Anti-Epstein-Barr Virus BNLF2b for Mass Screening for Nasopharyngeal Cancer.

BACKGROUND: Population screening of asymptomatic persons with Epstein-Barr virus (EBV) DNA or antibodies has improved the diagnosis of nasopharyngeal carcinoma and survival among affected persons. However, the positive predictive value of current screening strategies is unsatisfactory even in areas where nasopharyngeal carcinoma is endemic. METHODS: We designed a peptide library representing highly ranked B-cell epitopes of EBV coding sequences to identify novel serologic biomarkers for nasopharyngeal carcinoma. After a retrospective case-control study, the performance of the novel biomarker anti-BNLF2b total antibody (P85-Ab) was validated through a large-scale prospective screening program and compared with that of the standard two-antibody-based screening method (EBV nuclear antigen 1 [EBNA1]-IgA and EBV-specific viral capsid antigen [VCA]-IgA). RESULTS: P85-Ab was the most promising biomarker for nasopharyngeal carcinoma screening, with high sensitivity (94.4%; 95% confidence interval [CI], 86.4 to 97.8) and specificity (99.6%; 95% CI, 97.8 to 99.9) in the retrospective case-control study. Among the 24,852 eligible participants in the prospective cohort, 47 cases of nasopharyngeal carcinoma (38 at an early stage) were identified. P85-Ab showed higher sensitivity than the two-antibody method (97.9% vs. 72.3%; ratio, 1.4 [95% CI, 1.1 to 1.6]), higher specificity (98.3% vs. 97.0%; ratio, 1.01 [95% CI, 1.01 to 1.02]), and a higher positive predictive value (10.0% vs. 4.3%; ratio, 2.3 [95% CI, 1.8 to 2.8]). The combination of P85-Ab and the two-antibody method markedly increased the positive predictive value to 44.6% (95% CI, 33.8 to 55.9), with sensitivity of 70.2% (95% CI, 56.0 to 81.4). CONCLUSIONS: Our results suggest that P85-Ab is a promising novel biomarker for nasopharyngeal carcinoma screening, with higher sensitivity, specificity, and positive predictive value than the standard two-antibody method. (Funded by the National Key Research and Development Program of China and others; ClinicalTrials.gov number, NCT04085900.).

Antibody Profiling of Pan-Cancer Viral Proteome Reveals Biomarkers for Nasopharyngeal Carcinoma Diagnosis and Prognosis.

Diagnosing, predicting disease outcome, and identifying effective treatment targets for virus-related cancers are lacking. Protein biomarkers have the potential to bridge the gap between prevention and treatment for these types of cancers. While it has been shown that certain antibodies against EBV proteins could be used to detect nasopharyngeal carcinoma (NPC), antibodies targeting are solely a tiny part of the about 80 proteins expressed by the EBV genome. Furthermore, it remains unclear what role other viruses play in NPC since many diseases are the result of multiple viral infections. For the first time, this study measured both IgA and IgG antibody responses against 646 viral proteins from 23 viruses in patients with NPC and control subjects using nucleic acid programmable protein arrays. Candidate seromarkers were then validated by ELISA using 1665 serum samples from three clinical cohorts. We demonstrated that the levels of five candidate seromarkers (EBV-BLLF3-IgA, EBV-BLRF2-IgA, EBV-BLRF2-IgG, EBV-BDLF1-IgA, EBV-BDLF1-IgG) in NPC patients were significantly elevated than controls. Additional examination revealed that NPC could be successfully diagnosed by combining the clinical biomarker EBNA1-IgA with the five anti-EBV antibodies. The sensitivity of the six-antibody signature at 95% specificity to diagnose NPC was comparable to the current clinically-approved biomarker combination, VCA-IgA, and EBNA1-IgA. However, the recombinant antigens of the five antibodies are easier to produce and standardize compared to the native viral VCA proteins. This suggests the potential replacement of the traditional VCA-IgA assay with the 5-antibodies combination to screen and diagnose NPC. Additionally, we investigated the prognostic significance of these seromarkers titers in NPC. We showed that NPC patients with elevated BLLF3-IgA and BDLF1-IgA titers in their serum exhibited significantly poorer disease-free survival, suggesting the potential of these two seromarkers as prognostic indicators of NPC. These findings will help develop serological tests to detect and treat NPC in the future.

