not seen later until MUM1 was expressed in mature B cells. This expression pattern can be correlated with the GC pathway of B cells (Fig. 5). Expression of transcription factors in LA and PTGCs The expression of transcription factors in LA in our series reflects the maturation stage of the B cells in the different GC areas (Fig. 1). It is in agreement with numerous reports in the literature, based on murine models and on human tissue [21]. According to the observation of Saez et al., we found mature lymphocytes positive for Oct2 and BOB.1 in the T-cell area [39]. Previous investigations addressed the question whether PTGCs are precursor
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lesions of HL. However, immunohistochemical studies using an extended panel of antibodies against CD20, CD45RA, CD45RO, CD3, CD43, CD57, EMA, CD30 and CD21 could not show any relationship between the expression pattern of PTGCs and NLPHL [31]. Just as single-cell analyses did not reveal any evidence that PTGCs are clonally related to NLPHL [7]. Congruent with these data from the literature, we did not find evidence that could predict the evolution from PTGCs toward a HL in our study. Expression of BSAP in HL The Pax5 gene is expressed exclusively in the Blymphoid lineage within the hematopoetic system. BSAP functions both as a transcriptional activator and repressor of the Pax5 gene. It also seems to play a role in Ig class switching [32, 33, 34, 35, 36, 38]. Pax5 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 [3]. This finding might be explained by the fact that BSAP has an important repressor function for Ig heavy chain expression by suppressing the activity of the 3' alpha heavy chain enhancer. In NLPHL, all cases showed BSAP expression. In contrast, the LRCHL cases exhibited a mirror image with only few HRS cells in some cases presenting a moderately intense nuclear positivity for BSAP. In the other CHL, the number of positive cases was variable, ranging from 4 of 23 cases in NSHL to 3 of 12 in MCHL. Our findings in NSHL were similar to the study of Krenacs et al., but only in partial agreement with his results in NLPHL, where only 50% of cases were positive [21]. We found similar results in NLPHL to Foss et al. [10]. However, by using monoclonal antibodies, his group detected BSAP in a variable number of HRS cells in around 80% of CHL (NSHL, MCHL) [10]. To our knowledge, this is the first investigation of BSAP expression in LRCHL in a representative number of cases. On the genomic level, the mutation pattern of rearranged Ig genes of HRS cells in LRCHL is similar to that observed in other CHL, namely heavily mutated IgV genes and a fraction of cases with obviously crippling V gene mutations (Bruninger et al., unpublished observations). Our immunohistochemical investigations on the protein level underline the B-cell nature of both the classical and the lymphocyte predominant HLs. Based on the expression pattern of BSAP, LRCHL behaves more like CHL with similarities to MCHL. In addition, BSAP protein expression may facilitate the delineation between NLPHL and LRCHL. Expression of Oct2 and BOB.1 in HL BOB.1 protein is a B-cell specific co-activator of the Oct1 and Oct2 transcription factors [11, 42, 47]. In combination, the transcription factors Oct1 and Oct2 and their co-
activator BOB.1 regulate immunoglobulin gene transcription [16, 20, 24]. Whereas Oct1 is a ubiquitous transcription factor implicated in the control of genes expressed by all cells, Oct2 is largely B-lineage specific and has been implicated in the control of B-lymphocyte-specific gene expression [15, 40, 44, 45, 48]. Oct1 and Oct2 can replace each other, whereas BOB.1 as a specific cofactor is required to activate the immunoglobulin gene promoter [11, 25, 47]. For Oct2 and BOB.1, we observed three major groups of expression patterns: In the first group, a simultaneous expression of Oct2 and BOB.1 was seen. The percentage of positive tumor cells was high, ranging from 75% to 100%, accompanied by an intense staining confined to the nuclei, with some cases showing a faint cytoplasmic positivity. This first group was represented by NLPHL. These results are in agreement with recently published data for NLPHL by Stein et al. [46] and are in line with CD20 expression commonly observed in this entity. The findings are in accordance with Re et al. who detected Oct2 and BOB.1 transcripts in the lymphocyte-predominant-derived cell line DEV [37]. The second group was characterized by an exclusive expression