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.

A comprehensive predictive model for radiation-induced brain injury in risk stratification and personalized radiotherapy of nasopharyngeal carcinoma.

BACKGROUND AND PURPOSE: Radiation-induced brain injury (RBI) is a severe radiotoxicity for nasopharyngeal carcinoma (NPC) patients, greatly affecting their long-term life quality and survival. We aim to establish a comprehensive predictive model including clinical factors and newly developed genetic variants to improve the precision of RBI risk stratification. MATERIALS AND METHODS: By performing a large registry-based retrospective study with magnetic resonance imaging follow-up on RBI development, we conducted a genome-wide association study and developed a polygenic risk score (PRS) for RBI in 1189 NPC patients who underwent intensity-modulated radiotherapy. We proposed a tolerance dose scheme for temporal lobe radiation based on the risk predicted by PRS. Additionally, we established a nomogram by combining PRS and clinical factors for RBI risk prediction. RESULTS: The 38-SNP PRS could effectively identify high-risk individuals of RBI (P = 1.42 × 10-34). Based on genetic risk calculation, the recommended tolerance doses of temporal lobes should be 57.6 Gy for individuals in the top 10 % PRS subgroup and 68.1 Gy for individuals in the bottom 50 % PRS. Notably, individuals with high genetic risk (PRS > P50) and receiving high radiation dose in the temporal lobes (D0.5CC > 65 Gy) had an approximate 50-fold risk over individuals with low PRS and receiving low radiation dose (HR = 50.09, 95 %CI = 24.27-103.35), showing an additive joint effect (Pinteraction < 0.001). By combining PRS with clinical factors including age, tumor stage, and radiation dose of temporal lobes, the predictive accuracy was significantly improved with C-index increased from 0.78 to 0.85 (P = 1.63 × 10-2). CONCLUSIONS: The PRS, together with clinical factors, could improve RBI risk stratification and implies personalized radiotherapy.

The interaction and mediation effects between the host genetic factors and Epstein-Barr virus VCA-IgA in the risk of nasopharyngeal carcinoma.

Previous studies have demonstrated strong associations between host genetic factors and Epstein-Barr virus (EBV) VCA-IgA with the risk of nasopharyngeal carcinoma (NPC). However, the specific interplay between host genetics and EBV VCA-IgA on NPC risk is not well understood. In this two-stage case-control study (N = 4804), we utilized interaction and mediation analysis to investigate the interplay between host genetics (genome-wide association study-derived polygenic risk score [PRS]) and EBV VCA-IgA antibody level in the NPC risk. We employed a four-way decomposition analysis to assess the extent to which the genetic effect on NPC risk is mediated by or interacts with EBV VCA-IgA. We consistently found a significant interaction between the PRS and EBV VCA-IgA on NPC risk (discovery population: synergy index [SI] = 2.39, 95% confidence interval [CI] = 1.85-3.10; replication population: SI = 3.10, 95% CI = 2.17-4.44; all pinteraction < 0.001). Moreover, the genetic variants included in the PRS demonstrated similar interactions with EBV VCA-IgA antibody. We also observed an obvious dose-response relationship between the PRS and EBV VCA-IgA antibody on NPC risk (all ptrend < 0.001). Furthermore, our decomposition analysis revealed that a substantial proportion (approximately 90%) of the genetic effects on NPC risk could be attributed to host genetic-EBV interaction, while the risk effects mediated by EBV VCA-IgA antibody were weak and statistically insignificant. Our study provides compelling evidence for an interaction between host genetics and EBV VCA-IgA antibody in the development of NPC. These findings emphasize the importance of implementing measures to control EBV infection as a crucial strategy for effectively preventing NPC, particularly in individuals at high genetic risk.

Peptidome-wide association analysis of Epstein-Barr virus identifies epitope repertoires associated with nasopharyngeal carcinoma.

