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

Distribution of three LMP1 characteristics and EBNA1 subtypes in EBV isolates from NPC biopsies.

<p>Distribution of three LMP1 characteristics and EBNA1 subtypes in EBV isolates from NPC biopsies.</p

EBNA1 C-terminal nucleotide and amino acid changes found in three subtypes and seven subvariants identified in this study.

<p>EBNA1 C-terminal nucleotide and amino acid changes found in three subtypes and seven subvariants identified in this study.</p

Alignment of obtained LMP1 sequences isolated from UCNT biopsies.

<p>B95-8* represents aa sequence of the prototype LMP1. Seven characteristic aa positions for variant discrimination, described by Edwards et al. (1999) are underlined. China1 and NC isolates showed additional representative aa changes which were not listed in known classification: China1 (position 322 and 338) and NC (position 338). Of 85 aa substitutions which were identified at additional 58 positions, several were unique for single variant: H→R at 352 for Med-, E→Q at 328 and S at 309 for B95-8, and D→N at 250 for NC.</p

Phylogenetic trees of the C-termini of LMP1 and EBNA1.

<p>(a) Thirty five 506-bp fragments of LMP1 (from coordinates 168719–168213) NPC sequences available in GenBank/EMBL7DDBJ database with accession numbers: JF901794-JF901802, JN971085-JN971091 and KT820429-KT820448 and 13 sequences obtained from GenBank/EMBL7DDBJ database under the following accession numbers: V01555, AY493742, AY493743, AY337721, AY337722, AY493810, AY337723, AY493835, AY337724, AY493799, AY337725, AY337726 and X58140. (b) Forty 329-bp fragments of EBNA1 (from coordinates 109261–109590) NPC sequences available in GenBank/EMBL7DDBJ database with accession numbers KT820449-KT820488 and 10 sequences obtained from GenBank/EMBL7DDBJ database under the following accession numbers: V01555, GU475455, JN986939, AF192742, GU475448, AF192743, GU475431, AF192744, JN986947 and GU475442.</p

Combinations of TNM stages found in UCNT biopsies.

<p>Combinations of TNM stages found in UCNT biopsies.</p