Lymphoma

Expression pattern of intracellular leukocyte-associated proteins in primary mediastinal B cell lymphoma

Abstract

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.

Introduction

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 samples

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).

Immunohistologic analysis

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.

Western blotting

Proteins from cryostat sections of three different cases of primary mediastinal B cell lymphoma were solubilized and subjected to Western blotting as previously described.13, 14

Results

Immunohistologic labeling

The immunohistologic reactivity patterns of primary mediastinal B cell lymphoma and other tumor categories are detailed in Supplementary Table 1 and illustrated in Figures 1 and 2.

Figure 1
figure1

Expression of signaling molecules and transcription factor in primary mediastinal B cell lymphoma (PMBCL). (All immunostaining was performed on paraffin-embedded tissue sections by the immunoperoxidase technique.) Lyn kinase was expressed in almost all cases of mediastinal B cell lymphomas (image 1, × 40). The staining was associated with the cell membrane, and also frequently with the Golgi apparatus (as shown in the inset, × 60). In one case (image 2), the great majority of tumor cells were Lyn-negative ( × 40) but bystander normal cells (arrowed) were positive. Fyn kinase showed a granular cytoplasmic labeling pattern ( × 40). Syk kinase was found in the majority of cases, and labeling was cytoplasmic and sometimes weakly nuclear (image 1, × 60). However, in some cases, the tumor cells showed weaker staining, ranging from negative to weakly positive (image 2, × 40). The adapter molecule BLNK was also present in the majority of cases and staining was restricted to the cytoplasm (image 1, × 40). A BLNK-negative case is also shown (image 2, × 40). The phospholipase PLC-γ2 was strongly expressed as a cytoplasmic constituent in almost all cases (image 1). In a few cases (image 2), only scattered weakly positive tumor cells were present (arrowed, × 40). The transcription factor NFATc1 showed mainly cytoplasmic staining (image 1, × 60) in about half of cases. In the remainder, the tumor cells lacked NFATc1 (image 2) and were surrounded by NFATc1-positive reactive lymphocytes (arrowed, × 40).

Figure 2
figure2

Expression of intracellular molecules in mediastinal classical Hodgkin's disease and in diffuse large B cell lymphomas (DLBCL). (All immunostaining was performed on paraffin tissue sections by the immunoperoxidase technique.) In some cases of classical Hodgkin's disease, Reed–Sternberg cells showed Lyn positivity. Note the membrane- and Golgi-associated labeling in this group of tumor cells ( × 60). Fyn expression was also heterogeneous, Reed–Sternberg cells being Fyn-positive in some cases (image 1, × 60) and negative in others (image 2, × 40). Reed–Sternberg cells were consistently Syk-negative ( × 40) but reactive lymphocytes expressed Syk kinase strongly. Both the linker molecule BLNK ( × 40) and the phospholipase PLC-γ2 ( × 40) were constantly absent in Reed–Sternberg cells (in contrast to bystander lymphocytes). The transcription factor NFATc1 was also negative in Reed–Sternberg cells ( × 40, and inset × 60). Note normal lymphocytes with NFATc1 positivity. In diffuse large B cell lymphomas (DLBCL), Lyn showed cell membrane-associated labeling in all cases, as shown in this example ( × 40). Fyn stained the cytoplasm of the tumor cells in many cases (image 1, × 40), but a minority was Fyn-negative (image 2, × 40). Syk was present in the tumor cell at cytoplasmic level in the majority of the cases (image 1, × 20) but occasionally absent (image 2, × 40). The tumor cells were almost always BLNK-positive (image 1, × 40) but in one case were negative (image 2, × 40). Strong cytoplasmic PLC-γ2 staining of the tumor cells was seen in all cases ( × 40). Three different staining patterns were observed with NFATc1: (1) tumor cells expressing cytoplasmic NFATc1 (image 1, × 40); (2) nuclear and cytoplasmic NFATc1 (image 2, × 40); and (3) an NFATc1 negativity (image 3, × 40).

Mediastinal B cell lymphomas

Signaling molecules

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

Signaling molecules

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).

Figure 3
figure3

Western blotting for intracellular molecules in three cases of mediastinal B cell lymphoma. In cases 1 and 2, a band of the expected molecular weight is seen with antibodies to four signaling molecules, in keeping with immunostaining results. The third sample is similar, but only weak bands are seen with anti-BLNK and anti-PLC-γ2. However, this sample also gives a weaker reaction for BCL-2 (used as positive control), suggesting a lower quantity of protein.

Discussion

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.

