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Abstract Epstein-Barr virus (EBV) infects more than 95% of adults worldwide and is closely associated with various malignancies. Considering the complex life cycle of EBV, developing vaccines targeting key entry glycoproteins to elicit robust and durable adaptive immune responses may provide better protection. EBV gHgL-, gB- and gp42-specific antibodies in healthy EBV carriers contributed to sera neutralizing abilities in vitro, indicating that they are potential antigen candidates. To enhance the immunogenicity of these antigens, we formulate three nanovaccines by co-delivering molecular adjuvants (CpG and MPLA) and antigens (gHgL, gB or gp42). These nanovaccines induce robust humoral and cellular responses through efficient activation of dendritic cells and germinal center response. Importantly, these nanovaccines generate high levels of neutralizing antibodies recognizing vulnerable sites of all three antigens. IgGs induced by a cocktail vaccine containing three nanovaccines confer superior protection from lethal EBV challenge in female humanized mice compared to IgG elicited by individual NP-gHgL, NP-gB and NP-gp42. Importantly, serum antibodies elicited by cocktail nanovaccine immunization confer durable protection against EBV-associated lymphoma. Overall, the cocktail nanovaccine shows robust immunogenicity and is a promising candidate for further clinical trials.

Epstein–Barr virus (EBV) is the first reported human oncogenic virus and infects more than 95% of the human population worldwide. EBV latent infection in B lymphocytes is essential for viral persistence. Glycoprotein gp42 is an indispensable member of the triggering complex for EBV entry into B cells. The C-type lectin domain (CTLD) of gp42 plays a key role in receptor binding and is the major target of neutralizing antibodies. Here, we isolated two rabbit antibodies, 1A7 and 6G7, targeting gp42 CTLD with potent neutralizing activity against B cell infection. Antibody 6G7 efficiently protects humanized mice from lethal EBV challenge and EBV-induced lymphoma. Neutralizing epitopes targeted by antibodies 1A7 and 6G7 are distinct and novel. Antibody 6G7 blocks gp42 binding to B cell surface and both 1A7 and 6G7 inhibit membrane fusion with B cells. Furthermore, 1A7- and 6G7-like antibodies in immunized sera are major contributors to B cell neutralization. This study demonstrates that anti-gp42 neutralizing antibodies are effective in inhibiting EBV infection and sheds light on the design of gp42-based vaccines and therapeutics.

ABSTRACTReplicating SARS-CoV-2 has been shown to degrade HLA class I on target cells to evade the cytotoxic T-cell (CTL) response. HLA-I downregulation can be sensed by NK cells to unleash killer cell immunoglobulin-like receptor (KIR)-mediated self-inhibition by the cognate HLA-I ligands. Here, we investigated the impact of HLA and KIR genotypes and HLA-KIR combinations on COVID-19 outcome. We found that the peptide affinities of HLA alleles were not correlated with COVID-19 severity. The predicted poor binders for SARS-CoV-2 peptides belong to HLA-B subtypes that encode KIR ligands, including Bw4 and C1 (introduced by B*46:01), which have a small F pocket and cannot accommodate SARS-CoV-2 CTL epitopes. However, HLA-Bw4 weak binders were beneficial for COVID-19 outcome, and individuals lacking the HLA-Bw4 motif were at higher risk for serious illness from COVID-19. The presence of the HLA-Bw4 and KIR3DL1 combination had a 58.8% lower risk of developing severe COVID-19 (OR = 0.412, 95% CI = 0.187-0.904, p = 0.02). This suggests that HLA-Bw4 alleles that impair their ability to load SARS-CoV-2 peptides will become targets for NK-mediated destruction. Thus, we proposed that the synergistic responsiveness of CTLs and NK cells can efficiently control SARS-CoV-2 infection and replication, and NK-cell-mediated anti-SARS-CoV-2 immune responses being mostly involved in severe infection when the level of ORF8 is high enough to degrade HLA-I. The HLA-Bw4/KIR3DL1 genotype may be particularly important for East Asians undergoing COVID-19 who are enriched in HLA-Bw4-inhibitory KIR interactions and carry a high frequency of HLA-Bw4 alleles that bind poorly to coronavirus peptides.

Abstract Epstein-Barr virus (EBV), a γ-herpesvirus, is the first identified oncogenic virus, which establishes permanent infection in humans. EBV causes infectious mononucleosis and is also tightly linked to many malignant diseases. Various vaccine formulations underwent testing in different animals or in humans. However, none of them was able to prevent EBV infection and no vaccine has been approved to date. Current efforts focus on antigen selection, combination, and design to improve the efficacy of vaccines. EBV glycoproteins such as gH/gL, gp42, and gB show excellent immunogenicity in preclinical studies compared to the previously favored gp350 antigen. Combinations of multiple EBV proteins in various vaccine designs become more attractive approaches considering the complex life cycle and complicated infection mechanisms of EBV. Besides, rationally designed vaccines such as virus-like particles (VLPs) and protein scaffold-based vaccines elicited more potent immune responses than soluble antigens. In addition, humanized mice, rabbits, as well as nonhuman primates that can be infected by EBV significantly aid vaccine development. Innovative vaccine design approaches, including polymer-based nanoparticles, the development of effective adjuvants, and antibody-guided vaccine design, will further enhance the immunogenicity of vaccine candidates. In this review, we will summarize (i) the disease burden caused by EBV and the necessity of developing an EBV vaccine; (ii) previous EBV vaccine studies and available animal models; (iii) future trends of EBV vaccines, including activation of cellular immune responses, novel immunogen design, heterologous prime-boost approach, induction of mucosal immunity, application of nanoparticle delivery system, and modern adjuvant development.

