RESUMEN
PURPOSE: Antibodies to select Epstein-Barr virus (EBV) proteins can diagnose early-stage nasopharyngeal carcinoma (NPC). We have previously shown that IgA against EBV nuclear antigen 1 (EBNA1) can predict incident NPC in high- and intermediate-risk cohorts 4 years pre-diagnosis. Here, we tested EBNA1 variants, with mutants, to define the sequence requirements for an NPC risk assay. DESIGN: Mammalian-expressed constructs were developed to represent EBNA1 variants 487V and 487A which can differ by ≥15 amino acids in the N- and C-termini. Denatured lysates were evaluated by a refined IgA and IgG immunoblot assay in a case-control study using pre-diagnostic NPC sera from two independent cohorts in Singapore and Shanghai, P.R. China. RESULTS: At 95% sensitivity, 487V yielded a 94.9% specificity compared to 86.1% for 487A. EBNA1 deleted for the conserved glycine-alanine repeats (GAr) reduced false positives by 22.8%. NPC sera reacted more strongly to the C-terminus than healthy controls, but the C-terminal construct (a.a. 390-641) showed lower specificity (84.8%) than the EBNA1 GAr deleted construct (92.4%) at 95% sensitivity. CONCLUSION: Although EBNA1 IgA was present in healthy sera, most epitopes localized to the immunodominant GAr. We conclude that a refined EBNA1 antigen deleted for the GAr but with residues consistently detected in Southeast Asian NPC tumors is optimal for risk prediction with an extended sojourn time of 7.5 years. Furthermore, distinct EBNA1 serologic profiles enhanced the utility of the EBNA1 IgA assay for risk stratification. This illustrates the importance of serologically relevant EBNA1 sequences for NPC risk prediction and early detection.
RESUMEN
Genetic variants in Epstein-Barr virus (EBV) have been strongly associated with nasopharyngeal carcinoma (NPC) in South China. However, different results regarding the most significant viral variants, with polymorphisms in EBER2 and BALF2 loci, have been reported in separate studies. In this study, we newly sequenced 100 EBV genomes derived from 61 NPC cases and 39 population controls. Comprehensive genomic analyses of EBV sequences from both NPC patients and healthy carriers in South China were conducted, totaling 279 cases and 227 controls. Meta-analysis of genome-wide association study revealed a 4-bp deletion downstream of EBER2 (coordinates, 7188-7191; EBER-del) as the most significant variant associated with NPC. Furthermore, multiple viral variants were found to be genetically linked to EBER-del forming a risk haplotype, suggesting that multiple viral variants might be associated with NPC pathogenesis. Population structure and phylogenetic analyses further characterized a high risk EBV lineage for NPC revealing a panel of 38 single nucleotide polymorphisms (SNPs), including those in the EBER2 and BALF2 loci. With linkage disequilibrium clumping and feature selection algorithm, the 38 SNPs could be narrowed down to 9 SNPs which can be used to accurately detect the high risk EBV lineage. In summary, our study provides novel insight into the role of EBV genetic variation in NPC pathogenesis by defining a risk haplotype of EBV for downstream functional studies and identifying a single high risk EBV lineage characterized by 9 SNPs for potential application in population screening of NPC.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Genoma Viral , Herpesvirus Humano 4 , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Femenino , Humanos , Masculino , China/epidemiología , Pueblos del Este de Asia , Infecciones por Virus de Epstein-Barr/virología , Infecciones por Virus de Epstein-Barr/genética , Variación Genética , Estudio de Asociación del Genoma Completo , Herpesvirus Humano 4/genética , Carcinoma Nasofaríngeo/virología , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/virología , Neoplasias Nasofaríngeas/genética , Filogenia , Polimorfismo de Nucleótido SimpleRESUMEN
Nasopharyngeal carcinoma (NPC), a cancer that is etiologically associated with the Epstein-Barr virus (EBV), is endemic in Southern China and Southeast Asia. The scarcity of representative NPC cell lines owing to the frequent loss of EBV episomes following prolonged propagation and compromised authenticity of previous models underscores the critical need for new EBV-positive NPC models. Herein, we describe the establishment of a new EBV-positive NPC cell line, designated NPC268 from a primary non-keratinizing, differentiated NPC tissue. NPC268 can undergo productive lytic reactivation of EBV and is highly tumorigenic in immunodeficient mice. Whole-genome sequencing revealed close similarities with the tissue of origin, including large chromosomal rearrangements, while whole-genome bisulfite sequencing and RNA sequencing demonstrated a hypomethylated genome and enrichment in immune-related pathways, respectively. Drug screening of NPC268 together with six other NPC cell lines using 339 compounds, representing the largest high-throughput drug testing in NPC, revealed biomarkers associated with specific drug classes. NPC268 represents the first and only available EBV-positive non-keratinizing differentiated NPC model, and extensive genomic, methylomic, transcriptomic, and drug response data should facilitate research in EBV and NPC, where current models are limited. SIGNIFICANCE: NPC268 is the first and only EBV-positive cell line derived from a primary non-keratinizing, differentiated nasopharyngeal carcinoma, an understudied but important subtype in Southeast Asian countries. This model adds to the limited number of authentic EBV-positive lines globally that will facilitate mechanistic studies and drug development for NPC.