Kinetics of EBV antibody-based NPC risk scores in Taiwan NPC multiplex families.

Nasopharyngeal carcinoma (NPC) risk prediction models based on Epstein-Barr virus (EBV)-antibody testing have shown potential for screening of NPC; however, the long-term stability is unclear. Here, we investigated the kinetics of two EBV-antibody NPC risk scores within the Taiwan NPC Multiplex Family Study. Among 545 participants with multiple blood samples, we evaluated the stability of a 2-marker enzyme-linked immunosorbent assay score and 13-marker multiplex serology score using the intra-class correlation coefficient (ICC) by fitting a linear mixed model that accounted for the clustering effect of multiple measurements per subject and age. We also estimated the clustering of positive tests using Fleiss's kappa statistic. Over an average 20-year follow-up, the 2-marker score showed high stability over time, whereas the 13-marker score was more variable (p < .05). Case-control status is associated with the kinetics of the antibody response, with higher ICCs among cases. Positive tests were more likely to cluster within the same individual for the 2-marker score than the 13-marker score (p < .05). The 2-marker score had an increase in specificity from ~90% for single measurement to ~96% with repeat testing. The 13-marker score had a specificity of ~73% for a single measurement that increased to ~92% with repeat testing. Among individuals who developed NPC, none experienced score reversion. Our findings suggest that repeated testing could improve the specificity of NPC screening in high-risk NPC multiplex families. Further studies are required to determine the impact on sensitivity, establish optimal screening intervals, and generalize these findings to general population settings in high-risk regions.

Relevance of pyroptosis-associated genes in nasopharyngeal carcinoma diagnosis and subtype classification.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a highly aggressive and metastatic malignancy originating in the nasopharyngeal tissue. Pyroptosis is a relatively newly discovered, regulated form of necrotic cell death induced by inflammatory caspases that is associated with a variety of diseases. However, the role and mechanism of pyroptosis in NPC are not fully understood. METHODS: We analyzed the differential expression of pyroptosis-related genes (PRGs) between patients with and without NPC from the GSE53819 and GSE64634 datasets of the Gene Expression Omnibus (GEO) database. We mapped receptor operating characteristic profiles for these key PRGs to assess the accuracy of the genes for disease diagnosis and prediction of patient prognosis. In addition, we constructed a nomogram based on these key PRGs and carried out a decision curve analysis. The NPC patients were classified into different pyroptosis gene clusters by the consensus clustering method based on key PRGs, whereas the expression profiles of the key PRGs were analyzed by applying principal component analysis. We also analyzed the differences in key PRGs, immune cell infiltration and NPC-related genes between the clusters. Finally, we performed differential expression analysis for pyroptosis clusters and obtained differentially expressed genes (DEGs) and performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. RESULTS: We obtained 14 differentially expressed PRGs from GEO database. Based on these 14 differentially expressed PRGs, we applied least absolute shrinkage and selection operator analysis and the random forest algorithm to obtain four key PRGs (CHMP7, IL1A, TP63 and GSDMB). We completely distinguished the NPC patients into two pyroptosis gene clusters (pyroptosis clusters A and B) based on four key PRGs. Furthermore, we determined the immune cell abundance of each NPC sample, estimated the association between the four PRGs and immune cells, and determined the difference in immune cell infiltration between the two pyroptosis gene clusters. Finally, we obtained and functional enrichment analyses 259 DEGs by differential expression analysis for both pyroptosis clusters. CONCLUSIONS: PRGs are critical in the development of NPC, and our research on the pyroptosis gene cluster may help direct future NPC therapeutic approaches.