Abstract Background Epstein-Barr virus (EBV) is a wide-spread human herpesvirus that is highly associated with infectious mononucleosis and several malignancies. Evaluation of EBV neutralizing antibody titers is important for serological studies, vaccine development and monoclonal antibody screening. The traditional method based on antibody inhibition of EBV transformation of B cells is very time-consuming. A more practical flow cytometry-based (FCM) approach to evaluate neutralizing titers is not amenable to achieving high-throughput evaluation of large-scale samples. A high-throughput approach is urgently needed. Results Here, we present a rapid and high-throughput method based on high content imaging system (HCIS) analysis. EBV titers determined by the HCIS-based assay were similar to those obtained by the FCM-based assay. Neutralizing titers of sera and monoclonal antibodies measured by the HCIS-based assay strongly correlated with titers measured by the FCM-based assay. HCIS assays showed a strong correlation between B cell infection neutralizing titers and the anti-gp350 IgG titers in healthy EBV carriers and monkey sera. Finally, anti-gHgL IgG titers from sera of healthy EBV carriers significantly correlated with epithelial cell infection neutralizing titers. Conclusions This HCIS-based assay is a high-throughput assay to determine viral titers and evaluate neutralizing potentials of sera and monoclonal antibodies. This HCIS-based assay will aid the development of vaccines and therapeutic monoclonal antibody against EBV.

Polygenic Risk Scores (PRS) show promise for facilitating cancer risk stratification. Here, the authors performed a large genome-wide association study and develop a PRS for nasopharyngeal carcinoma, which could improve risk stratification and personalized screening.

Purpose. The purpose of the current study was to determine whether there is a difference between high levels of microsatellite instability (MSI-H) and microsatellite stability (MSS) in DNA mismatch repair-deficient (DMMR) colorectal cancer (CRC) patients. Methods. A total of 452 CRC patients with DMMR from December, 2014, to April, 2021, in our hospital were selected retrospectively. However, only 105 patients underwent Sanger or next-generation-sequencing (NGS) to confirm their microsatellite status. Ultimately, 55 MSI-H patients and 20 MSS patients with intact medical record information were included in this study. Results. The MSS group was associated with a higher mutation rate in the KRAS gene (P=0.011). Meanwhile, MSI-H was related to colon cancer (P

The glucose transporter GLUT1 is upregulated in multiple cancers and may contribute to tumour progression, but the underlying mechanisms are poorly understood. Here, the authors show that DHHC9-mediated GLUT1 palmitoylation at Cys207 is crucial for plasma membrane localisation of GLUT1 and for tumourigenesis in glioblastoma cells.

Epstein-Barr virus (EBV) is associated with high risk for nasopharyngeal carcinoma (NPC). Here, the authors develop a comprehensive risk score that combines risk effects of EBV and human genetics for NPC risk stratification.

Abstract It remains unknown for decades how some of the therapeutic fusion proteins positive in a small percentage of cancer cells account for patient outcome. Here, we report that osteosarcoma Rab22a-NeoF1 fusion protein, together with its binding partner PYK2, is sorted into exosomes by HSP90 via its KFERQ-like motif (RVLFLN142). The exosomal Rab22a-NeoF1 fusion protein facilitates the pulmonary pre-metastatic niche formation by recruiting bone marrow-derived macrophages. The exosomal PYK2 activates RhoA in its negative recipient osteosarcoma cells and induces signal transducer and activator of transcription 3 activation in its recipient macrophages to increase M2 phenotype. Consequently, lung metastases of its recipient osteosarcoma cells are promoted by this exosomal Rab22a-NeoF1 fusion protein, and this event can be targeted by disrupting its interaction with PYK2 using a designed internalizing RGD peptide.

Nasopharyngeal carcinoma is a diverse cancer characterised by a heterogeneous microenvironment. Here, the authors use single cell sequencing to analyse the tumour microenvironment in 10 nasopharyngeal carcinoma tumours and identify different cell types including immune-suppressive T regulatory, tolerogenic dendritic, and exhausted CD8 T cells.

Epstein-Barr virus (EBV) tegument proteins BBRF2 and BSRF1 have been suggested to form a hetero-complex. Here, He et al. provide the crystal structures of BBRF2 alone and in complex with BSRF1 and suggest that the complex tethers EBV nucleocapsids to the Golgi membrane, facilitating secondary envelopment.

Osteosarcoma is an aggressive tumour and little is known the mechanisms underpinning its highly metastatic nature. Here, the authors highlight a role for the CK1α/CBX4 axis in driving metastasis, suggesting that this pathway might be targeted for therapeutic benefit.