Human leukocyte antigen (HLA) molecules are essential for presenting Epstein-Barr virus (EBV) antigens and are closely related to nasopharyngeal carcinoma (NPC). This study aims to systematically investigate the association between HLA-bound EBV peptides and NPC risk through in silico HLA-peptide binding prediction. A total of 455 NPC patients and 463 healthy individuals in NPC endemic areas were recruited, and HLA-target sequencing was performed. HLA-peptide binding prediction for EBV, followed by peptidome-wide logistic regression and motif analysis, was applied. Binding affinity changes for EBV peptides carrying high-risk mutations were analyzed. We found that NPC-associated EBV peptides were significantly enriched in immunogenic proteins and core linkage disequilibrium (LD) proteins related to evolution, especially those binding HLA-A alleles (p = 3.10 × 10-4 for immunogenic proteins and p = 8.10 × 10-5 for core LD proteins related to evolution). These peptides were clustered and showed binding motifs of HLA supertypes, among which supertype A02 presented an NPC-risk effect (padj = 3.77 × 10-4 ) and supertype A03 presented an NPC-protective effect (padj = 4.89 × 10-4 ). Moreover, a decreased binding affinity toward risk HLA supertype A02 was observed for the peptide carrying the NPC-risk mutation BNRF1 V1222I (p = 0.0078), and an increased binding affinity toward protective HLA supertype A03 was observed for the peptide carrying the NPC-risk mutation BALF2 I613V (p = 0.022). This study revealed the distinct preference of EBV peptides for binding HLA supertypes, which may contribute to shaping EBV population structure and be involved in NPC development.

High-throughput identification of regulatory elements and functional assays to uncover susceptibility genes for nasopharyngeal carcinoma.

Large-scale genetic association studies have identified multiple susceptibility loci for nasopharyngeal carcinoma (NPC), but the underlying biological mechanisms remain to be explored. To gain insights into the genetic etiology of NPC, we conducted a follow-up study encompassing 6,907 cases and 10,472 controls and identified two additional NPC susceptibility loci, 9q22.33 (rs1867277; OR = 0.74, 95% CI = 0.68-0.81, p = 3.08 × 10-11) and 17q12 (rs226241; OR = 1.42, 95% CI = 1.26-1.60, p = 1.62 × 10-8). The two additional loci, together with two previously reported genome-wide significant loci, 5p15.33 and 9p21.3, were investigated by high-throughput sequencing for chromatin accessibility, histone modification, and promoter capture Hi-C (PCHi-C) profiling. Using luciferase reporter assays and CRISPR interference (CRISPRi) to validate the functional profiling, we identified PHF2 at locus 9q22.33 as a susceptibility gene. PHF2 encodes a histone demethylase and acts as a tumor suppressor. The risk alleles of the functional SNPs reduced the expression of the target gene PHF2 by inhibiting the enhancer activity of its long-range (4.3 Mb) cis-regulatory element, which promoted proliferation of NPC cells. In addition, we identified CDKN2B-AS1 as a susceptibility gene at locus 9p21.3, and the NPC risk allele of the functional SNP rs2069418 promoted the expression of CDKN2B-AS1 by increasing its enhancer activity. The overexpression of CDKN2B-AS1 facilitated proliferation of NPC cells. In summary, we identified functional SNPs and NPC susceptibility genes, which provides additional explanations for the genetic association signals and helps to uncover the underlying genetic etiology of NPC development.

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.

Association of HLA diversity with the risk of 25 cancers in the UK Biobank.

BACKGROUND: The human leukocyte antigen (HLA) is a highly polymorphic region, and HLA diversity may play a role in presenting tumour-associated peptides and inducing immune responses. However, the effect of HLA diversity on cancers has not been fully assessed. We aimed to explore the role of HLA diversity on cancer development. METHODS: A pan-cancer analysis was performed to evaluate the effect of HLA diversity, measured by HLA heterozygosity and HLA evolutionary divergence (HED), on the susceptibility of 25 cancers in the UK Biobank. FINDINGS: We observed that the diversity of HLA class II locus was associated with a lower risk of lung cancer (ORhetero = 0.94, 95% CI = 0.90-0.97, P = 1.29 × 10-4) and head and neck cancer (ORhetero = 0.91, 95% CI = 0.86-0.96, P = 1.56 × 10-3). Besides, a lower risk of non-Hodgkin lymphoma was associated with an increased diversity of HLA class I (ORhetero = 0.92, 95% CI = 0.87-0.98, P = 8.38 × 10-3) and class II locus (ORhetero = 0.89, 95% CI = 0.86-0.92, P = 1.65 × 10-10). A lower risk of Hodgkin lymphoma was associated with the HLA class I diversity (ORhetero = 0.85, 95% CI = 0.75-0.96, P = 0.011). The protective effect of HLA diversity was mainly observed in pathological subtypes with higher tumour mutation burden, such as lung squamous cell carcinoma (P = 9.39 × 10-3) and diffuse large B cell lymphoma (Pclass I = 4.12 × 10-4; Pclass Ⅱ = 4.71 × 10-5), as well as the smoking subgroups of lung cancer (P = 7.45 × 10-5) and head and neck cancer (P = 4.55 × 10-3). INTERPRETATION: We provided a systematic insight into the effect of HLA diversity on cancers, which might help to understand the etiological role of HLA on cancer development. FUNDING: This study was supported by grants from the National Natural Science Foundation of China (82273705, 82003520); the Basic and Applied Basic Research Foundation of Guangdong Province, China (2021B1515420007); the Science and Technology Planning Project of Guangzhou, China (201804020094); Sino-Sweden Joint Research Programme (81861138006); the National Natural Science Foundation of China (81973131, 81903395, 81803319, 81802708).