The serological diagnostic value of EBV-related IgA antibody panels for nasopharyngeal carcinoma: a diagnostic test accuracy meta-analysis.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is diagnosed relatively late and has a poor prognosis, requiring early detection to reduce the disease burden. This diagnostic test accuracy meta-analysis evaluated the serological diagnostic value of nine EBV-related IgA antibody panels (EBNA1-IgA, VCA-IgA, EA-IgA, Zta-IgA, EBNA1-IgA + VCA-IgA, VCA-IgA + EA-IgA, VCA-IgA + Rta-IgG, EBNA1-IgA + VCA-IgA + Zta-IgA and VCA-IgA + EA-IgA + Rta-IgG), aiming to identify suitable serological detection biomarkers for NPC screening. METHODS: PubMed, Embase, China National Knowledge Infrastructure and Chinese BioMedical Literature Database were searched from January 1st, 2000 to September 30th, 2023, with keywords nasopharyngeal carcinoma, IgA, screening, early detection, early diagnosis, sensitivity and specificity. Articles on the diagnostic value of serum EBV-related IgA antibody panels for NPC were included. Study selection, data extraction, and quality assessment were performed independently by two researchers, and a third researcher was consulted in the case of disagreement. Bivariate models were used for statistical analysis. The quality of included studies was evaluated through Quality Assessment of Diagnostic Accuracy Studies tool (QUADAS-2). RESULTS: A total of 70 articles were included, involving 11 863 NPC cases and 34 995 controls. Among the nine EBV-related IgA antibody panels, EBNA1-IgA + VCA-IgA [0.928 (0.898, 0.950)], VCA-IgA + Rta-IgG [0.925 (0.890, 0.949)], EBNA1-IgA + VCA-IgA + Zta-IgA [0.962 (0.909, 0.985)] and VCA-IgA + EA-IgA + Rta-IgG [0.945 (0.918, 0.964)] demonstrated higher pooled sensitivity (95%CI). In terms of diagnostic odds ratio (DOR) (95%CI), EBNA1-IgA + VCA-IgA [107.647 (61.173, 189.430)], VCA-IgA + Rta-IgG [105.988 (60.118, 186.857)] and EBNA1-IgA + VCA-IgA + Zta-IgA [344.450 (136.351, 870.153)] showed superior performance. Additionally, the SROC curves for EBNA1-IgA + VCA-IgA and VCA-IgA + Rta-IgG were more favorable. However, publication bias was detected for VCA-IgA (P = 0.005) and EBNA1-IgA + VCA-IgA (P = 0.042). CONCLUSIONS: In general, parallel detection of serum EBNA1-IgA, VCA-IgA and Zta-IgA antibodies using ELISA demonstrates better pooled sensitivity and DOR among the studied panels. In the cases where fewer indicators are used, serum VCA-IgA and EBNA1-IgA/Rta-IgG antibody panel exhibits a comparable performance. TRIAL REGISTRATION: The International Prospective Register of Systematic Reviews registration number: CRD42023426984, registered on May 28, 2023.

The plasma EBV DNA load with IL-6 and VEGF levels as predictive and prognostic biomarker in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is often diagnosed at a very advanced stage due to its location and non-specific initial symptoms. Moreover, no clinically useful serological marker has been established so far for early detection of NPC. In this study, we have investigated the clinical significance of plasma Epstein-Barr virus DNA load along with interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) levels to evaluate if these three all together can be useful as a strong serological marker for early detection and prediction of treatment response in patients with NPC. Plasma EBV DNA load, IL-6 level, VEGF expressions were measured in 24 patients with NPC at presentation and various time points during and after treatment. There was a positive correlation between high plasma EBV DNA load with higher IL-6 and VEGF expression, which was closely associated with therapeutic response as well. Persistent or recurrent plasma EBV load with higher IL-6 and VEGF levels can potentially predict disease progression and may be useful to select patients for additional therapy and longer follow-up.

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.

Labeling and Diagnostic Value of miRNA-28-3p in Patients with Nasopharyngeal Carcinoma.