Figure 4
figure4

Schematic comparison of signaling molecule and NFATc1 expression between primary mediastinal B cell lymphoma (PMBCL), mediastinal classical Hodgkin's disease (cHD) and diffuse large B cell lymphoma (DLBCL). The vertical axis indicates the percentage of cases for each marker that were positive (above the mid-line) or negative (below the mid-line).

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

References

  1. 1

    Guiter C, Dusanter-Fourt I, Copie-Bergman C, Boulland ML, Le Gouvello S, Gaulard P et al. Constitutive STAT6 activation in primary mediastinal large B-cell lymphoma. Blood 2004; 104: 543–549.

  2. 2

    Joos S, Otano-Joos MI, Ziegler S, Bruderlein S, du Manoir S, Bentz M et al. Primary mediastinal (thymic) B-cell lymphoma is characterized by gains of chromosomal material including 9p and amplification of the REL gene. Blood 1996; 87: 1571–1578.

  3. 3

    Bentz M, Barth TF, Bruderlein S, Bock D, Schwerer MJ, Baudis M et al. Gain of chromosome arm 9p is characteristic of primary mediastinal B-cell lymphoma (MBL): comprehensive molecular cytogenetic analysis and presentation of a novel MBL cell line. Genes Chromosomes Cancer 2001; 30: 393–401.

  4. 4

    Joos S, Kupper M, Ohl S, von Bonin F, Mechtersheimer G, Bentz M et al. Genomic imbalances including amplification of the tyrosine kinase gene JAK2 in CD30+ Hodgkin cells. Cancer Res 2000; 60: 549–552.

  5. 5

    Joos S, Menz CK, Wrobel G, Siebert R, Gesk S, Ohl S et al. Classical Hodgkin lymphoma is characterized by recurrent copy number gains of the short arm of chromosome 2. Blood 2002; 99: 1381–1387.

  6. 6

    Savage KJ, Monti S, Kutok JL, Cattoretti G, Neuberg D, De Leval L et al. The molecular signature of mediastinal large B-cell lymphoma differs from that of other diffuse large B-cell lymphomas and shares features with classical Hodgkin lymphoma. Blood 2003; 102: 3871–3879.

  7. 7

    Rosenwald A, Wright G, Leroy K, Yu X, Gaulard P, Gascoyne RD et al. Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma. J Exp Med 2003; 198: 851–862.

  8. 8

    Marafioti T, Pozzobon M, Hansmann ML, Delsol G, Pileri SA, Mason DY . Expression of intracellular signaling molecules in classical and lymphocyte predominance Hodgkin disease. Blood 2004; 103: 188–193.

  9. 9

    Jaffe SE, Harris LN, Stein H, Wardiman WJ . Mediastinal (thymic) large B-cell lymphoma. In: Jaffe ES, Harris NL, Stein H, Vardiman JW (eds). WHO Classification: Tumours of Haematopoietic and Lymphoid Tissues. IARC Press: Lyon (France), 2001, pp 175–176.

  10. 10

    Marafioti T, Jones M, Facchetti F, Diss TC, Du MQ, Isaacson PG et al. Phenotype and genotype of interfollicular large B cells, a subpopulation of lymphocytes often with dendritic morphology. Blood 2003; 102: 2868–2876.

  11. 11

    Mason DY, Micklem K, Jones M . Double immunofluorescence labelling of routinely processed paraffin sections. J Pathol 2000; 191: 452–461.

  12. 12

    Pozzobon M, Marafioti T, Hansmann ML, Natkunam Y, Mason DY . Intracellular signalling molecules as immunohistochemical markers of normal and neoplastic human leucocytes in routine biopsy samples. Br J Haematol 2004; 124: 519–533.

  13. 13

    Marafioti T, Pozzobon M, Hansmann M-L, Ventura R, Pileri SA, Roberton H et al. The NFATc1 transcription factor is widely expressed in white cells and translocates from the cytoplasm to the nucleus in a subset of human lymphomas. Br J Haem 2004; 128: 333–342.

  14. 14

    Pulford K, Falini B, Cordell J, Rosenwald A, Ott G, Muller-Hermelink HK et al. Biochemical detection of novel anaplastic lymphoma kinase proteins in tissue sections of anaplastic large cell lymphoma. Am J Pathol 1999; 154: 1657–1663.

  15. 15

    Cazals-Hatem D, Lepage E, Brice P, Ferrant A, d'Agay MF, Baumelou E et al. Primary mediastinal large B-cell lymphoma. A clinicopathologic study of 141 cases compared with 916 nonmediastinal large B-cell lymphomas, a GELA (‘Groupe d'Etude des Lymphomes de l'Adulte’) study. Am J Surg Pathol 1996; 20: 877–888.