Clinical and genome-wide association analysis of chemoradiation-induced hearing loss in nasopharyngeal carcinoma.

Chemoradiation-induced hearing loss (CRIHL) is one of the most devasting side effects for nasopharyngeal carcinoma (NPC) patients, which seriously affects survivors' long-term quality of life. However, few studies have comprehensively characterized the risk factors for CRIHL. In this study, we found that age at diagnosis, tumor stage, and concurrent cisplatin dose were positively associated with chemoradiation-induced hearing loss. We performed a genome-wide association study (GWAS) in 777 NPC patients and identified rs1050851 (within the exon 2 of NFKBIA), a variant with a high deleteriousness score, to be significantly associated with hearing loss risk (HR = 5.46, 95% CI 2.93-10.18, P = 9.51 × 10-08). The risk genotype of rs1050851 was associated with higher NFKBIA expression, which was correlated with lower cellular tolerance to cisplatin. According to permutation-based enrichment analysis, the variants mapping to 149 hereditary deafness genes were significantly enriched among GWAS top signals, which indicated the genetic similarity between hereditary deafness and CRIHL. Pathway analysis suggested that synaptic signaling was involved in the development of CRIHL. Additionally, the risk score integrating genetic and clinical factors can predict the risk of hearing loss with a relatively good performance in the test set. Collectively, this study shed new light on the etiology of chemoradiation-induced hearing loss, which facilitates high-risk individuals' identification for personalized prevention and treatment.

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.

Establishment and validation of a plasma oncofetal chondroitin sulfated proteoglycan for pan-cancer detection.

Various biomarkers targeting cell-free DNA (cfDNA) and circulating proteins have been tested for pan-cancer detection. Oncofetal chondroitin sulfate (ofCS), which distinctively modifies proteoglycans (PGs) of most cancer cells and binds specifically to the recombinant Plasmodium falciparum VAR2CSA proteins (rVAR2), is explored for its potential as a plasma biomarker in pan-cancer detection. To quantitate the plasma ofCS/ofCSPGs, we optimized an ELISA using different capture/detection pairs (rVAR2/anti-CD44, -SDC1, and -CSPG4) in a case-control study with six cancer types. We show that the plasma levels of ofCS/ofCSPGs are significantly higher in cancer patients (P values, 1.2 × 10-2 to 4.4 × 10-10). Validation studies are performed with two independent cohorts covering 11 malignant tumors. The individuals in the top decile of ofCS-CD44 have more than 27-fold cancer risk (OR = 27.8, 95%CI = 18.8-41.4, P = 2.72 × 10-62) compared with the lowest 20%. Moreover, the elevated plasma ofCS-CD44 could be detected at the early stage of pan-cancer with strong dose-dependent odds risk prediction.

Oral Microbiota Alteration and Roles in Epstein-Barr Virus Reactivation in Nasopharyngeal Carcinoma.

