Development and analytical validation of a novel nasopharynx swab-based Epstein-Barr virus C promoter methylation quantitative assay for nasopharyngeal carcinoma detection.

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.

Optimization and Local Cost-Effectiveness of Nasopharyngeal Carcinoma Screening Strategies in Southern China: Secondary Analysis of the Guangdong Randomized Trial.

BACKGROUND: Screening with anti-Epstein-Barr virus (EBV) serology and endoscopy decreased nasopharyngeal carcinoma (NPC) mortality in Guangdong in a randomized trial. We conducted a secondary analysis of this trial using local incidence and cost data to optimize screening programs, hypothesizing that screening could be cost-effective in southern China. METHODS: Screening costs and life-years after NPC diagnosis were obtained from the Guangdong trial's intent-to-screen population (men and women aged 30-69). Seropositive subjects were rescreened annually for 5 years. Thereafter, we evaluated 12 screening strategies in Guangdong and Guangxi using a validated model. Strategies used combinations of serology, nasopharyngeal swab PCR (NP PCR), endoscopy, and MRI from trial subcohorts. Incidence data and costs were obtained from local cancer registries and the provincial healthcare system. RESULTS: In the intent-to-screen population, screening with serology and endoscopy was cost-effective (¥42,366/life-year, 0.52 GDP per capita). Screening for 5 to 15 years between ages 35 and 59 years met a willingness-to-pay threshold of 1.5 GDP/quality-adjusted life-years in all modeled populations. Despite doubling costs, adding MRI could be cost-effective via improved sensitivity. NP PCR triage reduced endoscopy/MRI referrals by 37%. One-lifetime screen could reduce NPC mortality by approximately 20%. CONCLUSIONS: EBV-based serologic screening for NPC is likely to be cost-effective in southern China. Among seropositive subjects, the preferred strategies use endoscopy alone or selective endoscopy triaged by MRI with or without NP PCR. These data may aid the design of screening programs in this region. IMPACT: These findings support population-based screening in southern China by defining the target population, cost-effectiveness, and optimized screening approach.

Host genetic variants, Epstein-Barr virus subtypes, and the risk of nasopharyngeal carcinoma: Assessment of interaction and mediation.

Epstein-Barr virus (EBV) and human leukocyte antigen (HLA) polymorphisms are well-known risk factors for nasopharyngeal carcinoma (NPC). However, the combined effects between HLA and EBV on the risk of NPC are unknown. We applied a causal inference framework to disentangle interaction and mediation effects between two host HLA SNPs, rs2860580 and rs2894207, and EBV variant 163364 with a population-based case-control study in NPC-endemic southern China. We discovered the strong interaction effects between the high-risk EBV subtype and both HLA SNPs on NPC risk (rs2860580, relative excess risk due to interaction [RERI] = 4.08, 95% confidence interval [CI] = 2.03-6.14; rs2894207, RERI = 3.37, 95% CI = 1.59-5.15), accounting for the majority of genetic risk effects. These results indicate that HLA genes and the high-risk EBV have joint effects on NPC risk. Prevention strategies targeting the high-risk EBV subtype would largely reduce NPC risk associated with EBV and host genetic susceptibility.