Nasopharyngeal carcinoma risk prediction via salivary detection of host and Epstein-Barr virus genetic variants.
ABSTRACT: Genetic susceptibility and Epstein-Barr virus (EBV) infection are important etiological factors in nasopharyngeal carcinoma (NPC). In this study, in southern China, where NPC is endemic, a single nucleotide polymorphism (SNP) in the EBV-encoded RPMS1 gene (locus 155391: G > A [G155391A]) and seven host SNPs (rs1412829, rs28421666, rs2860580, rs2894207, rs31489, rs6774494, and rs9510787) were confirmed to be significantly associated with NPC risk in 50 NPC cases versus 54 hospital-based controls with throat washing specimens and 1925 NPC cases versus 1947 hospital-based controls with buffy coat samples, respectively. We established a strategy to detect the NPC-associated EBV and host SNPs using saliva samples in a single test that is convenient, noninvasive, and cost-effective and displays good compliance. The potential utility of this strategy was tested by applying a risk prediction model integrating these EBV and host genetic variants to a population-based case-control study comprising 1026 incident NPC cases and 1148 controls. Receiver operating characteristic (ROC) curve analysis revealed an area under the curve of the NPC risk prediction model of 0.74 (95% CI: 0.71-0.76). Net reclassification improvement (NRI) analysis showed that inclusion of the EBV SNP significantly improved the discrimination ability of the model (NRI = 0.30, P < 0.001), suggesting the promising value of EBV characteristics for identifying high-risk NPC individuals in endemic areas. Taken together, we developed a promising NPC risk prediction model via noninvasive saliva sampling. This approach might serve as a convenient and effective method for screening the population with high-risk of NPC.
Project description:BACKGROUND:Epstein-Barr virus (EBV) commonly infects the general population and has been associated with nasopharyngeal carcinoma (NPC), which has a high incidence in certain regions. This study aimed to address how EBV variations contribute to the risk of NPC. METHODS:Using logistic regression analysis and based on the sequence variations at EBV-encoded RPMS1, a multi-stage association study was conducted to identify EBV variations associated with NPC risk. A protein degradation assay was performed to characterize the functional relevance of the RPMS1 variations. RESULTS:Based on EBV-encoded RPMS1 variations, a single nucleotide polymorphism (SNP) in the EBV genome (locus 155391: G>A, named G155391A) was associated with NPC in 157 cases and 319 healthy controls from an NPC endemic region in South China [P < 0.001, odds ratio (OR) = 4.47, 95% confidence interval (CI) 2.71-7.37]. The results were further validated in three independent cohorts from the NPC endemic region (P < 0.001, OR = 5.20, 95% CI 3.18-8.50 in 168 cases vs. 241 controls, and P < 0.001, OR = 5.27, 95% CI 4.06-6.85 in 726 cases vs. 880 controls) and a non-endemic region (P < 0.001, OR = 7.52, 95% CI 3.69-15.32 in 58 cases vs. 612 controls). The combined analysis in 1109 cases and 2052 controls revealed that the SNP G155391A was strongly associated with NPC (P(combined) < 0.001, OR = 5.27, 95% CI 4.31-6.44). Moreover, the frequency of the SNP G155391A was associated with NPC incidence but was not associated with the incidences of other EBV-related malignancies. Furthermore, the protein degradation assay showed that this SNP decreased the degradation of the oncogenic RPMS1 protein. CONCLUSIONS:Our study identified an EBV variation specifically and significantly associated with a high risk of NPC. These findings provide insights into the pathogenesis of NPC and strategies for prevention.