of Oct2 or BOB.1, which means that we found either an expression of Oct2 or BOB.1 in the same case. The percentage of cases with positive signals for these factors was lower than in the first group. The staining intensity was as strong as in group one. This pattern was found in most LRCHL. As an exception to the abovementioned observations, we found three cases simultaneously positive for Oct2 and BOB.1 in LRCHL. These particular cases were negative for CD20. In the literature, there are only four cases of LRCHL investigated for the expression of Oct2 and BOB.1 by immunohistochemistry. In two cases, no positivity – neither for Oct2 nor BOB.1 – could be detected [37]. In the two other cases, only a faint staining for Oct2 and no or only moderate staining for BOB.1 was reported [39]. The third group showed no expression of Oct2 and BOB.1 in the majority of the cases. This is especially true for BOB.1. Nevertheless, about a quarter of these cases showed positivity for Oct2 in the majority of tumor cells. This group was represented by NSHL and MCHL. Our observations on the expression patterns of Oct2 and BOB.1 in HL are identical to the findings of Saez et al. [39]. Lack of immunoglobulin gene expression has been shown by in situ hybridization in cases of CHL, despite the detection of potentially functional IgVDJ gene rearrangements in most of these cases [27]. The unexpected finding on the lack of Oct2 and BOB.1 expression in primary HRS cells of CHL (2 of 20 cases of LRCHL) was previously discussed by Re et al., where a novel mechanism involved in transcriptional deregulation of immunoglobulin genes in CHL was suggested [37]. Likewise, downregulation of BOB.1 and Oct2 was described in CHL, but not in NLPHL and correlated with immunoglobulin transcription in these cases [37, 46].
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Expression of MUM1 in HL MUM1 is a myeloma-associated oncogene transcriptionally activated as a result of t(6;14)(p25,q32) chromosomal translocation and by virtue of its juxtaposition to the immunoglobulin heavy chain gene locus [17]. It belongs to the interferon regulatory factor family which is generally induced by interferon and therefore plays an important role in antiviral defense and in cellular proliferation [14, 30, 49]. The physiological function has not yet been elucidated, except that it cooperates with PU.1 as a transcriptional regulator in lymphoid cells [4, 51]. Findings in mice suggest a prerequisite role for MUM1 in lymphocyte activation and in terminal B-cell differentiation [28]. In normal reactive lymph nodes, the spectrum of MUM1 expression ranged from that of a centrocyte to a plasmablast/plasma cell [9]. The factor is reported to be strongly expressed in lymphoplasmacytoid lymphoma, multiple myeloma, diffuse large B-cell lymphoma as well as in a few cases of Hodgkin’s disease, but also in T-cell lymphomas including adult T-cell leukemia/ lymphoma and anaplastic large cell lymphoma [9]. In the present study, MUM1 was constitutively expressed in all of the CHL and exhibited a strong nuclear and a weaker cytoplasmic labeling. In LRCHL, the majority of HRS cells were positive, but not always in 100% of the malignant cells, as seen in MCHL and NSHL. In NLPHL, MUM1 expression was more inconsistent, with around 25% of cases lacking positivity or presenting only occasional cells as positive. Our results, although a little more heterogeneous, are in accordance with those of Falini et al. [9], who reported positivity in only two cases of his study. Furthermore, to our knowledge, only Tsuboi et al. have reported MUM1 expression in one case of LRCHL [50]. In conclusion, the immunohistochemical detection of the transcription factors BSAP, BOB.1 and Oct2 confirms the B-cell origin of HL. We show, that LRCHL is a distinct entity that shares some similarities with MCHL and NSHL and takes an intermediate position between NLPHL and MCHL. Though our data do not allow to clearly assign a HL type to a precise stage of B-cell maturation, LRCHL seems to exhibit a more mature phenotype than NLPHL, taking into consideration the MUM1 expression. Although, the expression of transcription factors in HL is very heterogeneous, their simultaneous implementation for differential diagnosis may be useful. Acknowledgements M.T. is supported by the Swiss National Science Foundation and the Janggen-Phn-Stiftung, Switzerland. We thank Ralf Lieberz and Anja Mottok for excellent technical assistance.
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