Characteristics of epstein barr virus variants associated with gastric carcinoma in Southern Tunisia

Abstract

Background: EBV-associated Gastric Carcinoma (EBVaGC) has a distinct clinical features and its prevalence is variable worldwide.

Results: To determine the prevalence of EBVaGC in Tunisia, EBV-encoded small RNA (EBER) expression was assessed in 81 gastric carcinoma (GC) specimens. The nuclear EBER expression was detected in 12 out of 81 GC cases (14.81%) and concordance between the score range of EBER staining and the number of EBV DNA copies as estimate by QPCR is observed. On the other hand, we found that EBVaGC strongly correlated with age at diagnosis, and weakly with tumor differentiation and venous invasion.Furthermore, the EBVaGC specimens were subjected to determine the EBV DNA polymorphisms. Our results show a unique genetic profile of the EBV strains regarding the A and D types, the F prototype, the retention of XhoI restriction site and the 30 bp del-LMP1 variant. According to our previous studies on nasopharyngeal carcinoma (NPC), we suggested that EBV strains associated to GC and NPC shared some similarities in Tunisian patients.

Conclusion: The prevalence of EBVaGC is of 14.81% in the southern Tunisia and that common EBV strain are associated with both NPC and GC which are likely to differ from Asian strains. Our findings support therefore a certain geographical distribution of EBV strains which is not restricted to EBV-associated malignancies.

Figure 1

Detection of Epstein-Barr virus encoded small RNAs (EBERs) by in-situ hybridization in gastric carcinoma tissues. A: H&E staining. B: Intense EBER positive staining (immuno-score 3+) in the nuclei of tumor cells (original magnification × 40). C: Moderate EBER positive staining (immuno-score 2+) in the nuclei of tumor cells (original magnification × 40). D: Weak EBER positive staining (immuno-score 1+) in the nuclei of tumor cells (original magnification × 40). E: Positive control represented by a known EBER-positive NPC tissues (original magnification × 40). F: Gastric carcinoma tissues negative for EBER (original magnification × 10).

Figure 2

Determination of EBV DNA copy number by Q-PCR. Representative examples of GC cases with high (GC10), intermediate (GC3) and low (GC8) EBV DNA copies number. The horizontal line marks the threshold used for assessment of C_t value.

Figure 3

Genotyping of EBVaGC strains. A: PCR -RFLP of the BamHI-F region showing the f variant (presence of two bands of 128 bp and 71 bp) for the control C666-1 cell line (lane 1) and one band of 199 bp for the F variant for cases GC2 to GC6 (lane 2 to 6). B: PCR-RFLP of the BamHI-W1/I1 region. DNA fragments were digested with BamHI giving two bands of 139 and 67 bp (type D) for GC2 to GC6 (lane 2 to 6) or one band of 245 bp (type C) for the control C666-1 cell line (lane 1). C: EBNA3C region. EBV strains of types A and B correspond to DNA fragment of 153 and 246 bp respectively. Case GC6 shows dual infection with both types A and B viruses. B95.8 is a type A virus that was used as a control (lane 1). D: XhoI polymorphism in exon 1 of the BNLF1 gene. PCR product was digested by XhoI to yield two bands of 343 bp and 154 bp in XhoI+ variant for GC2 to GC6 (lane 2 to 6). C666-1 cell line is a positive of loss-XhoI variant (lane 1).

Figure 4

Comparison of deduced amino acid sequences from Tunisian GC with B95.8 prototype and previously published LMP1 sequences: Cao: NPC cell line from Shangai [27]; C666-1 (GenBank accession No. ABV54173) NPC 10 from Hong Kong [28]and Tunisian NPC specimens: CV4, CV5, CV6 [29]. Symbols (---) indicate amino acid deletion.