Project description:BACKGROUND:Genetic loci within the major histocompatibility complex (MHC) have been associated with nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV)-associated cancer, in several GWAS. Results outside this region have varied. METHODS:We conducted a meta-analysis of four NPC GWAS among Chinese individuals (2,152 cases; 3,740 controls). Forty-three noteworthy findings outside the MHC region were identified and targeted for replication in a pooled analysis of four independent case-control studies across three regions in Asia (4,716 cases; 5,379 controls). A meta-analysis that combined results from the initial GWA and replication studies was performed. RESULTS:In the combined meta-analysis, rs31489, located within the CLPTM1L/TERT region on chromosome 5p15.33, was strongly associated with NPC (OR = 0.81; P value 6.3 × 10(-13)). Our results also provide support for associations reported from published NPC GWAS-rs6774494 (P = 1.5 × 10(-12); located in the MECOM gene region), rs9510787 (P = 5.0 × 10(-10); located in the TNFRSF19 gene region), and rs1412829/rs4977756/rs1063192 (P = 2.8 × 10(-8), P = 7.0 × 10(-7), and P = 8.4 × 10(-7), respectively; located in the CDKN2A/B gene region). CONCLUSIONS:We have identified a novel association between genetic variation in the CLPTM1L/TERT region and NPC. Supporting our finding, rs31489 and other SNPs in this region have been reported to be associated with multiple cancer sites, candidate-based studies have reported associations between polymorphisms in this region and NPC, the TERT gene has been shown to be important for telomere maintenance and has been reported to be overexpressed in NPC, and an EBV protein expressed in NPC (LMP1) has been reported to modulate TERT expression/telomerase activity. IMPACT:Our finding suggests that factors involved in telomere length maintenance are involved in NPC pathogenesis.
Project description:Epstein-Barr virus (EBV) establishes lifelong latent infection in humans and is associated with several lymphoid and epithelial cancers. In nasopharyngeal carcinoma (NPC), EBV expresses few viral proteins but elevated levels of Bam-HI A rightward transcripts (BARTs) RNA, which includes viral microRNAs and long non-coding RNAs (lncRNAs). BART lncRNAs localize within the nucleus of EBV-infected cells and knockdown of BART lncRNAs significantly affects the expression of genes associated with cell adhesion, oxidoreductase activity, inflammation, and immunity. Notably, downregulation of IKAROS family zinc finger 3 (IKZF3/Aiolos), which plays a role in lymphocyte development and cell attachment, occurred in NPC C666-1 cells following BART lncRNA-knockdown. Since Aiolos expression is normally restricted to lymphoid cells and rarely observed in epithelial cells, induction of Aiolos by BART lncRNA was confirmed by expressing the major BART lncRNA isoform, RPMS1, in EBV-positive and -negative cells. BART lncRNA associated with the CBP/p300 complex and RNA polymerase II (Pol II) in the nucleus, suggesting that BART lncRNAs may mediate epigenetic regulation of gene expression through interaction with the chromatin remodeling machinery. This contention is further supported by evidence that BART lncRNA appears to stall Pol II at the promoter region and may regulate IFNB1 and CXCL8 expression by inhibiting transcription by Pol II in NPC. We hypothesize that EBV BART lncRNA expression modulates host gene expression and maintains EBV latency by interfering with histone methylation and acetylation processes. Aberrant expression of affected host genes mediated by BART lncRNA may lead to immune evasion, progression, and metastasis of NPC.
Project description:To date, the only established model for assessing risk for nasopharyngeal carcinoma (NPC) relies on the sero-status of the Epstein-Barr virus (EBV). By contrast, the risk assessment models proposed here include environmental risk factors, family history of NPC, and information on genetic variants. The models were developed using epidemiological and genetic data from a large case-control study, which included 1,387 subjects with NPC and 1,459 controls of Cantonese origin. The predictive accuracy of the models were then assessed by calculating the area under the receiver-operating characteristic curves (AUC). To compare the discriminatory improvement of models with and without genetic information, we estimated the net reclassification improvement (NRI) and integrated discrimination index (IDI). Well-established environmental risk factors for NPC include consumption of salted fish and preserved vegetables and cigarette smoking (in pack years). The environmental model alone shows modest discriminatory ability (AUC?=?0.68; 95% CI: 0.66, 0.70), which is only slightly increased by the addition of data on family history of NPC (AUC?=?0.70; 95% CI: 0.68, 0.72). With the addition of data on genetic variants, however, our model's discriminatory ability rises to 0.74 (95% CI: 0.72, 0.76). The improvements in NRI and IDI also suggest the potential usefulness of considering genetic variants when screening for NPC in endemic areas. If these findings are confirmed in larger cohort and population-based case-control studies, use of the new models to analyse data from NPC-endemic areas could well lead to earlier detection of NPC.