Objective: To explore how miRNA-28-3p targets BIN1 and affects the biological behavior of nasopharyngeal carcinoma cells, and to evaluate its potential as a predictive biomarker for NPC. Methods: We studied 100 nasopharyngeal carcinoma patients who underwent surgery between January 2021 and January 2022. Tissues from tumors and adjacent normal areas were analyzed. Participants were categorized into two groups: one with nasopharyngeal carcinoma and another with adjacent normal tissue. Additionally, the nasopharyngeal carcinoma cell line CNE-2 was divided into three groups: an untreated control, a negative control with NC plasmid, and a test group treated with a miRNA-28-3p inhibitor. Key techniques included PCR, Western blotting, CCK-8, flow cytometry, focusing on the interactions between miRNA-28-3p and BIN1 and their predictive significance for NPC. Results: miRNA-28-3p levels were significantly higher in nasopharyngeal carcinoma tissues compared to adjacent normal tissues (P < .05). The predictive performance of miRNA-28-3p for NPC featured an AUC > 0.75 with sensitivity and specificity both exceeding 70% (P < .001). In nasopharyngeal carcinoma cells, miRNA-28-3p levels were significantly elevated compared to normal cells (P < .05). Transfection with the miRNA-28-3p inhibitor increased apoptosis and BIN1 protein levels while reducing cell proliferation, invasion, and migration significantly (P < .05). Conclusion: miRNA-28-3p is overexpressed in nasopharyngeal carcinoma and inhibits tumor cell proliferation, migration, and invasion, while promoting apoptosis by targeting BIN1. The level of miRNA-28-3p expression serves as a sensitive indicator for predicting nasopharyngeal carcinoma, affirming its potential as a diagnostic biomarker and underscoring the significance of these findings in enhancing understanding and clinical management of NPC.

Circulating tumor cells: a valuable indicator for locally advanced nasopharyngeal carcinoma.

BACKGROUND: Advancements in nasopharyngeal carcinoma (NPC) treatment have led to a focus on personalized treatment. Circulating tumor cells (CTCs) are important for liquid biopsies and personalized treatment but are not being fully utilized. This study examined how pre- and post-treatment CTC counts, EMT subtypes, clinical characteristics, and patient prognosis are related in order to support the use of liquid biopsy in managing NPC. METHODS: This retrospective study included 141 patients with locally advanced NPC. All patients underwent CanPatrol™ CTC detection pre- and post-treatment and were categorized into EMT subtypes: epithelial type, mixed type, and mesenchymal type. This study analyzed CTC enumeration, EMT subtypes, and their associations with clinical characteristics and survival outcomes. RESULTS: The results indicated a positive correlation between the pre-treatment detection rate of CTCs and N stage (P < 0.01), alongside a positive correlation with the TNM clinical stage (P = 0.02). Additionally, the detection rate of mesenchymal CTCs post-treatment is positively associated with the N stage (P = 0.02). The enumeration of CTCs pre- and post-treatment is negatively correlated with prognosis and has statistical significance. Additionally, an investigation into the EMT subtypes of CTCs revealed a significant association between the presence of mesenchymal CTCs pre- and post-treatment and decreased overall survival (OS) (P < 0.05). Furthermore, T stage, N stage, TNM clinical stage, and Epstein-Barr virus (EBV) DNA were also significantly correlated with OS. CONCLUSION: The study found that mesenchymal CTCs pre- and post-treatment, as well as the number of CTCs, were linked to a poor prognosis.

Ectopic Olfactory Neuroblastoma Arising in the Nasopharynx.

ABSTRACT: Olfactory neuroblastoma (ONB) is an uncommon malignant tumor typically located in the upper nasal cavity. Olfactory neuroblastoma originating in the nasopharynx is extremely rare and tends to be misdiagnosed. The authors describe a rare case of ONB arising ectopically in the nasopharynx. The patient was a 65-year-old woman with recurrent epistaxis and a feeling of fullness in the right ear. After evaluation, endoscopic surgery was performed. The pathological result proved to be ONB. Postoperative magnetic resonance imaging showed that the tumor was completely resected. The patient proceeded to have 66 Gy of postoperative intensity-modulated radiotherapy and was followed for 36 months without tumor recurrence. Olfactory neuroblastoma originating from the nasopharynx is more rare condition compared with ONB located in other areas in the literature. The symptoms of ONB ectopic to the nasopharynx are similar to those of other nasopharyngeal tumors, which were likely to be misdiagnosed. The treatment principle is the same as that of nonectopic ONB, which is surgery combined with radiotherapy. Surgery can be performed using an endoscopic transnasal approach.

