Supplementary information 1.
TMA construction & Immunohistochemical studies.
One TMA (tissue microarray) was constructed for extended immunohistochemical and FISH analysis. We used a tissue arrayer device (Beecher Instruments, Sun Prairie, WI) to construct TMA blocks, according to conventional protocols (Kallioniemi OP, Wagner U, Kononen J, Sauter G. Tissue microarray technology for high-throughput molecular profiling of cancer. Hum Mol Genet 2001;10:657-62). Standard tissue sections were also analyzed in some cases.Each case was represented per duplicate in the TMA.
Immunohistochemical staining was performed as follows: 2-4-μm-thick paraffin-embedded TMAs and whole-tissue sections were cut onto Dako slides (DAKO), and subsequently dewaxed, rehydrated and stained following automated protocols in slide processing instruments (DAKO Autostainer Plus and Leica Bond Max). Briefly, the slides were subjected to antigen retrieval by heating in 50 mM Tris (Trizma base)-1 mM EDTA (ethylenediaminetetraacetic acid) (Sigma Chemical) (pH 8) or 10 mMcitrate pH 6.5.. The slides were cooled and treated with peroxidase-blocking solution (DAKO) for 5 minutes. Sections were then immunostained with antibodies against CD20, CD30, CD15, EBV-LMP1, GCET1, MUM1, CD10, BCL6, FOXP1, p50, p52, BCL2, CD3, kappa, lambda and Ki67, and visualized using either FLEX DAKO polymers or Novolink detection systems. Then, were counterstained with hematoxylin and mounted. Incubations either omitting the specific antibody or containing unrelated antibodies were used as a negative control for the technique.Details about clones used, antigen retrieval and visualization methods are detailed in SuppTable2. (ISH) In situ hybridization for EBV-EBER was done using and EBER-PNA probe (DAKO). Briefly, slides were dewaxed, rehydrated and treated with proteinase K. After incubation with EBER-PNA probe the tissue sections were visualized using an antiFITC polyclonal antibody an revealed with HRP-FLEX DAKO polymers.
All cases (study and control series) were classified according to the cell of origin using the two currently established immunohistochemical algorithms by Hans and Choi (Hans et al. 2004; Choi et al. 2009). For this purpose the immunohistochemical expression of the different markers used (GCET1, CD10, MUM1, BCL6 and FOXP1) was scored by two independent pathologists (SMM and JADP) and the percentage of tumor-cell staining was estimated by visual inspection and categorized into the appropriate decile. Disagreements were resolved by joint review using a multihead microscope. Immunohistochemical expression of NFkB-related proteins p100/p52 and p105/p50 was considered positive only when there was nuclear staining (as a surrogate for the activation of alternative and classical NFkB pathways, respectively).
Fluorescent in situ hybridization (FISH) analysis.
Interphase FISH analysis was performed on TMA paraffin sections following conventional protocols.Abbot Molecular Dual color Break Apart DNA probes were used against BCL2 (05N51-20), BCL6 (01N23-020) and C-MYC (01N63-020). 100 cells were counted on each TMA core. Discordant duplicates were reevaluated by two observers (AB, SG).
Clonality analysis.
Clonality assays were performed by conventional methods using DNA extracted from FFPE tissue. In brief, DNA was extracted following conventional protocols and PCR was done in a Veriti 384-well Thermal Cycler (Applied Biosystems). Each case was DNA-extracted and amplified in duplicate. DNA quality was tested using a multiplex control gene set of primers that results in a ladder of five fragments (100, 200, 300, 400 and 60 bp). DNAs with a control gene set amplification ≤200bp were not further analyzed. Multiplex PCR for the detection of clonal VH-JH rearrangements was performed using standardizedBiomed2 primers, as fully described elsewhere(van Krieken et al. 2007). We analyzed IgH complete rearrangements (FR1, FR2, FR3), IgK (Vk-Jk and Kde) and IgL and TCR rearrangements (TCRG-A, TCRG-B, TCRB-A, TCRB-B).
Western blot
Protein from primary frozen diagnostic samples was extracted using RIPA lysis buffer (Sigma-Aldrich Inc., St. Louis, MO, USA) supplemented with protease inhibitors. Protein concentrations were measured using the RCDC Protein Assay (Bio-Rad, Hercules, CA, USA) and 40 μg of protein per well were separated by SDS-PAGE on 6% and 8% acrylamide gels, and Western blot was performed following standard protocols. The following primary antibodies were used: mouse mAb NF-κB p52 (#05-361, Upstate Biotechnology, Lake Placid, NY, USA), mouse mAb NF-κB p50 (sc-8414, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse mAb α-tubulin (Sigma-Aldrich Inc., St. Louis, MO, USA) and rabbit pAb α-tubulin (ab15246, Abcam plc, Cambridge, UK). Detection was carried out using fluorescent-labeled secondary antibodies (Alexa 680 and Alexa 800, Rockland, Gilbertsville, PA, USA) and an Odyssey Infrared System Scanner (LI-COR Biosciences, Lincoln, NE, USA). Western blots were quantified using the ImageJ program (National Institute of Health, Bethesda, MD, USA).