Multiplex assays reveal anti-EBV antibody profile and its implication in detection and diagnosis of nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) is detected in nearly 100% of nonkeratinizing nasopharyngeal carcinoma (NPC) and EBV-based biomarkers are used for NPC screening in endemic regions. Immunoglobulin A (IgA) against EBV nuclear antigen 1 (EBNA1) and viral capsid antigen (VCA), and recently identified anti-BNLF2b antibodies have been shown to be the most effective screening tool; however, the screening efficacy still needs to be improved. This study developed a multiplex serological assay by testing IgA and immunoglobulin G (IgG) antibodies against representative EBV antigens that are highly transcribed in NPC and/or function crucially in viral reactivation, including BALFs, BNLF2a/b, LF1, LF2, and Zta (BZLF1). Among them, BNLF2b-IgG had the best performance distinguishing NPC patients from controls (area under the curve: 0.951, 95% confidence interval [CI]: 0.913-0.990). Antibodies to lytic antigens BALF2 and VCA were significantly higher in advanced-stage than in early-stage tumors; in contrast, antibodies to latent protein EBNA1 and early lytic antigen BNLF2b were not correlated with tumor progression. Accordingly, a novel strategy combining EBNA1-IgA and BNLF2b-IgG was proposed and validated improving the integrated discrimination by 15.8% (95% CI: 9.8%-21.7%, p < .0001) compared with the two-antibody method. Furthermore, we found EBV antibody profile in patients was more complicated compared with that in healthy carriers, in which stronger correlations between antibodies against different phases of antigens were observed. Overall, our serological assay indicated that aberrant latent infection of EBV in nasopharyngeal epithelial cells was probably a key step in NPC initiation, while more lytic protein expression might be involved in NPC progression.

EBV DNA methylation profiles and its application in distinguishing nasopharyngeal carcinoma and nasal NK/T-cell lymphoma.

BACKGROUND: As an oncovirus, EBV is associated with multiple cancers, including solid tumors and hematological malignancies. EBV methylation plays an important role in regulating tumor occurrence. However, the EBV methylation profiles in EBV-associated tumor tissues are poorly understood. RESULTS: In this study, EBV methylation capture sequencing was conducted in several different tumor tissue samples, including NPC, EBVaGC, lung LELC and parotid LELC. Besides, EBV capture sequencing and following qMSP were performed on nasopharyngeal brushing samples from NPC and nasal NKTCL patients. Our results showed that the EBV genome among different types of tumors displayed specific methylation patterns. Among the four types of tumors from epithelial origin (NPC, EBVaGC, lung LELC and parotid LELC), the most significant differences were found between EBVaGC and the others. For example, in EBVaGC, all CpG sites within 1,44,189-1,45,136 bp of the EBV genome sequence on gene RPMS1 were hyper-methylated compared to the others. Differently, significant differences of EBV CpG sites, particularly those located on gene BILF2, were observed between NPC and nasal NKTCL patients in nasopharyngeal brushing samples. Further, the methylated level of BILF2 was further detected using qMSP, and a diagnostic model distinguishing NPC and nasal NKTCL was established. The AUC of the model was 0.9801 (95% CI 0.9524-1.0000), with the sensitivity and specificity of 98.81% (95% CI 93.63-99.94%) and 76.92% (95% CI 49.74-91.82%), respectively. CONCLUSIONS: Our study reveals more clues for further understanding the pathogenesis of EBV, and provides a possibility for distinguishing EBV-related tumor by detecting specific EBV CpG sites.

Detection of Epstein‒Barr virus DNA methylation as tumor markers of nasopharyngeal carcinoma patients in saliva, oropharyngeal swab, oral swab, and mouthwash.