[Analysis of cases of cervical cystic lymph node metastasis with an unknown primary misdiagnosed as branchial cleft carcinoma].

Objectives: To analyze the location, discovery time and possible causes of cases of cervical cystic lymph node metastasis with an unknown primary misdiagnosed as branchial cleft carcinoma. Methods: A retrospective analysis was performed on clinical and pathological data of 15 patients misdiagnosed as branchiogenic carcinoma at Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College between January 2000 and December 2020. Results: Among the 15 patients, 6 were nasopharyngeal squamous cell carcinoma, 4 tonsil squamous cell carcinoma, 2 tongue root squamous cell carcinoma, 2 hypopharyngeal squamous cell carcinoma and 1 thyroid papillary carcinoma. The median time from the diagnosis of branchial cleft carcinoma to the discovery of primary lesions was 3.58 months (0-76 months). The causes of misdiagnosis might be the lack of experience in the diagnosis and treatment of branchial cleft carcinoma, and not enough attention to comprehensive examination and close follow-up. Conclusions: Different from oropharyngeal cancer reported internationally, the proportion of misdiagnosed cases with nasopharyngeal carcinoma as the primary site in the current article is higher. As a country with a high incidence of nasopharyngeal carcinoma, the examination of nasopharynx should not be taken lightly. Most hidden cases can be found in the comprehensive examination in a short time, while a few cases need long-term follow-up. Finding the primary sites should not rely too much on imaging examination, and we cannot ignore the importance of clinical physical examination.

[Primary nasopharyngeal carcinoma with Sjögren's syndrome and positive cerebrospinal fluid Epstein-Barr virus: a case report and literature review].

The study presents an analysis of the diagnostic and treatment protocol for a patient with a first episode of nasopharyngeal carcinoma who also has Sjogren's syndrome and Epstein-Barr Virus (EBV) positive cerebrospinal fluid, as detected through metagenomic next-generation sequencing (mNGS). It reviews existing literature to examine the connections between EBV and various conditions including Sjogren's syndrome, encephalitis or meningitis, and nasopharyngeal carcinoma, emphasizing the importance of EBV positive cerebrospinal fluid. The study focuses on a case from the Eighth Medical Center of the General Hospital of the People's Liberation Army, where a patient was admitted with headaches as the primary symptom on March 3, 2021. This patient had a history of Sjogren's syndrome and was later diagnosed with nasopharyngeal carcinoma. The research involved reviewing both domestic and international databases for cases related to cerebrospinal fluid EBV positive encephalitis or meningitis, and nasopharyngeal carcinoma. It aimed to aggregate data on demographics, initial symptoms, treatment methods, and patient outcomes. Findings suggest that positive cerebrospinal fluid EBV is linked to autoimmune diseases, viral encephalitis or meningitis, and nasopharyngeal carcinoma, albeit infrequently in the context of Sjogren's syndrome. Notably, EBV positive cerebrospinal fluid is commonly associated with recurrent nasopharyngeal carcinoma rather than initial episodes. The study concludes that for patients with an immune condition, exhibiting symptoms like headaches or cranial nerve issues, or in cases where nasopharyngeal carcinoma is suspected, early testing through cerebrospinal fluid mNGS or EBV DNA is recommended. This approach facilitates risk assessment, prognosis determination, and the creation of individualized treatment plans.

Body mass index and body shape before treatment and nasopharyngeal carcinoma prognosis: A population-based patient cohort study in southern China.

