Plasma Epstein-Barr Virus (EBV) DNA is an established biomarker for endemic nasopharyngeal carcinoma (NPC). However, existing real-time PCR (qPCR) assays are limited by poor inter-laboratory reproducibility. This is a barrier to biomarker integration into staging systems and management. It was hypothesized that EBV digital PCR (dPCR) would have similar sensitivity but improved precision relative to qPCR. Using the WHO EBV standard and patient specimens, the NRG BamHI-W qPCR, two commercial EBNA-1 qPCR assays, and two laboratory-developed dPCR assays amplifying the BamHI-W, EBNA-1, and EBER targets were compared. Testing was conducted in the North American reference laboratory for the NRG-HN001 randomized trial. The EBV dPCR assays achieved similar performance compared with qPCR. Although dPCR does not require quantitation standards, different dPCR thresholding algorithms yielded significant qualitative and quantitative variation. This was most evident with low levels of EBV DNA. No-template control-informed thresholding (ddpcRquant) mitigated false positives/negatives. The NRG BamHI-W qPCR and laboratory-developed BamHI-W droplet dPCR offered higher sensitivity, lower limit of blank, higher precision at low plasma EBV DNA levels (=1500 IU/mL), and higher overall agreement with clinical specimens compared to single-copy qPCR/dPCR targets (EBNA-1/EBER). These data confirm the rationale for the use of the BamHI-W target to define prognostic thresholds, and indicate that both qPCR and dPCR methods harmonized to the WHO standard can provide the necessary analytical performance.
View details for DOI 10.1016/j.jmoldx.2023.03.007
View details for PubMedID 37068736