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

Abstract

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.

NCCN Guidelines Insights: Head and Neck Cancers, Version 1.2018

“…8,9 Workup for NPC may include EBV testing of both the tumor itself and the blood, particularly in the presence of nonkeratinizing and undifferentiated histology. [10][11][12] Testing methods for detection of EBV in the tumor include in situ hybridization for EBV-encoded RNA 13 and immunohistochemical staining for LMP1. 14 The former tends to be a more sensitive testing method for carcinomas, relative to LMP1 immunohistochemical staining.…”

The prognostic value of volumetric reduction of the target lesions after induction chemotherapy in locoregionally advanced nasopharyngeal carcinoma

“…6 Recently, many other kinds of prognostic factors have been found for NPC, including Epstein-Barr virus oncogene subvariant; leucinerich repeats and immunoglobulin-like domains gene status and its mRNA and protein expression levels; the maximum standard uptake value, metabolic tumor volume, and total lesion glycolysis of positron emission tomography/computed tomography; and tumor diameter. [7][8][9][10][11] However, the majority of these factors are not suitable for clinical practice for various reasons, such as having a high cost or low specificity. A more accurate appraisal system is urgently needed.…”

Epstein Barr Virus Associated Lymphomas and Epithelia Cancers in Humans

“…EBV can be classified into two main genotypes, A and B, or types 1 and 2, based on sequence divergence in the nuclear proteins, Epstein Barr Nuclear Antigen (EBNA)-1, -2, -3A, -3B, and 3C [22]. However, EBNA-2 is generally used for the classification because the protein has the least percentage of sequence homology between the two genotypes [23,24].…”

“…The two genotypes also vary in biological properties; EBV type 1 is more efficient in immortalizing B cells and the type 2 has a higher lytic ability [26,27]. The virus is further classified into seven different strains: China 1 (C1), China 2 (C2), China 3 (C3), Mediterranean with (Med+) or without (Med-) deletions, Alaskan (AL), and North Carolina, based on nucleotide polymorphism, 30 bp deletions, or signature amino acid changes in the LMP1 gene as compared to the sequence of a prototype EBV, B95-8 [22,28]. The different strains show diversity in tumorigenic activity; a 30 bp deletion at the carboxyl terminal, for example, has a higher tumorigenic activity and poor immunogenicity compared to the undeleted variant [29].…”

“…EBV, which is the main cause of UCNT, has been classified as group 1 carcinogenic agent by the International Agency for Research and Cancer (IARC) [22]. The viral infection in NPC epithelia cells is clonal in origin; developing from clonal proliferation of single EBV infected epithelia cell.…”