Objectives: Given the role of exposures related to residence in the development of nasopharyngeal carcinoma (NPC) has not been well explored, present study aims to investigate the magnitude and pattern of associations for NPC with lifelong residential exposures. Materials and Methods: We carried out a multi-center, population-based case-control study with 2533 incident NPC cases and 2597 randomly selected population controls in southern China between 2010 and 2014. We performed multivariate logistic regression to estimate odds ratios (ORs) with 95% confidence intervals (CIs) for the risk of NPC associated with residential exposures. Results: Compared with those living in a building over lifetime, risk of NPC was higher for individuals living in a cottage (OR: 1.56; 95% CI: 1.34–1.81) or in a boat (3.87; 2.07–7.21). NPC risk was also increased in individuals using wood (1.34; 1.03–1.75), coal (1.70; 1.17–2.47), or kerosene (3.58; 1.75–7.36) vs. using gas/electricity as cooking fuel; using well water (1.57; 1.34–1.83), river water (1.80; 1.47–2.21), or spring/pond/stream water (2.03; 1.70–2.41) vs. tap water for source of drinking water; living in houses with smaller-sized vs. larger windows in the bedroom (3.08; 2.46–3.86), hall (1.89; 1.55–2.31) or kitchen (1.67; 1.34–2.08); and increasing exposure to cooking smoke [(1.53; 1.20–1.94) for high exposure)] or burned incense [(1.59; 1.31–1.95) for daily use)]. Weighted Cox regression analysis corroborated these results. Conclusion: Poorer residential conditions and household air pollution are associated with an increased risk of NPC. Large-scale studies in other populations or longitudinal studies are warranted to further corroborate these findings.

Abstract Epstein‐Barr virus (EBV) reactivation, reflected by aberrantly increased levels of various serological antibodies, has been suggested to be an early indicator of nasopharyngeal carcinoma (NPC) onset and progression. We have previously suggested that certain lifestyle and dietary factors were associated with elevated serological levels of the antibody against various EBV antigens namely VCA, Zta, EBNA1, and oral EBV DNA loads among healthy population. It remains unclear whether these potential environmental factors would also influence EBV serological antibodies in NPC patients. We conducted an epidemiological study to evaluate the associations between such environmental factors and EBV antibody levels among 1701 NPC patients in South China. Pretreatment serums were collected and examined for VCA‐IgA and EA‐IgA by immunoenzymatic assays and antienzyme rate (AER) of EBV DNase‐specific neutralizing antibody. We found that consumption of Canton‐style herbal tea was significantly correlated with increased serological antibody levels of VCA‐IgA and EA‐IgA, with adjusted ORs of 1.35 (95% CI: 1.03‐1.76) and 1.32 (95% CI: 1.01‐1.73), respectively, in the weekly intake frequency stratum, while not related to AER of EBV DNase‐specific neutralizing antibody. Smoking was found to be not only an apparent risk factor for higher antibody levels of AER in stage III‐IV patients (OR = 1.60, 95% CI: 1.11‐2.30), but also associated closely with NPC stage at diagnosis (OR = 2.17, 95% CI: 1.47‐3.22), with dose‐response effects. In conclusion, we found consumption of Canton‐style herbal tea and cigarette smoking were in positive associations with elevated EBV antibodies in NPC patients, which may be of public health significance for the primary prevention of EBV‐associated diseases especially NPC.

Abstract The aim of this study was to use whole‐exome sequencing to derive a molecular classifier for nasopharyngeal carcinoma (NPC) and evaluate its clinical performance. We performed whole‐exome sequencing on 82 primary NPC tumors from Sun Yat‐sen University Cancer Center (Guangzhou cohort) to obtain somatic single‐nucleotide variants, indels, and copy number variants. A novel molecular classifier was then developed and validated in another NPC cohort (Hong Kong cohort, n = 99). Survival analysis was estimated by the Kaplan‐Meier method and compared using the log‐rank test. Cox proportional hazards model was adopted for univariate and multivariate analyses. We identified three prominent NPC genetic subtypes: RAS/PI3K/AKT (based on RAS, AKT1, and PIK3CA mutations), cell‐cycle (based on CDKN2A/CDKN2B deletions, and CDKN1B and CCND1 amplifications), and unclassified (based on dominant mutations in epigenetic regulators, such as KMT2C/2D, or the Notch signaling pathway, such as NOTCH1/2). These subtypes differed in survival analysis, with good, intermediate, and poor progression‐free survival in the unclassified, cell‐cycle, and RAS/PI3K/AKT subgroups, respectively, among the Guangzhou, Hong Kong, and combined cohorts (n = 82, P = 0.0342; n = 99, P = 0.0372; and n = 181, P = 0.0023; log‐rank test). We have uncovered genetic subtypes of NPC with distinct mutations and/or copy number changes, reflecting discrete paths of NPC tumorigenesis and providing a roadmap for developing new prognostic biomarkers and targeted therapies.

Abstract Whether the association between body size or shape and nasopharyngeal carcinoma (NPC) risk exists or varies by age‐specific body size indicators is unclear. In a population‐based case–control study conducted in Southern China between 2010 and 2014, self‐reported height, weight, and body shape at age 20 and 10 years before interview were collected from 2448 histopathologically confirmed NPC cases and 2534 population‐based controls. Body mass index (BMI) was categorized according to the World Health Organization guidelines for Asian populations: underweight (