Microbiota has recently emerged as a critical factor associated with multiple malignancies. Nasopharyngeal carcinoma (NPC) is highly associated with Epstein-Barr virus (EBV); the oncovirus resides and is transmitted in the oral cavity. However, the alternation of oral microbiota in NPC patients and its potential link to EBV reactivation and host cell response under the simultaneous existence of EBV and specific bacteria is largely unknown. Here, oral microbiota profiles of 303 NPC patients and controls with detailed clinical information, including serum EBV anti-virus capsid antigen (VCA) IgA level, were conducted. A distinct microbial community with lower diversity and imbalanced composition in NPC patients was observed. Notably, among enriched bacteria in patients, Streptococcus sanguinis was associated with anti-VCA IgA, an indicator of NPC risk and EBV reactivation. By measuring the concentration of its metabolite, hydrogen peroxide (H2O2), in the saliva of clinical patients, we found the detection rate of H2O2 was 2-fold increased compared to healthy controls. Further coculture assay of EBV-positive Akata cells with bacteria in vitro showed that S. sanguinis induced EBV lytic activation by its metabolite, H2O2. Host and EBV whole genome-wide transcriptome sequencing and EBV methylation assays showed that H2O2 triggered the host cell signaling pathways, notably tumor necrosis factor alpha (TNF-α) via NF-κB, and induced the demethylation of the global EBV genome and the expression of EBV lytic-associated genes, which could result in an increase of virus particle release and potential favorable events toward tumorigenesis. In brief, our study identified a characterized oral microbial profile in NPC patients and established a robust link between specific oral microbial alteration and switch of latency to lytic EBV infection status in the oral cavity, which provides novel insights into EBV's productive cycle and might help to further clarify the etiology of NPC. IMPORTANCE EBV is classified as the group I human carcinogen and is associated with multiple cancers, including NPC. The interplays between the microbiota and oncovirus in cancer development remain largely unknown. In this study, we investigate the interactions between resident microbes and EBV coexistence in the oral cavity of NPC patients. We identify a distinct oral microbial feature for NPC patients. Among NPC-enriched bacteria, we illustrated that a specific species, S. sanguinis, associated with elevated anti-IgA VCA in patients, induced EBV lytic activation by its by-product, H2O2, and activated the TNF-α/NF-κB pathway of EBV-positive B cells in vitro, together with increased detection rate of H2O2 in patients' oral cavities, which strengthened the evidence of bacteria-virus-host interaction in physiological circumstances. The effects of imbalanced microbiota on the EBV latent-to-lytic switch in the oral cavity might create the likelihood of EBV infection in epithelial cells at the nasopharynx and help malignant transition and cancer development.

Development and validation of a polygenic hazard score to predict prognosis and adjuvant chemotherapy benefit in early-stage non-small cell lung cancer.

Background: It remains controversial who would benefit from adjuvant chemotherapy (ACT) in patients with early-stage non-small cell lung cancer (NSCLC). We aim to construct a polygenic hazard score (PHS) to predict prognosis and ACT benefit among NSCLC patients. Methods: We conducted a retrospective study including 1,395 stage I-II NSCLC patients. We performed a genome-wide association study (GWAS) on overall survival (OS) in patients treated with ACT (SYSUCC ACT set, n=404), and then developed a PHS using LASSO Cox regression in a random subset (training, n=202) and tested it in the remaining set (test, n=202). The PHS was further validated in two independent datasets (SYSUCC surgery set, n=624; PLCO cohort, n=367). Results: The GWAS-derived PHS consisting of 37 single-nucleotide polymorphisms (SNPs) was constructed to classify patients into high and low PHS groups. For patients treated with ACT, those with low PHS had better clinical outcomes than high PHS (test set: HR =0.21, P<0.001; PLCO ACT set: HR =0.33, P=0.260). Similar results were found in the extended validation cohorts including patients with or without ACT (SYSUCC: HR =0.48, P<0.001; PLCO: HR =0.60, P=0.033). Within subgroup analysis by treatment or clinical factors, we further observed consistent results for the prognostic value of the PHS. Notably, ACT significantly improved OS in stage II patients with low PHS (HR =0.26, P<0.001), while there was no ACT survival benefit among patients with high PHS (HR =0.97, P=0.860). Conclusions: The PHS improved prognostic stratification and could help identify patients who were most likely to benefit from ACT in early-stage NSCLC.

Rare POLN mutations confer risk for familial nasopharyngeal carcinoma through weakened Epstein-Barr virus lytic replication.