A concern of reverse causation exists about the association between nasopharyngeal carcinoma (NPC) prognosis and body mass index (BMI) at diagnosis, while the prognostic impact of BMI measured years before diagnosis is unknown. Therefore, we investigated associations of prediagnosis and pretreatment BMI and body shape on NPC mortality. From a population-based patient cohort in southern China between 2010 and 2013, we included 2526 incident NPC cases with prospective follow-up through 2018. We assessed the associations of BMI and body shape at age 20 years, 10 years before diagnosis, and at diagnosis with NPC mortality, combining strategies of stratification and statistical adjustment to minimize reverse causation. We observed 25% lower all-cause mortality (hazard ratio [HR] 0.75, 95% confidence interval [CI]: 0.64-0.89) and 25% lower NPC-specific mortality (HR 0.75, 95% CI: 0.61-0.91) among overweight vs normal-weight NPC cases at diagnosis. Lean body shapes 1 and 2 at diagnosis were associated with 68% and 23% higher all-cause mortality, respectively, compared to normal body shape 3. No effect modification by cancer stage was detected for associations with all-cause or NPC-specific mortality. Associations with BMI and body shape 10 years before diagnosis were similar but attenuated, while body size and shape at age 20 were not associated with mortality. Being overweight at diagnosis decreased mortality, and thinner body shape increased mortality, compared to normal weight/body shape. These associations may be due to poorer nutrition and treatment intolerance, resulting in treatment discontinuation and worse survival outcomes.

Quantitative detection of Epstein-Barr virus DNA methylation in the diagnosis of nasopharyngeal carcinoma by blind brush sampling.

Detecting EBV DNA load in nasopharyngeal (NP) brushing samples for the diagnosis of nasopharyngeal carcinoma (NPC) has attracted widespread attentions. Currently, NP brush sampling mostly relies on endoscopic guidance, and there are few reports on diagnostic markers suitable for nonguided conditions (blind brush sampling), which is of great significance for extending its application. One hundred seventy nasopharyngeal brushing samples were taken from 98 NPC patients and 72 non-NPC controls under the guidance of endoscope, and 305 blind brushing samples were taken without endoscopic guidance from 164 NPC patients and 141 non-NPC controls (divided into discovery and validation sets). Among these, 38 cases of NPC underwent both endoscopy-guided NP brushing and blind brushing. EBV DNA load targeting BamHI-W region and EBV DNA methylation targeting 11029 bp CpG site located at Cp-promoter region were detected by quantitative polymerase chain reaction (q-PCR). EBV DNA load showed good classification accuracy for NPC in endoscopy-guided brushing samples (AUC = 0.984). However, in blind bushing samples, the diagnostic performance was greatly reduced (AUC = 0.865). Unlike EBV DNA load, the accuracy of EBV DNA methylation was less affected by brush sampling methods, whether in endoscopy-guided brushing (AUC = 0.923) or blind brushing (AUC = 0.928 in discovery set and AUC = 0.902 in validation set). Importantly, EBV DNA methylation achieved a better diagnostic accuracy than EBV DNA load in blind brushing samples. Overall, detection of EBV DNA methylation with blind brush sampling shows great potential in the diagnosis of NPC and may facilitate its use in nonclinical screening of NPC.

Saliva biopsy: Detecting the difference of EBV DNA methylation in the diagnosis of nasopharyngeal carcinoma.

Saliva sampling is a non-invasive method, and could be performed by donors themselves. However, there are few studies reporting biomarkers in saliva in the diagnosis of NPC. A total of 987 salivary samples were used in this study. First, EBV DNA methylation was profiled by capture sequencing in the discovery cohort (n = 36). Second, a q-PCR based method was developed and five representative EBV DNA CpG sites (11 029 bp, 45 849 bp, 57 945 bp, 66 226 bp and 128 102 bp) were selected and quantified to obtain the methylated density in the validation cohort1 (n = 801). Third, a validation cohort2 (n = 108) was used to further verify the differences of EBV methylation in saliva. A significant increase of EBV methylation was found in NPC patients compared with controls. The methylated score of EBV genome obtained by capture sequencing could distinguish NPC from controls (sensitivity 90%, specificity 100%). Further, the methylated density of EBV DNA CpG sites revealed by q-PCR showed a good diagnostic performance. The sensitivity and specificity of detecting a single CpG site (11 029 bp) could reach 75.4% and 99.7% in the validation cohort1, and 78.2% and 100% in the validation cohort2. Besides, the methylated density of the CpG site was found to decrease below the COV in NPC patients after therapy, and increase above the COV after recurrence. Our study provides an appealing alternative for the non-invasive detection of NPC without clinical setting. It paves the way for conducting a home-based large-scale screening in the future.

Deep Learning Fuzzy Inference: An Interpretable Model for Detecting Indirect Immunofluorescence Patterns Associated with Nasopharyngeal Cancer.

