Two microarray studies of mediastinal B cell lymphoma have shown that this disease has a distinct gene expression profile, and also that this is closest to the pattern seen in classical Hodgkin's disease. We reported previously an immunohistologic study in which the loss of intracellular B cell-associated signaling molecules in Reed–Sternberg cells was demonstrated, and in this study we have investigated the expression of the same components in more than 60 mediastinal B cell lymphomas. We report that these signaling molecules are frequently present, and in particular that Syk, BLNK and PLC-γ2 (absent from Reed–Sternberg cells) are present in the majority of mediastinal B cell lymphomas. The overall pattern of B cell signaling molecules in this disease is therefore closer to that of diffuse large B cell lymphoma than to Hodgkin's disease, and is consistent with a common cell of origin as an explanation of the similar gene expression profiles.
Primary mediastinal B cell lymphoma and classical Hodgkin's disease share several morphologic and clinical features. More recently, similarities at the molecular and genetic level have also been noted. For example, STAT-6 is activated in both diseases,1 and comparative genomic hybridization (CGH)-profiles show features in common (especially gains on chromosomes 2p and 9p).2, 3, 4, 5 Furthermore, two independent microarray studies have reported gene expression signatures in primary mediastinal B cell lymphoma that differ from those of diffuse large B cell lymphoma and are closest to Hodgkin's disease.6, 7
We have recently documented a marked reduction (or absence) of five intracellular B cell-associated signaling molecules in Reed–Sternberg cells.8 The present study aimed to investigate the same molecules (plus the NFATc1 transcription factor) in a cohort of more than 60 mediastinal B cell lymphomas. Here we show that three B cell-associated signaling molecules (Syk, BLNK and PLC-γ2) are frequently present in mediastinal B cell lymphomas but are constantly absent in classical Hodgkin's disease.
Materials and methods
Tissue arrays containing 1.0 mm cores from primary mediastinal B cell lymphoma biopsies were obtained from the authors' institutions in Frankfurt am Main (Germany), Bologna (Italy), Créteil (France), and Cleveland (USA), and in addition the former institution provided a tissue array comprising biopsies of classical Hodgkin's disease with primary mediastinal localization, and two tissue arrays of diffuse large B cell lymphoma. Paraffin-embedded tissue sections of 10 cases of mediastinal B cell lymphomas were also retrieved from the authors' institutions in Ulm (Germany) and Vancouver (Canada). The diagnosis of mediastinal B cell lymphomas in each case was based on accepted histopathologic criteria.9 Cases of primary mediastinal B cell lymphomas and classical Hodgkin's disease were also investigated for Rel and JAK2 amplification by the FISH technique (data not shown).
Conventional single immunohistochemistry and double immunofluorescence were performed as previously described.10, 11 All primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).8, 12, 13 All immunostaining was reviewed at least twice by pairs of observers to ensure consensus.
Mediastinal B cell lymphomas
Tumor cells in essentially all cases expressed the B cell-associated molecules Lyn and PLC-γ2, the former molecule usually being localized at the periphery of the tumor cells (Figure 1), and the latter diffusely within the cytoplasm (Figure 1). In most cases, the reactivity was comparable in intensity to that of normal B cells.
Syk kinase was present in the tumor cell cytoplasm in the majority of cases (Supplementary Table 1), although in 30% of the samples the staining appeared weaker than in normal B cells. The other two B cell signaling molecules studied (the linker molecule BLNK and the kinase Fyn) were present in about three quarters of all cases, and in a minority of the positive cases (30 and 20% respectively) it was noted that labeling was weaker than in normal cells (Supplementary Table 1).
NFATc1 transcription factor
This molecule, which is extensively expressed in normal leukocytes,13 was present in only about half of the cases (Supplementary Table 1). In most samples it was seen as a cytoplasmic constituent, but in two cases it was localized to nuclei. The intensity of staining when compared to that of normal lymphocytes ranged from very weak to moderate.
Mediastinal classical Hodgkin's disease and diffuse large B cell lymphoma
In keeping with our data in a previous series of Hodgkin's disease biopsies,8 the signaling molecules Syk, BLNK and PLC-γ2 were absent from Reed–Sternberg cells in all cases, whereas Lyn and Fyn were present in a proportion of cases (60 and 61%, respectively) (Supplementary Table 1, Figure 2). In contrast, all five B cell-associated signaling molecules were present in the majority of cases of diffuse large B cell lymphomas, in accordance with earlier observations (Figure 2).
NFATc1 transcription factor
Reed–Sternberg cells were consistently NFATc1-negative (Supplementary Table 1) whereas all bystander lymphocytes showed cytoplasmic staining (Figure 2). In contrast, the majority of the diffuse large B cell lymphomas (19 out of 24) were positive: immunostaining was cytoplasmic in most of these cases, but three showed nuclear localization (Supplementary Table 1).
Western blotting of signaling molecules
Western blotting of fresh tissues from three cases of primary mediastinal B cell lymphoma showed the presence of proteins of the expected molecular weight detected by antibodies to Lyn, Syk, BLNK and PLC-γ2 (Figure 3).