BACKGROUND: Nasopharyngeal carcinoma (NPC) exhibits significant familial aggregation; however, its susceptibility genes are largely unknown. Thus, this study aimed to identify germline mutations that might contribute to the risk of familial NPC, and explore their biological functions. METHODS: Whole-exome sequencing was performed in 13 NPC pedigrees with multiple cases. Mutations co-segregated with disease status were further validated in a cohort composed of 563 probands from independent families, 2,953 sporadic cases, and 3,175 healthy controls. Experimental studies were used to explore the functions of susceptibility genes and their disease-related mutations. FINDINGS: The three rare missense mutations in POLN (DNA polymerase nu) gene, P577L, R303Q, and F545C, were associated with familial NPC risk (5/576 [0·87%] in cases vs. 2/3374 [0·059%] in healthy controls with an adjusted OR of 44·84 [95% CI:3·91-514·34, p = 2·25 × 10-3]). POLN was involved in Epstein-Barr virus (EBV) lytic replication in NPC cells in vitro. POLN promoted viral DNA replication, immediate-early and late lytic gene expression, and progeny viral particle production, ultimately affecting the proliferation of host cells. The three mutations were located in two pivotal functional domains and were predicted to alter the protein stability of POLN in silico. Further assays demonstrated that POLN carrying any of the three mutations displayed reduced protein stability and decreased expression levels, thereby impairing its ability to promote complete EBV lytic replication and facilitate cell survival. INTERPRETATION: We identified a susceptibility gene POLN for familial NPC and elucidated its function. FUNDING: This study was funded by the National Key Research and Development Program of China (2021YFC2500400); the National Key Research and Development Program of China (2020YFC1316902); the Basic and Applied Basic Research Foundation of Guangdong Province, China (2021B1515420007); the National Natural Science Foundation of China (81973131); the National Natural Science Foundation of China (82003520); the National Natural Science Foundation of China (81903395).

Whole-Exome Sequencing Study of Familial Nasopharyngeal Carcinoma and Its Implication for Identifying High-Risk Individuals.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is closely associated with genetic factors and Epstein-Barr virus infection, showing strong familial aggregation. Individuals with a family history suffer elevated NPC risk, requiring effective genetic counseling for risk stratification and individualized prevention. METHODS: We performed whole-exome sequencing on 502 familial NPC patients and 404 unaffected relatives and controls. We systematically evaluated the established cancer predisposition genes and investigated novel NPC susceptibility genes, making comparisons with 21 other familial cancers in the UK biobank (N = 5218). RESULTS: Rare pathogenic mutations in the established cancer predisposition genes were observed in familial NPC patients, including ERCC2 (1.39%), TP63 (1.00%), MUTYH (0.80%), and BRCA1 (0.80%). Additionally, 6 novel susceptibility genes were identified. RAD54L, involved in the DNA repair pathway together with ERCC2, MUTYH, and BRCA1, showed the highest frequency (4.18%) in familial NPC. Enrichment analysis found mutations in TP63 were enriched in familial NPC, and RAD54L and EML2 were enriched in both NPC and other Epstein-Barr virus-associated cancers. Besides rare variants, common variants reported in the studies of sporadic NPC were also associated with familial NPC risk. Individuals in the top quantile of common variant-derived genetic risk score while carrying rare variants exhibited increased NPC risk (odds ratio = 13.47, 95% confidence interval = 6.33 to 28.68, P = 1.48 × 10-11); men in this risk group showed a cumulative lifetime risk of 24.19%, much higher than those in the bottom common variant-derived genetic risk score quantile and without rare variants (2.04%). CONCLUSIONS: This study expands the catalog of NPC susceptibility genes and provides the potential for risk stratification of individuals with an NPC family history.

Genomic landscape of Epstein-Barr virus in familial nasopharyngeal carcinoma.