Abstract Background The male predominance in the incidence of nasopharyngeal carcinoma (NPC) suggests the contribution of the X chromosome to the susceptibility of NPC. However, no X-linked susceptibility loci have been examined by genome-wide association studies (GWASs) for NPC by far. Methods To understand the contribution of the X chromosome in NPC susceptibility, we conducted an X chromosome-wide association analysis on 1615 NPC patients and 1025 healthy controls of Guangdong Chinese, followed by two validation analyses in Taiwan Chinese (n = 562) and Malaysian Chinese (n = 716). Results Firstly, the proportion of variance of X-linked loci over phenotypic variance was estimated in the discovery samples, which revealed that the phenotypic variance explained by X chromosome polymorphisms was estimated to be 12.63% (non-dosage compensation model) in males, as compared with 0.0001% in females. This suggested that the contribution of X chromosome to the genetic variance of NPC should not be neglected. Secondly, association analysis revealed that rs5927056 in DMD gene achieved X chromosome-wide association significance in the discovery sample (OR = 0.81, 95% CI 0.73–0.89, P = 1.49 × 10−5). Combined analysis revealed rs5927056 for DMD gene with suggestive significance (P = 9.44 × 10−5). Moreover, the female-specific association of rs5933886 in ARHGAP6 gene (OR = 0.62, 95%CI: 0.47–0.81, P = 4.37 × 10−4) was successfully replicated in Taiwan Chinese (P = 1.64 × 10−2). rs5933886 also showed nominally significant gender × SNP interaction in both Guangdong (P = 6.25 × 10−4) and Taiwan datasets (P = 2.99 × 10−2). Conclusion Our finding reveals new susceptibility loci at the X chromosome conferring risk of NPC and supports the value of including the X chromosome in large-scale association studies.

Abstract Our study presents the genetic landscape betel quid chewing‐associated tongue carcinomas (BQ‐TCs). We compared the genetic landscape and mutational signatures of 15 BQ‐TCs, five nonbetel quid chewing‐associated tongue carcinomas (nBQ‐TCs), and 82 tongue carcinomas in general population from the TCGA (TCGA‐TCs) project. The highlights of this research mainly include: (a) The genetic landscape of BQ‐TC was characterized with frequent mutations in RASA1 gene and in CpG islands throughout the genome. (b) The BQ‐TC had a distinct mutational signature from that of nBQ‐TC and tongue carcinomas in the general population, and this signature was associated with the mutations in RASA1 and in CpG islands. (c) Our study indicates that betel quid (BQ) chewing classifies a distinct group of tongue carcinoma. The BQ chewing might not contribute to the tumorigenesis of tongue carcinomas as a mutagen.

EBV latent infection contributes to the pathogenesis of epithelial malignancies by inducing angiogenesis. Here, the authors show EBV promotes vasculogenic mimicry in EBV associated epithelial cancers via AKT/HIF-1α pathway and combination therapy of HIF-1α and VEGF reduces tumour growth.

Abstract Metabolic reprogramming plays important roles in development and progression of nasopharyngeal carcinoma (NPC), but the underlying mechanism has not been completely defined. In this work, we found INSL5 was elevated in NPC tumor tissue and the plasma of NPC patients. Plasma INSL5 could serve as a novel diagnostic marker for NPC, especially for serum VCA‐IgA‐negative patients. Moreover, higher plasma INSL5 level was associated with poor disease outcome. Functionally, INSL5 overexpression increased, whereas knockdown of its receptor GPCR142 or inhibition of INSL5 reduced cell proliferation, colony formation, and cell invasion in vitro and tumorigenicity in vivo. Mechanistically, INSL5 enhanced phosphorylation and nuclear translocation of STAT5 and promoted glycolytic gene expression, leading to induced glycolysis in cancer cells. Pharmaceutical inhibition of glycolysis by 2‐DG or blockade of INSL5 by a neutralizing antibody reversed INSL5‐induced proliferation and invasion, indicating that INSL5 can be a potential therapeutic target in NPC. In conclusion, INSL5 enhances NPC progression by regulating cancer cell metabolic reprogramming and is a potential diagnostic and prognostic marker as well as a therapeutic target for NPC.

Abstract Germline polymorphisms are linked with differential survival outcomes in cancers but are not well studied in nasopharyngeal carcinoma (NPC). Here, a two‐phase association study is conducted to discover germline polymorphisms that are associated with the prognosis of NPC. The discovery phase includes two consecutive hospital cohorts of patients with NPC from Southern China. Exome‐wide genotypes at 246 173 single nucleotide polymorphisms (SNPs) are determined, followed by survival analysis for each SNP under Cox proportional hazard regression model. Candidate SNP is replicated in another two independent cohorts from Southern China and Singapore. Meta‐analysis of all samples (n = 5553) confirms that the presence of rs1131636‐T, located in the 3′‐UTR of RPA1, confers an inferior overall survival (HR = 1.33, 95% CI = 1.20–1.47, P = 6.31 × 10−8). Bioinformatics and biological assays show that rs1131636 has regulatory effects on upstream RPA1. Functional studies further demonstrate that RPA1 promotes the growth, invasion, migration, and radioresistance of NPC cells. Additionally, miR‐1253 is identified as a suppressor for RPA1 expression, likely through regulation of its binding affinity to rs1131636 locus. Collectively, these findings provide a promising biomarker aiding in stratifying patients with poor survival, as well as a potential drug target for NPC.