Saliva biopsy of nasopharyngeal carcinoma (NPC) has been developed in our latest study, indicating the application of oral sampling in NPC detection. Further exploration of the potential for self-sampling from the oral cavity is necessary. A total of 907 various samples from oral cavity, including saliva (n = 262), oropharyngeal swabs (n = 250), oral swabs (n = 210), and mouthwash (n = 185), were collected. Epstein‒Barr virus (EBV) DNA methylation at the 12,420 bp CpG site in EBV genome from the repeat-copy W promoter (Wp) region and at the 11,029 bp CpG site in the single-copy C promoter (Cp) region were simultaneously detected in these samples. A significant increase in EBV methylation, no matter at Wp or Cp region, was found in all types of samples from NPC patients. However, EBV DNA methylation in saliva and oropharyngeal swab showed a better diagnostic performance in detecting NPC. The combination of these two sample types and two markers could help to improve the detection of NPC. Our study further explored the optimal self-sampling methods and detection target in the detection of NPC and may facilitate the application of EBV DNA methylation detection in a home-based large-scale screening of NPC.

Polygenic risk-stratified screening for nasopharyngeal carcinoma in high-risk endemic areas of China: a cost-effectiveness study.

Background: Nasopharyngeal carcinoma (NPC) has an extremely high incidence rate in Southern China, resulting in a severe disease burden for the local population. Current EBV serologic screening is limited by false positives, and there is opportunity to integrate polygenic risk scores for personalized screening which may enhance cost-effectiveness and resource utilization. Methods: A Markov model was developed based on epidemiological and genetic data specific to endemic areas of China, and further compared polygenic risk-stratified screening [subjects with a 10-year absolute risk (AR) greater than a threshold risk underwent EBV serological screening] to age-based screening (EBV serological screening for all subjects). For each initial screening age (30-34, 35-39, 40-44, 45-49, 50-54, 55-59, 60-64, and 65-69 years), a modeled cohort of 100,000 participants was screened until age 69, and then followed until age 79. Results: Among subjects aged 30 to 54 years, polygenic risk-stratified screening strategies were more cost-effective than age-based screening strategies, and almost comprised the cost-effectiveness efficiency frontier. For men, screening strategies with a 1-year frequency and a 10-year absolute risk (AR) threshold of 0.7% or higher were cost-effective, with an incremental cost-effectiveness ratio (ICER) below the willingness to pay (¥203,810, twice the local per capita GDP). Specifically, the strategies with a 10-year AR threshold of 0.7% or 0.8% are the most cost-effective strategies, with an ICER ranging from ¥159,752 to ¥201,738 compared to lower-cost non-dominated strategies on the cost-effectiveness frontiers. The optimal strategies have a higher probability (29.4-35.8%) of being cost-effective compared to other strategies on the frontier. Additionally, they reduce the need for nasopharyngoscopies by 5.1-27.7% compared to optimal age-based strategies. Likewise, for women aged 30-54 years, the optimal strategy with a 0.3% threshold showed similar results. Among subjects aged 55 to 69 years, age-based screening strategies were more cost-effective for men, while no screening may be preferred for women. Conclusion: Our economic evaluation found that the polygenic risk-stratified screening could improve the cost-effectiveness among individuals aged 30-54, providing valuable guidance for NPC prevention and control policies in endemic areas of China.

Microbes translocation from oral cavity to nasopharyngeal carcinoma in patients.

The presence of oral microbes in extra-oral sites is linked to gastrointestinal cancers. However, their potential ectopically colonization in the nasopharynx and impact on local cancer development remains uncertain. Our study involving paired nasopharyngeal-oral microbial samples from nasopharyngeal carcinoma (NPC) patients and controls unveils an aberrant oral-to-nasopharyngeal microbial translocation associated with increased NPC risk (OR = 4.51, P = 0.012). Thirteen species are classified as oral-translocated and enriched in NPC patients. Among these, Fusobacterium nucleatum and Prevotella intermedia are validated through culturomics and clonal strain identification. Nasopharyngeal biopsy meta-transcriptomes confirm these microbes within tumors, influencing local microenvironment and cytokine response. These microbes correlate significantly with the Epstein-Barr virus (EBV) loads in the nasopharynx, exhibiting an increased dose-response relationship. Collectively, our study identifies oral microbes migrating to the nasopharynx, infiltrating tumors, impacting microenvironments and linking with EBV infection. These results enhance our understanding of abnormal microbial communication and their roles in carcinogenesis.