To better understand the genomic characteristics of Epstein-Barr virus (EBV) in familial nasopharyngeal carcinoma (NPC), we sequenced the EBV genomes by whole-genome capture in 38 unrelated patients with NPC family history in first-degree relatives and 47 healthy controls, including 13 with family history and 34 without. Compared with type 1 reference genome, mutation hotspots were observed in the latent gene regions of EBV in familial NPC cases. Population structure analysis showed that one cluster has a higher frequency in familial cases than in controls (OR=5.33, 95 % CI 2.50-11.33, P=1.42×10-5), and similar population structure composition was observed among familial and sporadic NPC cases in high-endemic areas. By genome-wide association analysis, four variants were found to be significantly associated with familial NPC. Consistent results were observed in the meta-analysis integrating two published case-control EBV sequencing studies in NPC high-endemic areas. High-risk haplotypes of EBV composed of 34 variants were associated with familial NPC risk (OR=13.85, 95 % CI 4.13-46.44, P=2.06×10-5), and higher frequency was observed in healthy blood-relative controls with NPC family history (9/13, 69.23 %) than those without family history (16/34, 47.06%). This study suggested the potential contribution of EBV high-risk subtypes to familial aggregation of NPC.

Genomic Landscapes of Epstein-Barr Virus in Pulmonary Lymphoepithelioma-Like Carcinoma.

Epstein-Barr virus (EBV) infection is associated with multiple malignancies, including pulmonary lymphoepithelioma-like carcinoma (pLELC), a particular subtype of primary lung cancer. However, the genomic characteristics of EBV related to pLELC remain unclear. Here, we obtained the whole-genome data set of EBV isolated from 78 pLELC patients and 37 healthy controls using EBV-captured sequencing. Compared with the reference genome (NC_007605), a total of 3,995 variations were detected across pLELC-derived EBV sequences, with the mutational hot spots located in latent genes. Combined with 180 published EBV sequences derived from healthy people in Southern China, we performed a genome-wide association study and identified 32 variations significantly related to pLELC (P < 2.56 × 10-05, Bonferroni correction), with the top signal of single nucleotide polymorphism (SNP) coordinate T7327C (OR = 1.22, P = 2.39 × 10-15) locating in the origin of plasmid replication (OriP). The results of population structure analysis of EBV isolates in East Asian showed the EBV strains derived from pLELC were more similar to those from nasopharyngeal carcinoma (NPC) than other EBV-associated diseases. In addition, typical latency type-II infection were recognized for EBV of pLELC at both transcription and methylation levels. Taken together, we defined the global view of EBV genomic profiles in pLELC patients for the first time, providing new insights to deepening our understanding of this rare EBV-associated primary lung carcinoma. IMPORTANCE Pulmonary lymphoepithelioma-like carcinoma (pLELC) is a rare, distinctive subtype of primary lung cancer closely associated with Epstein-Barr virus (EBV) infection. Here, we gave the first overview of pLELC-derived EBV at the level of genome, methylation and transcription. We obtained the EBV sequences data set from 78 primary pLELC patients, and revealed the sequences diversity across EBV genome and detected variability in known immune epitopes. Genome-wide association analysis combining 217 healthy controls identifies significant variations related to the risk of pLELC. Meanwhile, we characterized the integration landscapes of EBV at the genome-wide level. These results provided new insight for understanding EBV's role in pLELC tumorigenesis.

Glycogenes in Oncofetal Chondroitin Sulfate Biosynthesis are Differently Expressed and Correlated With Immune Response in Placenta and Colorectal Cancer.

Oncofetal chondroitin sulfate expression plays an important role in the development of tumors and the pathogenesis of malaria in pregnancy. However, the biosynthesis and functions of these chondroitin sulfates, particularly the tissue-specific regulation either in tumors or placenta, have not been fully elucidated. Here, by examining the glycogenes availability in chondroitin sulfate biosynthesis such as xylosytransferase, chondroitin synthase, sulfotransferase, and epimerase, the conserved or differential CS glycosylation in normal, colorectal cancer (CRC), and placenta tissue were predicted. We found that the expression of seven chondroitin sulfate biosynthetic enzymes, namely B4GALT7, B3GALT6, B3GAT3, CHSY3, CHSY1, CHPF, and CHPF2, were significantly increased, while four other enzymes (XYLT1, CHST7, CHST15, and UST) were decreased in the colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) patients. In the human placenta, where the distinct chondroitin sulfate is specifically bound with VAR2CSA on Plasmodium parasite-infected RBC, eight chondroitin sulfate biosynthesis enzymes (CSGALNACT1, CSGALNACT2, CHSY3, CHSY1, CHPF, DSE, CHST11, and CHST3) were significantly higher than the normal colon tissue. The similarly up-regulated chondroitin synthases (CHSY1, CHSY3, and CHPF) in both cancer tissue and human placenta indicate an important role of the proteoglycan CS chains length for Plasmodium falciparum VAR2CSA protein binding. Interestingly, twelve highly expressed chondroitin sulfate enzymes were significantly correlated to worse outcomes (prognosis) in both COAD and READ. Furthermore, we showed that the levels of chondroitin sulfate enzymes are significantly correlated with the expression of immuno-regulators and immune infiltration levels in CRCs and placenta, and involved in multiple essential pathways, such as extracellular matrix organization, epithelial-mesenchymal transition, and cell adhesion. Our study provides novel insights into the oncofetal chondroitin sulfate biosynthesis regulation and identifies promising targets and biomarkers of immunotherapy for CRC and malaria in pregnancy.

