Comparison of Multiplex Real-Time PCR and Droplet Digital PCR for Detection of Plasma Epstein-Barr Virus DNA in Nasopharyngeal Carcinoma

Abstract

Plasma Epstein-Barr Virus (EBV) DNA is an established biomarker for endemic nasopharyngeal carcinoma (NPC) and EBV-associated lymphoproliferative disorders. The ongoing NRG-HN001 randomized trial is testing one biomarker-directed treatment approach using real-time PCR (qPCR). Existing qPCR assays are limited by inter-laboratory imprecision secondary to external calibrators, and development of a standardized assay would facilitate reproducible quantitation. We hypothesized that droplet digital PCR (ddPCR) would have similar analytical sensitivity, precision, and accuracy relative to qPCR.We designed a multiplex ddPCR assay to detect two single-copy EBV targets (EBNA-1, EBER), a variable-repeat EBV target (BamHI-W), and a human internal control (RNaseP) in plasma across two reactions. This was compared against the NRG-HN001 qPCR assay, which detects EBNA-1, BamHI-W, and an internal control across two reactions. Quantitation for the ddPCR assay was based on Poisson statistics and automated threshold determination without external calibrators. Quantitation for the qPCR assay was based on two external calibrators traceable to the WHO EBV standard. Assays were compared by measuring sensitivity, specificity, 95% lower limit of detection (LLOD), lower limit of quantitation (LLOQ), accuracy (difference from nominal concentration), and precision (reproducibility). The WHO EBV standard was used as control material. Clinical specimens sent for routine plasma EBV testing were collected from patients with EBV-associated neoplasms. For clinical specimens, a composite reference standard was defined as positivity by at least two targets.The 95% LLOD for BamHI-W, EBNA-1, and EBER ddPCR were 1.49, 13.26, and 9.77 international units (IU) per well, respectively. Analytical sensitivity was not significantly different than BamHI-W qPCR (1.75 IU/well, P = 0.99) or EBNA-1 qPCR (7.58 IU/well, P = 0.60). The LLOQ for both assays was 100 IU/mL. Across the range of prognostic cutoffs for plasma EBV DNA copy number (1,500-40,000 IU/mL), within-laboratory precision was highest with BamHI-W qPCR (0.04-0.06) and ddPCR (0.04-0.08) and lower with EBNA-1 qPCR (0.04-0.14). Across this range, ddPCR had modestly improved accuracy (absolute mean difference from nominal, BamHI-W: 1104 IU/mL; EBER: 1206 IU/mL) relative to qPCR (BamHI-W: 1565 IU/mL; EBNA-1: 1874 IU/mL, P = 0.77 across platforms). Sensitivity (100% vs. 95%, P = 0.50) and specificity (100% vs. 100%) were similar between BamHI-W ddPCR and BamHI-W qPCR for clinical specimens. Sensitivity was lower for all single-copy targets (80-85%).A multiplex ddPCR amplifying single- and variable-copy EBV targets in plasma offers similar analytical performance as the existing NRG-HN001 qPCR assay without the need for external calibrators. Further research will inform whether inter-laboratory precision is superior with ddPCR, which may facilitate standardization of EBV DNA quantitation.