We examined 30 samples of primary gastric carcinoma resected from randomly selected Korean patients including 21 males and 9 females whose ages ranged from 34~76 years old. The positive and negative controls used for EBV gene expression were EBV positive B lymphoblastoid cell line and the gastric carcinoma cell line, MKN45, respectively. Cells were cultured in RPMI 1640 medium (Gibco BRL) containing 10% fetal bovine serum (Gibco BRL), 100 U/ml of penicillin and 100 µg/ml of streptomycin (Gibco BRL).

DNA PCR was performed using the primers SL18 (5'-GGCGCACCTGGAGGTGGTCC-3') and SL19 (5'-TTTCCAGCAGAGTCGCTAGG-3') to the variable number of tandem repeat (VNTR) in a mixture of 2 µl of 5 mM dNTP, 1.25 U Taq polymerase (Perkin Elmer, Branchburg, NJ) and 10 pmol primer set. PCR was done in a thermal cycler (Perkin Elmer) by carrying out the reaction in 35 cycles of 30 sec at 94℃, 30 sec at 55℃, and 30 sec at 72℃ with 10 min. extension at 72℃. The resulting PCR product was electrophoresed in 2% agarose gel and observed using an UV transilluminator after staining with ethidium bromide.

EBV-encoded small nonpolyadenylated RNAs (EBERs) constitutively expressed in EBV infected cells were used as the probe for in situ hybridization. Each of the 6 µm tissue samples was placed on a sialine coated slide, left for 30 min at 56℃, and placed in xylene twice for 3 min each to remove paraffin. The tissue sample was then placed twice in 99% and 95% ethyl alcohol solutions for 3 min each and hydrated by leaving the sample in distilled water. It was then reacted in Proteinase K (Sigma, St. Louis, MO) at 37℃ for 10 min, washed in distilled water, and completely dried by exposing to air for 20 min. After 40~100 µl FITC-labeled EBERs probe (Dako, Denmark) was placed on the dried tissue sample, it was reacted at 37℃ for 2 h. The sample was placed in tris buffered saline (0.1 M Tris in normal saline; TBS) for 5 min and washed 3 times in TBS containing 0.1% Triton X-100. After 100 µl anti-FITC (Dako) was placed onto the sample slide, the sample was reacted for 30 min at room temperature, washed with TBS, reacted with alkaline phosphatase-conjugated anti-mouse Ig for 30 min, and stained with BCIP/NBT.

In order to determine the type of EBV, PCR was performed using EBNA3C specific primers (sense: 5'-AGAAGGGGAGCGTGTGTTGT-3', antisense: 5'-GGCTCGTTTTTGACGTCGGC-3'). The EBV type was determined based on the fact that type 1 EBV would have an internal deletion at 93 bp, unlike type 2 EBV, so the PCR product of type 1 EBV would be 153 bp, while that of type 2 EBV would be 246 bp (17).

Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to determine the expression of EBV specific genes in gastric cancer tissues. RNA was extracted using the RNeasy kit (Qiagen, Valencia, CA). A 30 µl reaction mixture was prepared by adding 5 µg RNA, 2 µg oligo-dT primer (Pharmacia, Uppsala, Sweden), 2 µl 10 mM dNTP (Boehringer Mannheim, Mannheim, Germany), 6 µl 5× reverse transcriptase buffer, 200 U M-MLV reverse transcriptase (Gibco BRL). cDNA was synthesized at 42℃ for 60 min. RT-PCR was performed using the synthesized cDNA as the template and the primer specific for each gene (18).

Immunohistochemistry was performed using anti-CD4, anti-CD8, and anti-CD20 antibodies to confirm the distribution of infiltrating T lymphocytes and B lymphocytes into gastric carcinoma in paraffin-embedded tissue sections. After leaving 6 µm of gastric carcinoma tissue sample on a sialine-coated slide, paraffin was removed. Add each primary antibody conjugated to alkaline phosphatase was placed onto the slide, the sample was reacted at 37℃ for 1 h and stained with BCIP/NBT.

For TCR Vβ CDR3 RT-PCR, the β chain constant region (Cβ: 5'-TTCTGATGGCTCAAACAC-3') was used as the antisense primer and a specific primer corresponding to each Vβ family was used as the sense primer (19,20). PCR was performed using 4 µl 1.25 mM dNTP, 1.25 U Taq polymerase (Perkin Elmer), and 10 pmol Vβ family specific primer and Cβ primer using cDNA synthesized from RNA extracted from gastric carcinoma tissues. PCR was performed by carrying out the reaction in 30 cycles of 30 sec at 94℃, 30 sec at 55℃, and 30 sec at 72℃, and extension was done at 72℃ for 10 min. After confirming the PCR product by electrophoresis in a 2% agarose gel, it was used as the nested-PCR template to determine the TCR Vβ family. Nested-PCR was performed using 1 µl TCR Vβ PCR product, 1 pmol sense Vβ primer, and ³²P-labelled antisense Cβ primer. After electrophoresis of the PCR product in a sequencing gel, the gel was dried and exposed to Fuji Super HR-G X-ray film (Fuji Photo Film Co., Tokyo, Japan). The band intensity of each Vβ specific RT-PCR product was measured using the image plate and the predominant band was defined when the intensity of a single band was more than 1/3 of the total intensity. The predominant bands considered to be specific T lymphocyte clonal expansion were cut and extract DNA. PCR was performed with the extracted DNA using the same Vβ primer and Cβ primer, and the PCR products were confirmed using electrophoresis in a 2% agarose gel. PCR products of DNA extracted from the gel were cloned in pT7Blue-T vector (Novagen, La Jolla, CA), and the nucleotide sequences were analyzed. At the same time, direct sequencing analysis was done using the PCR product by using the AmpliCycle sequencing kit (Perkin Elmer). The nucleotide sequences of at least 4 of the clones that were cloned into the pT7 Blue-T vector were analyzed. Clonal expansion of a specific T lymphocyte was defined when the clone showing the same result as the direct sequencing of the PCR product was present in more than 2/3 of the total clones. The BLAST server by NCBI was used for Nucleotide database searching and computation.