The detection of serum Epstein-Barr virus antibodies by immunofluorescence assay (IFA) is considered the gold standard screening test for nasopharyngeal cancer (NPC) in high-risk populations. Given the high survival rate after early detection in asymptomatic patients, compared to the poor prognosis in patients with late-stage NPC, screening using IFA has tremendous potential for saving lives in the general population. However, IFA requires visual interpretation of cellular staining patterns by trained pathology staff, making it labor intensive and hence nonscalable. In this study, an automated fuzzy inference (FI) system achieved high agreement with a human IFA expert in identifying cellular patterns associated with NPC (κ = 0.82). The integration of a deep learning module into FI further improved the performance of FI (κ = 0.90) and reduced the number of uncertain cases that required manual evaluation. The performance of the resulting hybrid model, termed deep learning FI (DeLFI), was then evaluated with a separate testing set of clinical samples. In this clinical validation, DeLFI outperformed human evaluation on the area under the curve (0.926 versus 0.821) and closely matched human performance on Youden J index (0.81 versus 0.80). Data from this study indicate that the combination of deep learning with FI in DeLFI has the potential to improve the scalability and accuracy of NPC detection.

Cytoplasmic poly(A)-binding protein 1 (PABPC1) is a prognostic biomarker to predict survival in nasopharyngeal carcinoma regardless of chemoradiotherapy.

BACKGROUND: Nasopharyngeal carcinoma (NPC), especially the nonkeratinizing type, is a malignant tumor primarily occurring in southern China and Southeast Asia. Chemotherapy (CT) and combined radiotherapy (RT) is used to treat NPC. However, the mortality rate is high in recurrent and metastatic NPC. We developed a molecular marker, analyzed its correlation with clinical characteristics, and assessed the prognostic value among NPC patients with or without chemoradiotherapy. METHODS: A total of 157 NPC patients were included in this study, with 120 undergoing treatment and 37 without treatment. EBER1/2 expression was investigated using in situ hybridization (ISH). Expression of PABPC1, Ki-67, and p53 was detected with immunohistochemistry. The correlations of EBER1/2 and the expression of the three proteins having clinical features and prognosis were evaluated. RESULTS: The expression of PABPC1 was associated with age, recurrence, and treatment but not with gender, TNM classification, or the expression of Ki-67, p53, or EBER. High expression of PABPC1 was associated with poor overall survival (OS) and disease-free survival (DFS) and was an independent predictor depending on multivariate analysis. Comparatively, no significant correlation was observed between the expression of p53, Ki-67, and EBER and survival. In this study, 120 patients received treatments and revealed significantly better OS and DFS than the untreated 37 patients. PABPC1 high expression was an independent predictor of shorter OS in the treated (HR = 4.012 (1.238-13.522), 95% CI, p = 0.021) and the untreated groups (HR = 5.473 (1.051-28.508), 95% CI, p = 0.044). However, it was not an independent predictor of shorter DFS in either the treated or the untreated groups. No significant survival difference was observed between patients with docetaxel-based induction chemotherapy (IC) + concurrent chemoradiotherapy (CCRT) and those with paclitaxel-based IC + CCRT. However, when combined with treatment and PABPC1 expression, patients with paclitaxel-added chemoradiotherapy plus PABPC1 low expression had significantly better OS than those who underwent chemoradiotherapy (p = 0.036). CONCLUSIONS: High expression of PABPC1 is associated with poorer OS and DFS among NPC patients. Patients with PABPC1 having low expression revealed good survival irrespective of the treatment received, indicating that PABPC1 could be a potential biomarker for triaging NPC patients.

Pre-diagnostic anti-EBV antibodies and primary liver cancer risk: a population-based nested case-control study in southern China.