Abstract Background Metastasis is the major cause of treatment failure in patients with nasopharyngeal carcinoma (NPC). We previously reported that TEL2, a negative regulator of SERPINE1, could inhibit NPC metastasis to lymph nodes. Method A series of in vivo and in vitro assays were performed to elucidate the regulation between Snail and TEL2. TEL2 expression was analyzed in three representative NPC cell lines expressing low levels of Snail (S26, 6-10B, HK1) and two cell lines expressing high levels of Snail (S18, 5-8F). Luciferase and chromatin immunoprecipitation assays were used to analyze the interaction between Snail and TEL2. The roles of the Snail/TEL2 pathway in cell migration and invasion of NPC cells were examined using transwell assays. Metastasis to the lungs was examined using nude mouse receiving NPC cells injection through the tail vein. Results Ectopic Snail expression down-regulated TEL2 at the mRNA and protein levels, whereas knockdown of Snail using short hairpin RNA up-regulated TEL2. Luciferase and chromatin immunoprecipitation assays indicated that Snail binds directly to the TEL2 promoter. Ectopic Snail expression enhanced migration and invasion of NPC cells, and such effects were mitigated by TEL2 overexpression. TEL2 overexpression also attenuated hypoxia-induced cell migration and invasion, and increased the number of metastatic pulmonary nodules. Snail overexpression reduced the number of metastatic pulmonary nodules. Conclusions TEL2 is a novel target of Snail and suppresses Snail-induced migration, invasion and metastasis in NPC.

Abstract The link of nasopharyngeal carcinoma (NPC) with Epstein‐Barr virus (EBV) has been established for decades. Although an abnormal high level of EBV sero‐antibody spectrum and cell‐free circulating EBV DNA loads were exhibited in NPC patients, oral EBV DNA loads, which are primarily responsible for the EBV transmission, has not been previously studied in NPC patients. We conducted an epidemiological study to measure the oral EBV loads, viral components, and the relationship with the serum antibody titers in a large case‐control population (968 cases and 1656 controls) and a family‐based population (91 cases and 165 unaffected family members). EBV DNA loads were detected by quantitative PCR approach targeting the BamHI‐W region. Although a large individualized variation existed, we still observed a decreased oral EBV DNA loads in the population of NPC patients compared to that of healthy controls (ORs were 1.00, 0.69, 0.62, 0.33 classified by the quartiles of viral loads, Ptrend

Our previous study found that smoking was associated with an elevated level of the antibody against VCA in the Epstein-Barr virus (EBV) lytic phase, which was an important predictive marker of the risk of nasopharyngeal carcinoma (NPC). It remained unknown whether environmental factors were associated with the levels of other EBV antibodies, such as Zta-IgA, EA-IgA, EBNA1-IgA, and LMP1-IgA, in the lytic and latent infection periods. We aimed to investigate the possible environmental inducers that could affect EBV antibody levels in two independent healthy male populations from endemic NPC areas in South China (N = 1498) and non-endemic NPC areas in North China (N = 1961). We performed ELISA and immunoenzymatic assays to test the levels of antibodies specific to the EBV antigens. The seropositive rates of antibodies against the antigens expressed in both the EBV latent and lytic infection periods, namely, LMP1-IgA, EBNA1-IgA, and Zta-IgA, in endemic areas (28.65%, 5.43% and 14.49%, respectively) were significantly higher than those in non-endemic areas (14.43%, 1.07% and 6.32%, respectively). Smoking was associated with higher seropositivity for EBNA1-IgA (OR = 1.47, 95% CI = 1.12–1.93) and Zta-IgA (OR = 1.28, 95% CI = 0.99–1.66), with dose-response effects, while not associated with the levels of LMP1-IgA. In conclusion, smoking was an important environmental factor, which associated with increased levels of EBNA1-IgA, and Zta-IgA. Keywords: Anti-Epstein-Barr virus antibodies, Asymptomatic males, Cigarette smoking, Nasopharyngeal carcinoma, China

Humanized mouse models are used as comprehensive small-animal models of EBV infection. Previously, infectious doses of EBV used in vivo have been determined mainly on the basis of TD50 (50% transforming dose), which is a time-consuming process. Here, we determined infectious doses of Akata-EBV-GFP using green Raji units (GRUs), and characterized dose-dependent effects in humanized mice. We defined two outcomes in vivo, including an infection model and a lymphoma model, following inoculation with low or high doses of Akata-EBV-GFP, respectively. Inoculation with a low dose induced primary B cells to become lymphoblastoid cell lines in vitro, and caused latent infection in humanized mice. In contrast, a high dose of Akata-EBV-GFP resulted in primary B cells death in vitro, and fatal B cell lymphomas in vivo. Following infection with high doses, the frequency of CD19+ B cells decreased, whereas the percentage of CD8+ T cells increased in peripheral blood and the spleen. At such doses, a small part of activated CD8+ T cells was EBV-specific CD8+ T cells. Thus, GRUs quantitation of Akata-EBV-GFP is an effective way to quantify infectious doses to study pathologies, immune response, and to assess (in vivo) the neutralizing activity of antibodies raised by immunization against EBV.

Neuroblastoma cell differentiation is regulated by Id proteins. Here, the authors show that CaMKII-mediated phosphorylation of Beclin 1 can activate K63-linked ubiquitination and autophagic degradation of Id proteins uncovering a role for autophagy in cell differentiation.