Project description:Circulating RNAs in serum, plasma or other body liquid have emerged as useful and highly promising biomarkers for noninvasive diagnostic application. Herein, we aimed to establish a serum long non-coding RNAs (lncRNAs) signature for diagnosing nasopharyngeal carcinoma (NPC). In this study, we recruited a cohort of 101 NPC patients, 20 patients with chronic nasopharyngitis (CN), 20 EBV carriers (EC) and 101 healthy controls. qRT-PCR was performed with NPC cells and serum samples to screen a pool of 38 NPC-related lncRNAs obtained from the LncRNADisease database. A profile of three circulating lncRNAs (MALAT1, AFAP1-AS1 and AL359062) was established for NPC diagnosis. By Receiver Operating Characteristic (ROC) curve analysis, this three-lncRNA signature showed high accuracy in discriminating NPC from healthy controls (AUC = 0.918), CN (AUC = 0.893) or EC (AUC = 0.877). Furthermore, high levels of these three lncRNAs were closely related to advanced NPC tumor node metastasis stages and EBV infection. Serum levels of these three lncRNAs declined significantly in patients after therapy. Our present study indicates that circulating MALAT1, AFAP1-AS1 and AL359062 may represent novel serum biomarkers for NPC diagnosis and prognostic prediction after treatment.
Project description:EBV is a key risk factor for many malignancy diseases such as nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL). EBV integration has been reported, but its scale and impact to cancer development is remains unclear. C666-1 (NPC cell line) and Raji (BL cell line) are commonly studied EBV-positive cancer cells. A rare few EBV integration sites in Raji were found in previous research by traditional methods. To deeply survey EBV integration, we sequenced C666-1 and Raji whole genomes by the next generation sequencing (NGS) technology and a total of 909 breakpoints were detected in the two cell lines. Moreover, we observed that the number of integration sites was positive correlated with the total amount of chromosome structural variations (SVs) and copy number structural variations (CNVs), and most breakpoints located inside or nearby genome structural variations regions. It suggested that host genome instability provided an opportunity for EBV integration on one hand and the integration aggravated host genome instability on the other hand. Then, we respectively assembled the C666-1 and Raji EBV strains which would be useful resources for EBV-relative studies. Thus, we report the most comprehensive characterization of EBV integration in NPC cell and BL cell, and EBV shows the wide range and random integration to increase the tumorigenesis. The NGS provides an incomparable level of resolution on EBV integration and a convenient approach to obtain viral strain compared to any research technology before.
Project description:Nuclear antigen-1 (NA1) protein of Epstein-Barr virus (EBV) is expressed in EBV-infected cells in the microenvironment of cancer. Since immune cells infiltrate abundantly in nasopharyngeal carcinoma (NPC) tumor tissues, we hypothesized that the local tumor microenvironment may perform an important role in the production of antibodies directed at NA1. Furthermore, we hypothesized that anti-NA1 antibody originating in the local microenvironment could be secreted into the saliva of patients with NPC. In the present study, 20 healthy controls and 39 patients with NPC treated with intensity-modulated radiation therapy were recruited for the study. Saliva and serum samples were collected from the NPC patients, and nasopharyngeal tissue samples from the patients with NPC. The titers of anti-NA1 antibody [immunoglobulin A (IgA)] were determined by ELISA. Expression of NA1, human leukocyte antigen-antigen D related (HLA-DR), cluster of differentiation (CD)80, CD86, CD3, CD4, CD19 and IgA was detected by immunohistochemical staining on paraffin-embedded nasopharyngeal tissue sections. Anti-NA1 antibodies were detected in the serum and saliva samples of the patients with NPC. In infiltrating cells, expression of HLA-DR, CD80, CD86, CD3, CD4, CD19 and IgA was detected, indicating that dendritic cells, T lymphocytes and B lymphocytes were all present in the local tumor tissues. Furthermore, expression of EBNA1 protein was detected on the membrane of the NPC tumor cells. Therefore, the NPC tumor microenvironment has the potential to initiate a humoral response to EBNA1 by producing IgA antibodies.