PCR was performed using DNA extracted from gastric carcinoma tissue samples to confirm the presence of the EBV genome using EBV VNTR specific primers. The EBV genome was contained in gastric tissue samples obtained from 4 (#967, #974, #1010, and #1016) out of 30 patients (13.3%) (Fig. 1A). The fact that the VNTR PCR products of 4 tissue samples were seen as a single band (Fig. 1B) means that each case of gastric carcinoma was infected with a single EBV clonotype. As for VNTR, each EBV strain could have a different number of tandem repeats. Gastric carcinoma tissues with detected EBV genomes were undifferentiated adenocarcinomas according to H-E staining in which severe infiltration of lymphocytes was seen around the gastric carcinoma cells (Fig. 2A). Two (#967, #1016) of the 4 samples with the EBV genome were confirmed as LELC. According to the EBV EBER-1 in situ hybridization results, EBV genomes were observed in the nuclei of gastric carcinoma cells but not in the connective tissues and infiltrating lymphocytes (Fig. 2B).

To investigate the types of EBV infecting gastric carcinoma tissue samples with detectable EBV genomes, PCR was performed using EBNA3C specific primers. The EBNA3C gene shows the most significant difference between Type 1 and Type 2, in which an internal deletion is present in Type 1 EBV (17). The PCR product of Type 1 EBV was153 bp, and that of Type 2 EBV was 246 bp. The PCR results from all four samples showed the same size PCR product as that isolated from the B95.8 Type 1 EBV (Fig. 3), suggesting that all 4 samples were infected with Type 1 EBV.

To determine the type of latent infection present in gastric carcinoma tissues, which contain the EBV genome, the expression profile of EBV latent genes was investigated using RT-PCR. EBNA1, which is the most important for the maintenance of the EBV episome, was expressed in all 4 samples. The EBNA1, which is the gene that is expressed in all types of latent infection and that controls DNA replication of the EBV episome, was present in all samples. BARTs, which are usually expressed during EBV infection was detected in all 4 samples. LMP2a was expressed in one sample (#967) out of 4 samples. EBNA2 was not expressed in all 4 samples, suggesting that the C and W promoters were not activated. Because EBNA2, EBNA-LP, EBNA3A, EBNA3B, and EBNA3C expression is controlled by the C and W promoters, they were not expressed, either. LMP-1, expressed during Type 2 EBV latent infection, was not expressed (Fig. 4).

Immunohistochemistry was performed using anti-CD4, anti-CD8 and anti-CD20 antibodies to investigate the distribution of lymphocytes infiltrating EBV-positive gastric carcinoma tissue samples. Most lymphocytes were stained by anti-CD8 antibodies (Fig. 5A) but not with anti-CD4 and anti-CD20 antibodies (Fig. 5B and C), suggesting that most lymphocytes infiltrating gastric carcinoma tissues were CD8⁺ T cells.

Since the lymphocytes infiltrating gastric carcinoma tissues that contain the EBV genome were CD8⁺ T lymphocytes, TCR Vβ chain usage was analyzed to determine the presence of clonal expansion according to each Vβ family in order to determine whether single clonal or oligoclonal T lymphocytes recognizing a specific antigen were expanded. After performing RT-PCR with Vβ family specific primers, electrophoresis was done in a sequencing gel and CDR3 was sorted according to the different sizes (Fig. 6). After the band intensity of each RT-PCR product was measured according to each Vβ family, the predominant band was defined when the intensity of a single band was more than 1/3 of the total intensity. The predominant band was observed in Vβ family 2, 6 and 11 of T lymphocytes infiltrating sample #967; Vβ family 12 and 14 of sample #974; Vβ family 3, 6, 8, and 15 in sample #1010; and in Vβ family 8 of sample #1016. DNA was extracted from a total of 10 predominant bands and its nucleotide sequence was determined by using PCR-direct sequencing and also by cloning the PCR product with pT7 Blue-T vector at the same time. The two results were then compared. The nucleotide sequence of at least 4 clones that were cloned with pT7 Blue-T vector was analyzed. A specific T cell clone was expanded when the clone was more than 2/3 of the total clones showing the same result as the direct sequencing of the PCR product. Among the selected predominant bands, the nucleotide sequence obtained by direct sequencing was the same as the sequence obtained from the pT7 Blue-T vector with Vβ CDR3 cloned into it in all cases except for Vβ 12 of sample #974; Vβ 3, 6, and 15 of sample #1010; and Vβ 8 of sample #1016. The amino acid sequences deduced from these samples are shown in Table I. The length of CDR3 was determined by subtracting 4 amino acids from the number of amino acids present between the C-terminal cysteine in the Vβ region and the first glycine present on glycine/X/glycine in the J region. The CDR3 length was between 8 and 11, falling in the normal range of TCR Vβ CDR3. Its average was 9.6, which is similar to 9.5, the average length of TCR Vβ. When the Vβ family gene was searched using BLAST to confirm Jβ and C family, the result showed 3 clones using the same Jβ 2.3 TCR. Arginine was synthesized by N-addition in the V-D region of TCR Vβ of T lymphocytes infiltrating gastric carcinoma tissue samples #974 and #1010.