Abstract Background Small hepatocellular carcinoma (sHCC) is a unique variant of HCC that is characterized by small tumor size (maximum tumor diameter ≤3 cm) and favorable long-term outcomes. The present study aimed to define clinicopathologic factors that predict survival in patients with sHCC. Methods The study population consisted of 335 patients who underwent hepatectomy for solitary sHCC between December 1998 and 2010. Prognostic factors were evaluated using Kaplan–Meier curves and Cox proportional hazard models. Results The 5-year overall survival (OS) and recurrence-free survival (RFS) rates were 77.7% and 59.9%, respectively. Kaplan–Meier curves showed that tumor size and vascular invasion had prognostic significance within this relatively selected cohort (P

This genome-wide association study on persistent hepatitis B virus (HBV) infection among Chinese confirms previously associated genetic loci while discovering a novel protective locus at 8p21.3. The study also demonstrates the nearby gene INST10 suppresses HBV replication in vitro.

Abstract Background The current World Health Organization (WHO) classification of nasopharyngeal carcinoma (NPC) conveys little prognostic information. This study aimed to propose an NPC histopathologic classification that can potentially be used to predict prognosis and treatment response. Methods We initially developed a histopathologic classification based on the morphologic traits and cell differentiation of tumors of 2716 NPC patients who were identified at Sun Yat-sen University Cancer Center (SYSUCC) (training cohort). Then, the proposed classification was applied to 1702 patients (retrospective validation cohort) from hospitals outside SYSUCC and 1613 patients (prospective validation cohort) from SYSUCC. The efficacy of radiochemotherapy and radiotherapy modalities was compared between the proposed subtypes. We used Cox proportional hazards models to estimate hazard ratios (HRs) with 95% confidence intervals (CI) for overall survival (OS). Results The 5-year OS rates for all NPC patients who were diagnosed with epithelial carcinoma (EC; 3708 patients), mixed sarcomatoid-epithelial carcinoma (MSEC; 1247 patients), sarcomatoid carcinoma (SC; 823 patients), and squamous cell carcinoma (SCC; 253 patients) were 79.4%, 70.5%, 59.6%, and 42.6%, respectively (P

Epstein-Barr virus (EBV) is a human cancer-related virus closely associated with lymphoid and epithelial malignancies, and EBV glycoprotein B (gB) plays an essential role in viral entry into both B cells and epithelial cells by promoting cell-cell fusion. EBV gB is exclusively modified with high-mannose-linked N-glycans and primarily localizes to the endoplasmic reticulum (ER) with low levels on the plasma membrane (PM). However, the mechanism through which gB is regulated within host cells is largely unknown. Here, we report the identification of F-box only protein 2 (FBXO2), an SCF ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans and attenuates EBV infectivity by targeting N-glycosylated gB for degradation. gB possesses seven N-glycosylation sites, and FBXO2 directly binds to these high-mannose moieties through its sugar-binding domain. The interaction promotes the degradation of glycosylated gB via the ubiquitin-proteasome pathway. Depletion of FBXO2 not only stabilizes gB but also promotes its transport from the ER to the PM, resulting in enhanced membrane fusion and viral entry. FBXO2 is expressed in epithelial cells but not B cells, and EBV infection up-regulates FBXO2 levels. In summary, our findings highlight the significance of high-mannose modification of gB and reveal a novel host defense mechanism involving glycoprotein homeostasis regulation.

Epstein–Barr virus is a ubiquitous virus and is associated with several human malignances, including the significant subset of gastric carcinoma, Epstein–Barr virus–associated gastric carcinoma. Some Epstein–Barr virus–associated diseases are uniquely prevalent in populations with different geographic origins. However, the features of the disease and geographically associated Epstein–Barr virus genetic variation as well as the roles that the variation plays in carcinogenesis and evolution remain unclear. Therefore, in this study, we sequenced 95 geographically distinct Epstein–Barr virus isolates from Epstein–Barr virus–associated gastric carcinoma biopsies and saliva of healthy donors to detect variants and genes associated with gastric carcinoma and population structure from a genome-wide spectrum. We demonstrated that Epstein–Barr virus revealed the population structure between North China and South China. In addition, we observed population stratification between Epstein–Barr virus strains from gastric carcinoma and healthy controls, indicating that certain Epstein–Barr virus subtypes are associated with different gastric carcinoma risks. We identified that the BRLF1, BBRF3, and BBLF2/BBLF3 genes had significant associations with gastric carcinoma. LMP1 and BNLF2a genes were strongly geographically associated genes in Epstein–Barr virus. Our study provides insights into the genetic basis of oncogenic Epstein–Barr virus for gastric carcinoma, and the genetic variants associated with gastric carcinoma can serve as biomarkers for oncogenic Epstein–Barr virus.

N6-methyladenosine (m6A) is the most abundant epitranscriptomic mark found on mRNA and has important roles in various physiological processes. Despite the relatively high m6A levels in the brain, its potential functions in the brain remain largely unexplored. We performed a transcriptome-wide methylation analysis using the mouse brain to depict its region-specific methylation profile. RNA methylation levels in mouse cerebellum are generally higher than those in the cerebral cortex. Heterogeneity of RNA methylation exists across different brain regions and different types of neural cells including the mRNAs to be methylated, their methylation levels and methylation site selection. Common and region-specific methylation have different preferences for methylation site selection and thereby different impacts on their biological functions. In addition, high methylation levels of fragile X mental retardation protein (FMRP) target mRNAs suggest that m6A methylation is likely to be used for selective recognition of target mRNAs by FMRP in the synapse. Overall, we provide a region-specific map of RNA m6A methylation and characterize the distinct features of specific and common methylation in mouse cerebellum and cerebral cortex. Our results imply that RNA m6A methylation is a newly identified element in the region-specific gene regulatory network in the mouse brain.

