Expression pattern of intracellular leukocyte-associated proteins in primary mediastinal B cell lymphoma T Marafioti 1 , M Pozzobon 1 , M-L Hansmann 2 , P Gaulard 3 , TF Barth 4 , C Copie-Bergman 3 , H Roberton 1 , R Ventura 1 , JI Martı ´n-Subero 5 , RD Gascoyne 6 , SA Pileri 7 , R Siebert 5 , ED Hsi 8 , Y Natkunam 9 , P Mo¨ ller 4,10 and DY Mason 1,10 1 Leukaemia Research Fund Immunodiagnostics Unit, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, UK; 2 Senckenberg Pathology Institute, Johann Wolfgang Goethe-University Clinic Frankfurt am Main, Germany; 3 Department of Pathology, CHU Henri Mondor, University of Cre´teil, France; 4 Department of Pathology, University of Ulm, Germany; 5 Institute of Human Genetics, University Hospital Schleswig-Holstein Campus Kiel, Germany; 6 Department of Pathology, British Columbia Cancer Agency, Vancouver, BC, Canada; 7 Department of Pathology and Unit of Haematopathology, Institute of Haematology ‘L&A Seragnoli’, University of Bologna, Italy; 8 Division of Pathology and Laboratory Medicine, Cleveland Clinic Foundation, OH, USA; and 9 Department of Pathology, Stanford University School of Medicine, CA, USA 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 immunohistolo- gic study in which the loss of intracellular B cell-associated signaling molecules in Reed–Sternberg cells was demon- strated, 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-c2 (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. Leukemia (2005) 19, 856–861. doi:10.1038/sj.leu.2403702 Published online 3 March 2005 Keywords: signaling molecules; lymphomas; immunophenotype; Western blotting 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). 2–5 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 transcrip- tion 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-g2) 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), Cre´teil (France), and Cleveland (USA), and in addition the former institution provided a tissue array comprising biopsies of classical Hodgkin’s disease with primary mediastinal localiza- tion, 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 immuno- fluorescence were performed as previously described. 10,11 All primary antibodies were obtained from Santa Cruz Biotechnol- ogy (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. Received 20 August 2004; accepted 18 January 2005; Published online 3 March 2005 Correspondence: Dr T Marafioti, Leukaemia Research Fund Immuno- diagnostics Unit, Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, Oxford OX3 9DU, UK; Fax: þ 44 1865 740811; E-mail: teresa.marafioti@ndcls.ox.ac.uk 10 These authors contributed equally to this work. Leukemia (2005) 19, 856–861 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu