The sequence analysis of Epstein-Barr virus EBNA1 gene: could viral screening markers for nasopharyngeal carcinoma be identified?

Epstein-Barr virus (EBV) has been identified as a group 1 carcinogenic agent, particularly for nasopharyngeal carcinoma (NPC). The sequence diversity of EBV nuclear antigen 1 (EBNA1) reflects region-restricted polymorphisms, which may be associated with the development of certain malignancies. The aims of the present study were to evaluate EBV EBNA1 gene polymorphisms circulating in NPC, infectious mononucleosis, and isolates from patients with transplanted organs to determine if EBNA1 sequence specificities are useful as viral biomarkers for NPC. Forty biopsies of undifferentiated carcinoma of nasopharyngeal type (UCNT), 31 plasma samples from patients with mononucleosis syndrome, and 16 plasma samples from patients after renal transplantation were tested in this study. The EBNA1 gene was amplified by nested PCR. Further investigation included sequencing, phylogenetic, and statistical evaluations. Eighty-seven sequences were identified as one of the four EBNA1 subtypes, P-Ala, P-Thr, V-Val, and V-Ala, with further classification into ten subvariants. Of these, P-Thr-sv-1 and P-Thr-sv-3 have never been identified in Europe, while V-Val-sv-1 was newly discovered. Statistical analysis revealed significant differences in the distribution of EBNA1 P-Thr subvariants between the three groups of patients, with noticeable clustering of P-Thr-sv-5 in NPC isolates (p < 0.001). EBV EBNA1 showed no sequence specificity in primary infection. This research revealed a newly discovered EBNA1 subvariant. Importantly, EBNA1 P-Thr-sv-5 showed carcinoma-specific EBNA1 variability. Thus, identification of this subvariant should be considered as a viral screening marker for NPC or UCNT.

Characterization of the Variability of Epstein-Barr Virus Genes in Nasopharyngeal Biopsies: Potential Predictors for Carcinoma Progression.

Epstein-Barr virus (EBV) infection is a significant factor in the pathogenesis of nasopharyngeal carcinoma, especially in the undifferentiated carcinoma of nasopharyngeal type (UCNT, World Health Organization type III), which is the dominant histopathological type in high-risk areas. The major EBV oncogene is latent membrane protein 1 (LMP1). LMP1 gene shows variability with different tumorigenic and immunogenic potentials. EBV nuclear antigen 1 (EBNA1) regulates progression of EBV-related tumors; however, the influence of EBNA1 sequence variability on tumor pathogenesis is controversial. The aims of this study were to characterize polymorphisms of EBV genes in non-endemic nasopharyngeal carcinoma biopsies and to investigate potential sequence patterns that correlate with the clinical presentation of nasopharyngeal carcinoma. In total, 116 tumor biopsies of undifferentiated carcinoma of nasopharyngeal type (UCNT), collected from 2008 to 2014, were evaluated in this study. The genes EBNA2, LMP1, and EBNA1 were amplified using nested-PCR. EBNA2 genotyping was performed by visualization of PCR products using gel electrophoresis. Investigation of LMP1 and EBNA1 included sequence, phylogenetic, and statistical analyses. The presence of EBV DNA was significantly distributed between TNM stages. LMP1 variability showed six variants, with the detection of the first China1 and North Carolina variants in European nasopharyngeal carcinoma biopsies. Newly discovered variants Srb1 and Srb2 were UCNT-specific LMP1 polymorphisms. The B95-8 and North Carolina variants are possible predictors for favorable TNM stages. In contrast, deletions in LMP1 are possible risk factors for the most disfavorable TNM stage, independent of EBNA2 or EBNA1 variability. A newly discovered EBNA1 subvariant, P-thr-sv-5, could be a potential diagnostic marker, as it represented a UCNT-specific EBNA1 subvariant. A particular combination of EBNA2, LMP1, and EBNA1 polymorphisms, type 1/Med/P-thr was identified as a possible risk factor for TNM stage IVB or progression to the N3 stage.