Determining the complex relationships between diseases, polymorphisms in human genes and environmental factors is challenging. Multifactor dimensionality reduction (MDR) has been proven to be capable of effectively detecting the statistical patterns of epistasis, although classification accuracy is required for this approach. The imbalanced dataset can cause seriously negative effects on classification accuracy. Moreover, MDR methods cannot quantitatively assess the disease risk of genotype combinations. Hence, we introduce a novel weighted risk score-based multifactor dimensionality reduction (WRSMDR) method that uses the Bayesian posterior probability of polymorphism combinations as a new quantitative measure of disease risk. First, we compared the WRSMDR to the MDR method in simulated datasets. Our results showed that the WRSMDR method had reasonable power to identify high-order gene-gene interactions, and it was more effective than MDR at detecting four-locus models. Moreover, WRSMDR reveals more information regarding the effect of genotype combination on the disease risk, and the result was easier to determine and apply than with MDR. Finally, we applied WRSMDR to a nasopharyngeal carcinoma (NPC) case-control study and identified a statistically significant high-order interaction among three polymorphisms: rs2860580, rs11865086 and rs2305806.

Kelch proteins are implicated in the pathogenesis of many human diseases, including cancer. Nasopharyngeal carcinoma (NPC) is a rare malignancy in most countries, but prevalent in southern China and certain areas of Southeast Asia. In this study, we identified Kelch Domain Containing 4 (KLHDC4), an orphan member of the kelch repeat superfamily, as a prognosis marker for NPC. We examined the expression of KLHDC4 in 168 NPC cases by immunohistochemical staining and found a substantially higher level of KLHDC4 in NPC biopsies compared to adjacent normal nasopharyngeal mucosa. KLHDC4 expression was significantly related to the T classification (P

Targeted therapy based on adjustment of microRNA (miRNA)s activity takes great promise due to the ability of these small RNAs to modulate cellular behavior. However, the efficacy of miR-101 replacement therapy to hepatocellular carcinoma (HCC) remains unclear. In the current study, we first observed that plasma levels of miR-101 were significantly lower in distant metastatic HCC patients than in HCCs without distant metastasis, and down-regulation of plasma miR-101 predicted a worse disease-free survival (DFS, P

Anterior chamber depth (ACD) is a key anatomical risk factor for primary angle closure glaucoma (PACG). We conducted a genome-wide association study (GWAS) on ACD to discover novel genes for PACG on a total of 5,308 population-based individuals of Asian descent. Genome-wide significant association was observed at a sequence variant within ABCC5 (rs1401999; per-allele effect size = -0.045 mm, P = 8.17 × 10(-9)). This locus was associated with an increase in risk of PACG in a separate case-control study of 4,276 PACG cases and 18,801 controls (per-allele OR = 1.13 [95% CI: 1.06-1.22], P = 0.00046). The association was strengthened when a sub-group of controls with open angles were included in the analysis (per-allele OR = 1.30, P = 7.45 × 10(-9); 3,458 cases vs. 3,831 controls). Our findings suggest that the increase in PACG risk could in part be mediated by genetic sequence variants influencing anterior chamber dimensions.

BACKGROUND:The International Union Against Cancer/American Joint Committee on Cancer (UICC/AJCC) TNM staging system of nasopharyngeal carcinoma (NPC) is the most important system for survival prediction. The TNM 7th edition UICC/AJCC TNM staging system for NPC was adopted in January 2009, and is now internationally recommended. In comparison with the TNM 6th edition, there were several revisions in the new edition staging system. This study aims to evaluate the prognostic value of the TNM 7th edition for NPC patients in comparison with the TNM 6th edition. METHOD:Clinical data of 2,629 NPC patients from the Sun Yat-sen University Cancer Center between January 2006 and December 2010 were retrospectively collected and all the patients were restaged according to the criteria of the TNM 6th edition and TNM 7th edition UICC/AJCC staging manual. Univariate and multivariate COX proportional hazards analyses were applied to evaluate the prognostic values between adjacent stage categories of the TNM 6th edition and TNM 7th edition. RESULTS:In comparison with the TNM 6th edition, a significant alteration of the distribution of N categories was observed when the TNM 7th edition was applied (χ2 = 20.589, P

BACKGROUND: Epstein-Barr Virus (EBV) is a globally prevalent herpesvirus associated with infectious mononucleosis and many malignancies. The survey on EBV prevalence appears to be important to study EBV-related diseases and determine when to administer prophylactic vaccine. The purpose of this retrospective study was to collect baseline information about the prevalence of EBV infection in Chinese children. METHODOLOGY/PRINCIPAL FINDING: We collected 1778 serum samples from healthy children aged 0 to 10, who were enrolled in conventional health and nutrition examinations without any EBV-related symptom in 2012 and 2013 in North China (n = 973) and South China (n = 805). We detected four EBV-specific antibodies, i.e., anti-VCA-IgG and IgM, anti-EBNA-IgG and anti-EA-IgG, by ELISA, representing all of the phases of EBV infection. The overall EBV seroprevalence in samples from North and South China were 80.78% and 79.38% respectively. The EBV seropositivity rates dropped slightly at age 2, and then increased gradually with age. The seroprevalence became stabilized at over 90% after age 8. In this study, the seroprevalence trends between North and South China showed no difference (P>0.05), and the trends of average antibody concentrations were similar as well (P>0.05). CONCLUSIONS/SIGNIFICANCE: EBV seroprevalence became more than 50% before age 3 in Chinese children, and exceed 90% after age 8. This study can be helpful to study the relationship between EBV and EBV-associated diseases, and supportive to EBV vaccine development and implementation.