Project description:BACKGROUND:Epstein-Barr virus (EBV) infection is a crucial risk factor for nasopharyngeal carcinoma (NPC), but the mechanism for its elevated activation level in NPC endemic areas remains unclear. This study aims to identify the EBV natural variations contributed to the different reactivation potential between NPC endemic and non-endemic areas. METHODS:1030 subjects were recruited in China, including 303 healthy individuals from two NPC non-endemic areas, 483 healthy people from three endemic areas and 244 NPC patients. Among which, saliva DNA samples from 244 participants were sequenced for the EBV immediate early (IE) genes of BRLF1 and BZLF1, their promoters were included; the rest 786 subjects were used for the validation of significant variations among three different populations. Haplotype and population structure analysis were conducted. Dual-luciferase assay was used to detect the promoter activity. RESULTS:A total of 246 distinct variations were detected, 29 showed significant difference in the frequencies between healthy people from NPC endemic area and non-endemic area. Population structure analysis clustered EBV strains into 9 subgroups mostly in accordance with the geographical origin of samples. Interestingly, two EBV genotypes, Rp-V1 and Rp-V2, were identified according to the linkage relationship of the variations in BRLF1 promoter (Rp). Rp-V1 has higher frequency in NPC endemic areas than in non-endemic areas (52.38% vs 18.15%, P?=?2.07?×?10-14), and was associated with higher oral EBV DNA levels (adjusted OR?=?1.64, 95% CI =?1.21-2.24, P?=?.002), suggesting a more powerful activation ability of Rp-V1 than that of the prototype Rp-of the EBV strain; On the contrary, Rp-V2 has higher frequency in NPC non-endemic areas than in endemic areas (18.48% vs 0.38%, P?=?1.17?×?10-7), might represent a reduced activation potential of EBV. Further dual-luciferase assay showed Rp-V1 has higher promoter activity while compared with Rp-V2 (P?<?.0001). Notably, Rp-V1 impaired the transcription repression effect of YY1 while Rp-V2 strengthened the transcription repression effect of EBF1 on Rp. In addition, significant differences of Rta 393-407 CTL epitope which may influence the recognition of Rta by CD8+ T cells were detected between healthy people from NPC endemic area and non-endemic area. CONCLUSIONS:This study identified natural variations in cis-acting elements (YY1 and EBF1) of EBV Rp altering Rp transcription activities, which may contribute to the elevated EBV activation level in NPC endemic areas than non-endemic areas.
Project description:Exosomes have emerged as novel vehicles for proteins and other contents in cancer progression. Cyclophilin A (CYPA) is a pivotal member of immunophilin family. Whether CYPA can be detected in sera of nasopharyngeal carcinoma (NPC) patients remains to be explored. Epstein-Barr virus (EBV) is the first identified human tumor virus and is a causative agent of NPC. The antibody of EBV capsid antigen immunoglobulin A (EBV-VCA-IgA) is a known biomarker of NPC, with a proportion of no more than 70% being detected positively. Hence, novel biomarkers need to be discovered for early diagnosis, prognosis, and monitoring of EBV-associated NPC. A total of 110 NPC and 36 normal control serum samples were collected. Exosomes from these samples were extracted. The mRNA and protein expression levels of the above samples were validated by reverse transcription -quantitative polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay (ELISA). Finally, the results demonstrated that both the serum and exosomal CYPA levels of NPC patients were significantly higher than that of normal cases. In addition, exosomal CYPA had a much higher level than that in the whole sera. The positive rate of EBV-VCA-IgA antibody was 68.2% in NPC sera, and noticeably, among the cases with EBV-VCA-IgA negative, 80% of them presented high levels of CYPA above the standard (cutoff value). In particular, CYPA in exosomes was uniformly with higher significance than that in whole sera. Combined analysis of CYPA protein and EBV-VCA-IgA antibody showed a greatly higher discriminatory ability in diagnosis of NPC. Moreover, exosomal CYPA level had a positive correlation with that of the EBV-encoded latent membrane protein 1 (LMP1) in exosomes. EBV-positive cancer cells secreted significantly higher levels of exosomal CYPA. This study established the utility of circulating exosomal CYPA as a potential noninvasive diagnostic biomarker for EBV-associated NPC.