Association between HLA alleles and Epstein-Barr virus Zta-IgA serological status in healthy males from southern China.

BACKGROUND: Nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV) associated cancer, exhibits an extremely high incidence in southern Chinese. Given that human leukocyte antigen (HLA) plays critical roles in antigen presentation and relates to NPC susceptibility, it is speculated that certain HLA variants may affect EBV reactivation, which is a key pathogenic factor of NPC. Therefore, we attempted to identify HLA alleles associated with the indicator of EBV reactivation, Zta-IgA, in healthy males from NPC endemic area. METHODS: HLA alleles of 1078 healthy males in southern China from the 21-RCCP study were imputed using genome-wide single nucleotide polymorphism data. EBV Zta-IgA in blood samples were measured using an enzyme-linked immunosorbent assay. Multiple logistic regression analysis was used to evaluate the effect of HLA allele on Zta-IgA serological status and its potential joint association with smoking. The binding affinity for Zta-peptide was predicted using NetMHCIIpan 4.0. RESULTS: HLA-DRB1*09:01 was found to be associated with a higher risk of Zta-IgA seropositivity (odds ratio = 1.80, 95% confidence interval = 1.32-2.45; p = 1.82 × 10-4 ). Compared with non-smokers without HLA-DRB1*09:01, the effect size increased to 2.19- and 3.70-fold for the light and heavy smokers carrying HLA-DRB1*09:01, respectively. Furthermore, HLA-DRB1*09:01 showed a stronger binding affinity to Zta peptide than other HLA-DRB1 alleles. CONCLUSIONS: Our study highlighted the pivotal role of genetic HLA variants in EBV reactivation and the etiology of NPC. Smokers with HLA-DRB1*09:01 have a significantly higher risk of being Zta-IgA seropositive, which indicates the necessity of smoking cessation in certain high-risk populations and also provide clues for further research on the etiology of NPC.

Nasopharyngeal Epstein-Barr virus DNA loads in high-risk nasopharyngeal carcinoma families: Familial aggregation and host heritability.

Nasopharyngeal carcinoma (NPC), the most common head and neck cancer, is characterized by distinct geographic distribution and familial aggregation. Multiple risk factors, including host genetics, environmental factor, and EBV infection, have been linked to the development of NPC, particularly in the familial clustering cases. However, the cause of NPC endemicity remains enigmatic due possibly to the complicated interplay between these risk factors. Recently, positive Epstein-Barr virus (EBV) DNA loads at nasopharyngeal (NP) cavity has been found to reflect NPC development and applied in NPC screening. To examine whether the increased NP EBV loads could aggregate in the families and be affected by host genetics and environmental factor, EBV loads were obtained by 510 NP brushing samples from eligible unaffected individuals, who have two or more relatives affected with NPC, in 116 high-risk NPC families. The correlation of relative pairs was estimated using S.A.G.E. (version 6.4, 2016), and host heritability of NP EBV loads was calculated with variance component models using SOLAR (version 8.4.2, 2019). In result, significant correlations of EBV loads were observed between parent-offspring pairs and sibling-sibling pairs (P < .001), but not in distant kin relationship pairs. Interestingly, after excluding the shared environmental factor within families, host genetics contributes significantly to NP EBV loads with a heritability of 56.41% (P = 1.00 × 10-7 ), and its effect was slightly elevated (68.86%, P = 3.40 × 10-6 ) in families with more NPC cases (≥3). These findings indicate that additional host-genetic variants involved in the EBV local NP mucosal behavior may be especially important for the development of NPC.