Primary mediastinal B cell lymphoma differs from diffuse large B cell lymphoma in that it arises most commonly in younger female patients.9, 15, 16, 17 It is characterized clinically by aggressive tumor growth in the mediastinum with frequent lung involvement and a tendency when disseminated to involve extranodal sites.16 Histologically it comprises large tumor cells with abundant pale cytoplasm, and it has been noted that the morphology of these cells and the presence in some cases of a sclerotic background are features reminiscent of classical Hodgkin's disease (which also often involves the mediastinum).9, 18 However, other aspects of Hodgkin's disease (eg an inflammatory eosinophil-rich infiltrate) are lacking.
The immunophenotype of primary mediastinal B cell lymphoma is unusual in that B cell markers such as CD20 and CD79a are expressed but immunoglobulin is absent or present at low levels.19, 20, 21 Mediastinal B cell lymphomas are CD10-negative,6 and rarely have BCL-2 and/or BCL-6 gene rearrangement,9, 22 thus distinguishing them from diffuse large B cell lymphoma of germinal center type. Two protein markers characteristic of this tumor have been reported, namely MAL and FIG1.23, 24 The former is almost specific,25 whereas FIG1 protein can be found in a significant minority of non-mediastinal diffuse large B cell lymphoma6, 23, 25
Reed–Sternberg cells in the great majority of the cases of classical Hodgkin's disease are also clearly of B lymphoid origin but they show extensive disruption of the ‘B cell program’, that is, loss of B cell-associated molecules.26, 27, 28, 29, 30 We recently provided further evidence for this phenomenon in an immunohistologic labeling study of five B cell-associated intracellular signaling molecules (BLNK, Syk, PLC-γ2, Fyn and Lyn) in Hodgkin's disease.8 These molecules are expressed in normal B cells,12 but we noted that Reed–Sternberg cells in the great majority of cases lacked BLNK, Syk and PLC-γ2, and that Fyn and Lyn were present in a substantial number.8
Two independent gene expression microarray studies in 2003 reported that mediastinal B cell lymphoma shows a profile similar to that of Hodgkin's disease.6, 7 The present histologic study was undertaken to see if this similarity extended to the loss of B cell-associated intracellular signaling molecules that we had observed in Hodgkin's disease. Our findings (Supplementary Table 1, Figure 4) show a pattern in primary mediastinal B cell lymphoma quite different from that of Hodgkin's disease and closer to that of other diffuse large B cell lymphomas, since the three B cell signaling molecules, Syk, BLNK and PLC-γ2, were consistently absent from Reed–Sternberg cells but expressed in the majority of cases of mediastinal lymphoma. A similar trend was seen in the case of Lyn and Fyn, but the difference was not so marked since these kinases are also found in a substantial number of Hodgkin's samples.
An obvious question is whether these results correlate with the published microarray data on mediastinal B cell lymphoma, that is, whether levels of mRNA encoding Syk, BLNK and PLC-γ2 were found to be low in those studies.6, 7 The paper by Savage et al6 reported that expression of BLNK was reduced, but this was by a factor of only 1.5-fold. Although not specifically stated, Syk and PLC-γ2 mRNA levels appear not to have been significantly reduced. Rosenwald et al7 make no mention of Syk, BLNK and PLC-γ2 gene expression, but it is of interest that they report that levels of mRNA encoding several mature B cell genes (namely CD19, CD22, CD79 and OCT-2) were normal, in contrast to their reduction in Hodgkin's disease. This implies that much of the similarity in gene expression between the two diseases might arise, for example, from genes comprising the ‘lymph node’ signature, other than those associated with the B cell lineage.7
These findings indicate that the similarities previously noted between classical Hodgkin's disease and primary mediastinal B cell lymphoma do not extend to the intracellular signaling molecules investigated in this study. Differences between the two diseases are of course also found in their histomorphologic features, the most pronounced difference being the marked chronic inflammatory reaction typical of classical Hodgkin's disease which is not seen in mediastinal B cell lymphoma. Thus, molecular cytogenetics and expression profiling data may be interpreted as reflecting a common ancestor and/or early oncogenic events shared by both diseases, but differing secondary genetic events that subsequently lead to diverging programs in the two diseases. If the ‘common cell of origin’ explanation for the gene expression similarity between primary mediastinal B cell lymphoma and classical Hodgkin's disease is valid, it may imply that both diseases arise from the scattered ‘asteroid’ B cells found in the thymic medulla (since it is accepted that this cell represents the precursor of mediastinal B cell lymphoma).31, 32 Classical Hodgkin's disease very often involves the mediastinum but is most often diagnosed from cervical nodes and this may reflect early nodal spread (which is another important difference from mediastinal B cell lymphoma which spreads extra-nodally) or possibly a derivation from the recently described putative nodal equivalent of the thymic B cell.10
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Three grants supported this work: LRF (Ref Nos. 9970 and 0382) and the Julian Starmer-Smith Lymphoma Fund to DYM, Deutsche Krebshilfe to RS and Deutsche Krebshilfe (Ref No. 106367) to PM and TFB. We are grateful to Mr Ralf Lieberz and MS Roxanne Steinle for their help in preparing the tissue arrays, to Mrs Bridget Watson for her help in preparing the manuscript and to Dr Zheng Zhao for his invaluable advice concerning the presentation of the data.
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu).
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Marafioti, T., Pozzobon, M., Hansmann, M. et al. Expression pattern of intracellular leukocyte-associated proteins in primary mediastinal B cell lymphoma. Leukemia 19, 856–861 (2005) doi:10.1038/sj.leu.2403702
- signaling molecules
- Western blotting
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