A study conducted from January 2014 to June 2024 assessed circulating Epstein-Barr virus (EBV) DNA (cfEBV DNA) in nasopharyngeal carcinoma (NPC) patients at three hospitals in China. Researchers used two methods—concentrated precipitation (CP) and magnetic bead (MB)—to extract nucleic acids from plasma, followed by RT-qPCR for detection. Cohort 1 (2014–2021) utilized the CP method, while Cohort 2 (2022–2024) employed MB. Both cohorts included age-sex matched healthy controls. The study evaluated the sensitivity, positivity rates, and diagnostic values of cfEBV DNA检测 in NPC, as well as its clearance dynamics. Additionally, inter-laboratory consistency and digital PCR for low-concentration samples were analyzed. Statistical methods included ROC curves, Mann-Whitney U-tests, and half-life calculations. The research was approved by the institutional ethics committee and adhered to the Declaration of Helsinki.
From January 2014 to June 2024, a retrospective study was conducted on treatment-naïve nasopharyngeal carcinoma (NPC) patients at three medical institutions in China: Sichuan Cancer Hospital, West China Hospital, and Chongqing University-Three Gorges Hospital. The study included patients diagnosed with NPC through histopathological examination and confirmed by pre-treatment plasma Epstein-Barr virus (EBV) DNA detection using concentrated precipitation (CP) or magnetic bead (MB) based reverse transcription quantitative polymerase chain reaction (RT-qPCR). Clinical data, treatment details, and dynamic changes in cell-free EBV (cfEBV) DNA were retrieved from electronic medical records.
Prior to initiating anticancer therapy, all participants underwent comprehensive physical examinations and laboratory analyses. Patients diagnosed with two or more types of malignant cancers were excluded. Additionally, cfEBV DNA was detected in age- and sex-matched healthy controls using the same CP- or MB-based RT-qPCR methods.
The study design incorporated two distinct cohort studies, as illustrated in Figure 1. Cohort 1, a multicenter study performed from January 2014 to December 2021, utilized CP-based RT-qPCR to detect cfEBV DNA. This cohort included 1,405 NPC patients and 244 healthy controls. In contrast, Cohort 2, conducted from January 2022 to June 2024, consisted of 683 NPC patients and 303 healthy controls, with cfEBV DNA detected using MB-based RT-qPCR. Both cohorts employed identical detection protocols for healthy controls. The study aimed to evaluate the diagnostic sensitivity of cfEBV DNA, its positive detection rate, and the comparative clinical utility between the two cohorts.
Nucleic acid extraction from plasma samples was performed using either the CP or MB method, following the manufacturer’s instructions for EBV viral nucleic acid extraction kits (Sansure Biotech Co. Ltd., Hunan, China). For CP-based RT-qPCR, nucleic acid extraction involved blending plasma samples with a concentration agent, centrifugation, and subsequent addition of a releasing agent. In contrast, MB-based extraction utilized magnetic beads and a series of steps involving nucleic acid extraction solutions, proteinase K, and washing solutions to isolate cfEBV DNA.
Following extraction, RT-qPCR was performed to amplify the BamHI-W fragment region of the EBV genome, using specific primers and a fluorescent probe labeled with FAM. The reaction was conducted in an ABI7500 real-time PCR system (Thermo Fisher Scientific, USA), with thermal cycling parameters optimized for sensitivity and specificity. Standard curves for cfEBV DNA quantification were generated as previously described.
To ensure interlaboratory consistency, plasma samples from 45 NPC patients were divided and analyzed independently by Sichuan Cancer Hospital and Sun Yat-Sen University Cancer Center using MB-based extraction, assessing correlation and consistency across a wide range of cfEBV concentrations.
For samples with RT-qPCR results below 400 copies/mL, digital PCR was employed to achieve higher sensitivity. This process utilized the EBV Nucleic Acid Quantification Test Kit (Rainsure Scientific, China) and SG-2000 PCR Amplifying Apparatus, with reactions run in a droplet-based format. Results were analyzed using specialized software (GeneCount Analysis System v1.63.0222).
Statistical analyses included categorical variable comparisons using the Chi-square test, continuous variable comparisons via the Mann-Whitney U-test, and construction of receiver operating characteristic (ROC) curves to determine sensitivity, specificity, and area under the curve (AUC) for predictive performance. The percentage of cfEBV DNA clearance following treatment (PEC) was calculated based on the proportion of patients achieving undetectable cfEBV DNA levels. The plasma EBV DNA half-life (t1/2) was calculated using the formula [t1/2 = 0.693/k], derived from the exponential decay model of plasma EBV DNA concentration over time. All statistical analyses were performed using GraphPad Prism 8.0.2 or SPSS 27.0 (IBM, USA), with statistical significance set at P < 0.05.
The study was approved by the Institutional Ethics Committee for Medical Research and New Medical Technology of Sichuan Cancer Hospital (SCCHEC-02-2019-10) and conducted in accordance with the Declaration of Helsinki principles.
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