Formalin-fixed, paraffin-embedded tissue blocks from 59 invasive breast carcinomas were retrieved from the files of the Institute for Pathology, Friedrich-Alexander University, Erlangen, Germany. These included 30 invasive ductal carcinomas, 26 invasive lobular carcinomas, two medullary carcinomas and one undifferentiated carcinoma. Among these 59 carcinomas, 50 cases were classified as grade 2, seven cases as grade 3 and two cases were identified as grade 1. The age of the patients ranged from 30 to 89 years, and all patients were female.
For control, paraffin sections from EBV-positive, undifferentiated nasopharyngeal carcinomas were used.
Plasmids, probes, and in situhybridisation
The plasmids harbouring inserts specific for the small EBV-encoded nuclear RNAs (EBER1 and EBER2) have been described previously . Single-stranded anti-sense and sense RNA probes were obtained from these plasmids by in vitro transcription in the presence of 35S-labelled UTP . EBER in situ hybridisation was carried out as described in detail elsewhere . In brief, paraffin sections were dewaxed and rehydrated through graded ethanols, treated with 0.2 NHCl for 20 min, digested with 0.5 mg/ml pronase (Boehringer, Mannheim, Germany) for 10 min, fixed in cold 4% paraformaldehyde for 20 min, and acetylated using a freshly prepared 1:400 solution of acetic anhydride in 0.1 M triethanolamine (pH8.0) for 10 min. Sections were then dehydrated through graded ethanols and air dried. Approximately 50 μl hybridisation mixture (50% deionised formamide/10% dextran sulfate/2 × standard saline citrate [SSC]/0.2 mg/ml yeast tRNA/10 mM dithiothreitol [DTT]), including 1 × 105 cpm labelled probe, were applied to each section. After hybridisation overnight at 50°C, sections were washed in 50% formamide/1 × SSC/10 mM DTT at 52°C for 4 hours. To remove nonspecifically bound probe, sections were subjected to RNase A digestion (Sigma, Steinheim, Germany; 20 μg/ml, 30 min, 37°C). Sections were then rinsed in 2 × SSC and 0.1 × SSC, dehydrated through graded ethanols and dipped into Ilford G5 emulsion (Ilford, Mobberley, Cheshire, UK).
For the detection of EBV DNA, a plasmid harbouring the BamHI W internal repetitive fragment of the EBV genome was used . Total plasmid DNA was labelled with 35S-dCTP by nick translation using a commercially available kit (Amersham Pharmacia Biotech, Freiburg, Germany). Following the nick translation reaction, labelled DNA was separated from unincorporated nucleotides using Sephadex columns (Nick columns; Amersham Pharmacia Biotech) according to the supplier's instructions. Prehybridisation treatment was essentially the same as for RNA in situ hybridisation, with minor modifications as described previously . Approximately 50 μl hybridisation mixture (50% deionised formamide/10% dextran sulfate/2 × SSC/30 μg/ml herring sperm DNA/10 mM DTT) containing 40–50 ng/ml labelled probe were added per slide. Sections were covered with siliconised coverslips. Probe and cellular DNA were denatured by placing slides onto a 90°C heat block for 3 min. Hybridisation was carried out overnight at 37°C in an atmosphere of 50% formamide. Slides were washed in 50% formamide/0.1 × SSC/10 mM DTT at 37°C for 4 hours, followed by 2 × SSC/10 mM DTT and 0.1 × SSC/10 mM DTT, both for 30 min at room temperature. Sections were dehydrated through graded ethanols, air dried and dipped as already described. For the detection of EBERs and EBV DNA, sections were exposed at 4°C for 3–12 days.
Paraffin sections were dewaxed in xylene for 30 min at room temperature and rehydrated through graded ethanols (5 min each in 100%, 96% and 70% ethanol and water). The EBNA1-specific rat monoclonal antibodies, 1H4 and 2B4, were kindly provided by Elisabeth Kremmer, Munich . Bound primary antibodies were detected using a biotinylated rabbit antiserum specific for rat immunoglobulins, streptavidin biotinylated peroxidase complex ABC–PO and a tyramide signal amplification protocol followed by detection of precipitated biotin using alkaline phosphatase-labelled ABC (all Dako, Hamburg, Germany). Fast Red (Sigma, Steinheim, Germany) served as a chromogen.
EBV DNA PCR
For PCR, one 10 μm paraffin section from each case was dewaxed in xylene for 30 min at room temperature, rehydrated through graded ethanols (5 min each in 100%, 96% and 70% ethanol) and air dried. Tissues were incubated in 200 μl buffer (50 mM KCl, 10 mM Tris-HCl [pH8.3], 2.5 mM MgCl, 0.1 mg/ml gelatine, 0.45% NP-40, 0.45% Tween-20) and 0.2 mg/ml proteinase K overnight at 56°C, followed by heat inactivation of proteinase K.
Five microlitre aliquots of DNA extracts were subjected to PCR: 5 min denaturation at 94°C, 10 cycles of 20 s at 94°C, 20 s at 58.3°C and 30 s at 72°C, followed by 25 cycles of 20 s at 94°C, 20 s at 53.3°C and 30 s at 72°C, completed by 150 s at 72°C. The reaction volume was 50 μ l containing 5 μ l DNA, 1 × polymerase buffer, 1.5 mM MgCl, 200 μ M dNTP, 1 U Taq-polymerase and 400 nM primer mix (5'-CAC TTT AGA GCT CTG GAG GA-3' and 5'-TAA AGA TAG CAG CAG CAC AG-3'). The primers resulted in the amplification of a 153 base pair fragment of the EBV BamHI W internal repetitive fragment. For control, a 303 base pair fragment of the β-globin gene was amplified using published primers. The PCR products were analysed by electrophoresis using 2% agarose gels.