Abstract
Objectives
Epstein-Barr virus (EBV) C promoter (Cp) hypermethylation, a crucial factor for EBV latent infection of nasopharyngeal epithelial cells, has been recognized as a promising biomarker for nasopharyngeal carcinoma (NPC) detection. In this study, we develop a novel EBV Cp methylation quantification (E-CpMQ) assay and evaluate its diagnostic performance for NPC detection.
Methods
A novel qPCR assay for simultaneous quantification of methylated- and unmethylated EBV Cp was developed by the combinational modification of MethyLight and QASM, with an innovative calibrator to improve the detection accuracy and consistency. The NP swab samples and synthetic standards were used for the analytical validation of the E-CpMQ. The diagnostic efficacy of the developed E-CpMQ assay was validated in 137 NPC patients and 137 non-NPC controls.
Results
The E-CpMQ assay can detect the EBV Cp methylation ratio in one reaction system under 10 copies with 100 % recognition specificity, which is highly correlated to pyrosequencing with a correlation coefficient over 0.99. The calibrated E-CpMQ assay reduces the coefficient of variation by an average of 55.5 % with a total variance of less than 0.06 units standard deviation (SD). Linear methylation ratio detection range from 4.76 to 99.01 %. The sensitivity and specificity of the E-CpMQ respectively are 96.4 % (95 % CI: 91.7–98.8 %), 89.8 % (95 % CI: 83.5–94.3 %).
Conclusions
The developed E-CpMQ assay with a calibrator enables accurate and reproducible EBV Cp methylation ratio quantification and offers a sensitive, specific, cost-effective method for NPC early detection.
Funding source: The national Key Research and Development Program of China/Award Number
Award Identifier / Grant number: 2020YFC1316905
Funding source: National Natural Science Foundation of China
Award Identifier / Grant number: 81872700
Award Identifier / Grant number: 82073625
Funding source: Natural Science Foundation of Guangdong Province
Award Identifier / Grant number: 2023A1515010221
Acknowledgments
We thank Dr. Yu-Jie Zhang (Guangdong Province Key Laboratory of Molecular Tumor Pathology, Southern Medical University) for the assistance of analytical validation experiments. We thank Ms. Xue-Li (Guangzhou University of Chinese Medicine) for the assistance of the experiment. We thank the kind donation of the Raji cell line from Prof. Mu-sheng Zeng (Sun Yat-sen University Cancer Center). We thank Dr. Zhiwei Liu (National Cancer Institute of the United States) for the assistance in writing.
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Research ethics: This study has been approved by the Ethical Committee of the Sun Yat-sen University Cancer Center.
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Informed consent: Informed consent was obtained from all individuals included in this study.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Competing interests: Authors state no conflict of interest.
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Research funding: The national Key Research and Development Program of China/Award Number: 2020YFC1316905, National Natural Science Foundation of China, Grant/Award Numbers: 81872700, 82073625; Natural Science Foundation of Guangdong, Grant/Award Numbers: 2023A1515010221.
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Patents: A patent owned by Cao SM, Wu ZC, Xie SH, Lin DF was granted by the State Intellectual Property Office of China (NO. 2022101828343); A patent owned by Cao SM, Wu ZC, Hu X, Li F, Ge YY was granted by the State Intellectual Property Office of China (NO. 2023202411559).
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Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2023-0510).
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