BACKGROUND: We aimed to investigate associations between pre-diagnostic anti-Epstein-Barr virus (EBV) antibodies, including interactions with hepatitis B virus (HBV), and risk of primary liver cancer in southern China. METHODS: In a population-based nested case-control study, we measured pre-diagnostic immunoglobulin A (IgA) against EBV nuclear antigen 1 (EBNA1) and viral capsid antigen (VCA) in 125 primary liver cancer cases and 2077 matched controls. We also explored the interaction between HBV surface antigen (HBsAg) and anti-EBV antibodies. RESULTS: Participants with positive EBNA1-IgA, positive VCA-IgA or single-positive anti-EBV antibodies had two-fold odds of developing liver cancer, compared with seronegative subjects. The odds ratios (ORs) between the relative optical density of EBNA1-IgA and VCA-IgA and primary cancer, controlling for age and HBsAg, were 1.59 (95% confidence interval (CI): 1.17, 2.14) and 1.60 (95% CI: 1.07, 2.41), respectively. Subjects with both HBsAg and anti-EBV antibody seropositivity were at 50-fold increased risk compared with those negative for both biomarkers (OR: 50.67, 95% CI: 18.28, 140.46), yielding a relative excess risk due to interaction of 30.81 (95% CI: 3.42, 114.93). CONCLUSION: Pre-diagnostic seropositivity for EBNA1-IgA and/or VCA-IgA was positively associated with primary liver cancer risk, especially in combination with HBsAg positivity. EBV may interact with HBV in the development of primary liver cancer, and anti-EBV antibodies might be potential biomarkers for primary liver cancer in this high-risk population.

EBV antibody and gastric cancer risk: a population-based nested case-control study in southern China.

BACKGROUND: We aim to clarify the controversial associations between EBV-related antibodies and gastric cancer risk. METHODS: We analysed the associations between serological Epstein-Barr nuclear antigen 1 immunoglobulin A (EBNA1-IgA) and viral capsid antigen immunoglobulin A (VCA-IgA) by enzyme-linked immunosorbent assay and the risk of gastric cancer in a nested case-control study originated from a population-based nasopharyngeal carcinoma (NPC) screening cohort in Zhongshan, a city of southern China, including 18 gastric cancer cases and 444 controls. Conditional logistic regression was used to calculate the odds ratios (ORs) and corresponding 95% confidence intervals (CIs). RESULTS: All the sera of cases were sampled before diagnosis and the median time interval was 3.04 (range: 0.04, 7.59) years. Both increased relative optical density (rOD) values of EBNA1-IgA and VCA-IgA were associated with higher risks of gastric cancer with age adjusted ORs of 1.99 (95%CI: 1.07, 3.70) and 2.64 (95%CI: 1.33, 5.23), respectively. Each participant was further classified as high or medium/low risk based on a combination of two anti-EBV antibody levels. Participants in the high-risk group had substantially higher odds of developing gastric cancer than that in the medium/low risk group with an age adjusted OR of 6.53 (95%CI: 1.69, 25.26). CONCLUSIONS: Our research reveals positive associations between EBNA1-IgA and VCA-IgA and gastric cancer risk in southern China. We thus postulate that EBNA1-IgA and VCA-IgA might appear to be potential biomarkers for gastric cancer. More research to further validate the results among diverse populations and investigate its underlying biological mechanism is needed.

Classification of multiple cancer types by combination of plasma-based near-infrared spectroscopy analysis and machine learning modeling.

BACKGROUND AND AIM: Near-infrared spectroscopy (NIRS) is a non-invasive and convenient tool, which gains features related to chemical components in biological samples. Machine learning (ML) has been popularized in medical diagnosis. This study aimed at investigating a novel cancer diagnosis strategy using NIRS data based ML modeling. METHODS: Plasma samples were collected from a total of 247 participants, including lung cancer, cervical cancer, nasopharyngeal cancer, and healthy control, and were randomly split into train set and test set. After performing NIRS analysis, the train dataset was utilized to train ML models, including partial least-squares (PLS), random forest (RF), gradient boosting machine (GBM), and support-vector machine (SVM). Subsequently, these models were tested for their prediction performance by the test set. RESULTS: All ML models demonstrated high prediction performance in differentiating cancers from controls, and SVM had high prediction accuracy for different types of cancers. SVM was considered as the most suitable model for its minimal computational cost and high accuracies for both binary and quaternary classification. CONCLUSIONS: This strategy coupling NIRS with ML is insightful that may aid in clinic cancer diagnosis, while further studies should test our results in a larger cohort with better representativeness.