Formalin-fixed and paraffin-embedded blocks from eighty-seven surgically resected gastric specimens (40 cases with gastric adenocarcinoma, 34 normal tissue adjacent to tumor, 13 metastatic lymph nodes) were studied. Clinical data were obtained from clinical records.

A total of 40 cases (30 men and 10 women) were studied. All patients were diagnosed with gastric cancer between April 1995 and April 1997 at Kangnam St. Mary’s hospital. Median age was 60 years. 29 cases (72.5%) were in the antrum and 7 cases (17.5%) in the cardia. Most lesions were located at the lesser curvature (77.5%) and 5 cases in the greater curvature (Table 1). According to the Lauren classification⁷⁾, 40 cases consisted of 20 intestinal type, 10 diffuse type, and 10 mixed type. 22 patients had moderate differentiated adenocarcinoma, and 14 patients had poorly differentiated adenocarcinoma. Lymphatic invasion was found in 34 cases (85%), and vascular invasion was found in 8 cases (20%) (Table 2).

We obtained sixteen benign ulcer tissues from gastrofiberscopic biopsy specimen at St. Mary’s hospital between February 2000 and March 2000 as the control group, and subjected to these specimens to the same procedure.

Paraffin-embedded blocks were cut into 10 μm thick sections and H-E stained for microdissection of the tumor tissue under light microscopy. These microdissected tumor tissues were put into 1.5 mL microcentrifuge tubes, and were treated with 0.5 mL TaKaRa DEXPAT™ (cat. #9091, TaKaRa, Japan) to extract DNA as the manufacturers’ instructions. After incubation at 100°C for 10 minutes, the samples were centrifuged at 12,000 rpm for 10 minutes. The supernatant containing DNA was stored at −20°C and then used as the template for the PCR. The primer for PCR was a 129 bp region of the internal repeat, EBNA-1 (Epstein-Barr virus nuclear antigen-1), described in Table 3. The template DNA 1 μg was mixed with 10 X buffer (100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl₂), 10 mM dNTP mixture, and 1.5 μU Taq polymerase (TaKaRa, Japan). We added the primer to this mixture, and then performed PCR (MJ Research Inc. USA). The prepared samples were denatured at 95°C for 5 minutes and were subjected to 35 cycles of amplification. Each cycle consisted of 30 seconds of denaturation at 95°C, 30 seconds of annealing at 58°C, and 1 minute of extension at 72°C. Finally, the mixture samples were extended at 72°C for 5 minutes. We used the B95-8 cell line (EBV-transformed leukocytes) as a positive control, and a mixture of samples without DNA served as a negative control. The products of PCR were electrophoresed in a 2% agarose gel, and then stained with ethidium bromide.

We used CS1-4 monoclonal antibodies (DAKO, clone CS1-4, Denmark) for the LMP1, DO-7 monoclonal antibodies (DAKO, clone DO-7, Japan) for p53, and the variant-specific MAbs (R&D system, Abingdon, UK) for CD44. Formalin-fixed, paraffin-embedded sections were dewaxed and rehydrated in graded alcohol. After ending endogenous peroxidase activity with H₂O₂, sections were microwave-irradiated for 10 minutes in pH 6.0 citric acid buffer. After incubating for 120 minutes at room temperature, sections were mixed with pepsin for 10 min, and then added to acid alcohol. A primary antibody was applied for 30 minutes at 40°C and then rinsed with automaffin buffer. After the secondary antibody was applied for 10 minutes at 40°C, the avidin-alkaline phosphate procedure was applied for 15 minutes at 45°C. Finally, the sections were visualized by chromogen at 45°C and counterstained by Hematoxylin. The antibody for LMP1 was diluted 1:100 and other antibodies were diluted 1:50. The result was interpreted by two pathologists separately.

EBV was detected in 4 of 40 cases (10%) of gastric adenocarcinoma by PCR (Figure 1), but not in the benign ulcer cases.

With regard to LMP1, CD44 and p53 expression, all 4 EBV-positive cases were negative, but 9 and 15 cases were positive for p53 and CD44, respectively, in the 36 EBV-negative group (Table 4). However, EBV was not detected in 34 adjacent normal tissues and all these cases were negative in immunohistochemical staining. EBV was detected in only 1 of 13 metastatic lymph nodes, and that was from an EBV-positive case (Table 5).

In the 4 EBV-positive cases, 3 patients were men and other 1 patient was a woman. The lesions were located at the lesser curvature (2 antrum and 2 cardia). Histologically, 2 cases were moderately differentiated carcinoma and the other 2 cases were poorly differentiated carcinoma with lymphoepithelioma-like lesions (Table 6).