Multiplex Epstein-Barr Virus Genotyping PCR Detects High-Risk Variants in Plasma for Population-Level Nasopharyngeal Carcinoma Screening

Abstract

<h3>Purpose/Objective(s)</h3> Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) is unusually geographically and ethnically restricted. Without screening, most patients present at an advanced stage. Population-level screening in high-risk populations can detect most cases at an early stage, but existing serologic or molecular diagnostics are limited by low positive predictive value secondary to benign EBV reactivation. In contrast to human genome-wide association studies, recent EBV genome-wide association studies have identified polymorphisms with much greater attributable risk for NPC. We hypothesized that a noninvasive molecular diagnostic could detect high-risk EBV haplotypes in plasma to triage patients who screen positive for plasma EBV DNA. <h3>Materials/Methods</h3> We designed a multiplex allele-specific real-time polymerase chain reaction (qPCR) genotyping assay to detect three non-synonymous polymorphisms within the EBV BALF2 gene (162215C > A, 162476T > C, 163364C > T). Two dsDNA gene fragments served as either the high-risk or low-risk variant controls. Supernatant from the EBV-infected B95-8 cell line served as an additional wild-type whole-virus control. Positive clinical specimens sent for routine plasma EBV EBNA-1 testing were collected from patients with a variety of benign and neoplastic EBV-associated disorders. Three sets of conserved primers flanked the polymorphisms, with one allele-specific hydrolysis probe for each of the three high-risk alleles. A fourth allele-specific probe (162215-mt) served as an internal control. The 95% lower limit of detection (LLOD) was assessed in replicates of 20; linearity was assessed from 0.0-6.0 log10 cp/µL. Mutant allele frequencies from 0-100% were assessed by mixing dsDNA controls. <h3>Results</h3> The assay's limit of quantitation and 95% LLOD were 50 and 5 copies per well, respectively. At 100 copies per well, the assay could detect mutant allele frequencies as low as 5%, below the intra-host heterozygosity threshold. Wild-type B95-8 DNA confirmed assay specificity. Forty EBV-positive plasma specimens were genotyped from patients with non-transplant-associated viremia, post-transplant lymphoproliferative disorders, EBV-associated lymphomas (diffuse large-cell, Hodgkin, NK/T-cell, peripheral T-cell), and EBV-positive NPC. High-risk haplotypes were more common among patients with NPC (64% vs. 10%, <i>P</i> = 0.002). Compared to screening with plasma BamHI-W PCR alone, triaging screen-detected EBV DNA with this genotyping assay modestly reduced screening sensitivity (97% to 91%) but could decrease false positives and unnecessary nasoendoscopies by 63% in a modeled high-risk population. <h3>Conclusion</h3> Triaging screen-detected plasma EBV DNA with a multiplex BALF2 genotyping assay has the potential to decrease false positives and resource utilization in high-risk populations screened for NPC. This may be a low-cost and accessible alternative to higher-complexity sequencing assays in low- and middle-income countries.