  16. 16

    Lazzarino M, Orlandi E, Paulli M, Strater J, Klersy C, Gianelli U et al. Treatment outcome and prognostic factors for primary mediastinal (thymic) B-cell lymphoma: a multicenter study of 106 patients. J Clin Oncol 1997; 15: 1646–1653.

  17. 17

    Barth TF, Dohner H, Werner CA, Stilgenbauer S, Schlotter M, Pawlita M et al. Characteristic pattern of chromosomal gains and losses in primary large B-cell lymphomas of the gastrointestinal tract. Blood 1998; 91: 4321–4330.

  18. 18

    Lamarre L, Jacobson JO, Aisenberg AC, Harris NL . Primary large cell lymphoma of the mediastinum. A histologic and immunophenotypic study of 29 cases. Am J Surg Pathol 1989; 13: 730–739.

  19. 19

    Kanavaros P, Gaulard P, Charlotte F, Martin N, Ducos C, Lebezu M et al. Discordant expression of immunoglobulin and its associated molecule mb-1/CD79a is frequently found in mediastinal large B cell lymphomas. Am J Pathol 1995; 146: 735–741.

  20. 20

    Barth TF, Leithauser F, Joos S, Bentz M, Moller P . Mediastinal (thymic) large B-cell lymphoma: where do we stand? Lancet Oncol 2002; 3: 229–234.

  21. 21

    Pileri SA, Gaidano G, Zinzani PL, Falini B, Gaulard P, Zucca E et al. Primary mediastinal B-cell lymphoma: high frequency of BCL-6 mutations and consistent expression of the transcription factors OCT-2, BOB.1, and PU.1 in the absence of immunoglobulins. Am J Pathol 2003; 162: 243–253.

  22. 22

    Tsang P, Cesarman E, Chadburn A, Liu YF, Knowles DM . Molecular characterization of primary mediastinal B cell lymphoma. Am J Pathol 1996; 148: 2017–2025.

  23. 23

    Copie-Bergman C, Gaulard P, Maouche-Chretien L, Briere J, Haioun C, Alonso MA et al. The MAL gene is expressed in primary mediastinal large B-cell lymphoma. Blood 1999; 94: 3567–3575.

  24. 24

    Copie-Bergman C, Boulland ML, Dehoulle C, Moller P, Farcet JP, Dyer MJ et al. Interleukin 4-induced gene 1 is activated in primary mediastinal large B-cell lymphoma. Blood 2003; 101: 2756–2761.

  25. 25

    Copie-Bergman C, Plonquet A, Alonso MA, Boulland ML, Marquet J, Divine M et al. MAL expression in lymphoid cells: further evidence for MAL as a distinct molecular marker of primary mediastinal large B-cell lymphomas. Mod Pathol 2002; 15: 1172–1180.

  26. 26

    Ruprai AK, Pringle JH, Angel CA, Kind CN, Lauder I . Localization of immunoglobulin light chain mRNA expression in Hodgkin's disease by in situ hybridization. J Pathol 1991; 164: 37–40.

  27. 27

    Lauritzen AF, Pluzek KJ, Kristensen LE, Nielsen HW . Detection of immunoglobulin light chain mRNA in nodular sclerosing Hodgkin's disease by in situ hybridization with biotinylated oligonucleotide probes compared with immunohistochemical staining with poly- and monoclonal antibodies. Histopathology 1992; 21: 353–358.

  28. 28

    Hell K, Pringle JH, Hansmann ML, Lorenzen J, Colloby P, Lauder I et al. Demonstration of light chain mRNA in Hodgkin's disease. J Pathol 1993; 171: 137–143.

  29. 29

    Marafioti T, Hummel M, Foss HD, Laumen H, Korbjuhn P, Anagnostopoulos I et al. Hodgkin and Reed–Sternberg cells represent an expansion of a single clone originating from a germinal center B-cell with functional immunoglobulin gene rearrangements but defective immunoglobulin transcription. Blood 2000; 95: 1443–1450.

  30. 30

    Schwering I, Brauninger A, Klein U, Jungnickel B, Tinguely M, Diehl V et al. Loss of the B-lineage-specific gene expression program in Hodgkin and Reed–Sternberg cells of Hodgkin lymphoma. Blood 2003; 101: 1505–1512.

  31. 31

    Addis BJ, Isaacson PG . Large cell lymphoma of the mediastinum: a B-cell tumour of probable thymic origin. Histopathology 1986; 10: 379–390.

  32. 32

    Isaacson PG, Norton AJ, Addis BJ . The human thymus contains a novel population of B lymphocytes. Lancet 1987; 2: 1488–1491.

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Acknowledgements

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.

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Correspondence to T Marafioti.

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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

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Keywords

  • signaling molecules
  • lymphomas
  • immunophenotype
  • Western blotting

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