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 paraffinembedded 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 mM citrate 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 standardized Biomed2
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).