MicroRNA-214 (MiR-214) is aberrantly expressed in several human tumors such as ovarian cancer and breast cancer. However, the role of miR-214 in nasopharyngeal carcinoma (NPC) is still unknown. In this study, we report that miR-214 was overexpressed in NPC cell lines and tissues. Silencing of miR-214 by LNA-antimiR-214 in NPC cells resulted in promoting apoptosis and suppressing cell proliferation in vitro, and suppressed tumor growth in nude mice in vivo. Luciferase reporter assay was performed to identify Bim as a direct target of miR-214. Furthermore, this study showed that low Bim expression in NPC tissues correlated with poor survival of NPC patients. Taken together, our findings suggest that miR-214 plays an important role in NPC carcinogenesis.

BACKGROUND:Nasopharyngeal carcinoma (NPC) is an endemic neoplasm in southern China. Although NPC sufferers are sensitive to radiotherapy, 20-30% of patients finally progress with recurrence and metastases. Elevated lymphocyte-to-monocyte ratio (LMR) has been reported to be associated with favorable prognosis in some hematology malignancies, but has not been studied in NPC. The aim of this study was to evaluate whether LMR could predict the prognosis of NPC patients. METHODS:A retrospective cohort of 1,547 non-metastatic NPC patients was recruited between January 2005 and June 2008. The counts for peripheral lymphocyte and monocyte were retrieved, and the LMR was calculated. Receiver operating characteristic curve analysis, univariate and multivariate COX proportional hazards analyses were applied to evaluate the associations of LMR with overall survival (OS), disease-free survival (DFS), distant metastasis-free survival (DMFS) and loco-regional recurrence-free survival (LRRFS), respectively. RESULTS:Univariate analysis revealed that higher LMR level (≥ 5.220) was significantly associated with superior OS, DFS and DMFS (P values

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.

It has been suggested that autophagy-related Beclin 1 plays a critical role in the regulation of tumor development and/or progression, but its prognostic significance and relationship with Bcl-xL expression in ovarian carcinoma are unclear.In the present study, the methods of Western blotting and immunohistochemistry (IHC) were utilized to investigate the expression status of Beclin 1 and Bcl-xL in fresh ovarian tissues and paraffin-embedded epithelial ovarian tumor tissues. Decreased expression of Beclin 1 was examined by IHC in 8.3% of normal ovaries, in 15.4% of cystadenomas, in 20.0% of borderline tumors, and in 55.6% of ovarian carcinomas, respectively. In ovarian carcinomas, decreased expression of Beclin 1 was correlated closely with ascending histological grade, later pT/pN/pM status and/or advanced clinical stage (P

Genome-wide association studies (GWAS) have recently identified KIF1B as susceptibility locus for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). To further identify novel susceptibility loci associated with HBV-related HCC and replicate the previously reported association, we performed a large three-stage GWAS in the Han Chinese population. 523,663 autosomal SNPs in 1,538 HBV-positive HCC patients and 1,465 chronic HBV carriers were genotyped for the discovery stage. Top candidate SNPs were genotyped in the initial validation samples of 2,112 HBV-positive HCC cases and 2,208 HBV carriers and then in the second validation samples of 1,021 cases and 1,491 HBV carriers. We discovered two novel associations at rs9272105 (HLA-DQA1/DRB1) on 6p21.32 (OR = 1.30, P = 1.13×10⁻¹⁹) and rs455804 (GRIK1) on 21q21.3 (OR = 0.84, P = 1.86×10⁻⁸), which were further replicated in the fourth independent sample of 1,298 cases and 1,026 controls (rs9272105: OR = 1.25, P = 1.71×10⁻⁴; rs455804: OR = 0.84, P = 6.92×10⁻³). We also revealed the associations of HLA-DRB1*0405 and 0901*0602, which could partially account for the association at rs9272105. The association at rs455804 implicates GRIK1 as a novel susceptibility gene for HBV-related HCC, suggesting the involvement of glutamate signaling in the development of HBV-related HCC.

BACKGROUND:Accurate prognostication of locally advanced nasopharyngeal carcinoma (NPC) will benefit patients for tailored therapy. Here, we addressed this issue by developing a mathematical algorithm based on support vector machine (SVM) through integrating the expression levels of multi-biomarkers. METHODOLOGY/PRINCIPAL FINDINGS:Ninety-seven locally advanced NPC patients in a randomized controlled trial (RCT), consisting of 48 cases serving as training set and 49 cases as testing set of SVM models, with 5-year follow-up were studied. We designed SVM models by selecting the variables from 38 tissue molecular biomarkers, which represent 6 tumorigenesis signaling pathways, and 3 EBV-related serological biomarkers. We designed 3 SVM models to refine prognosis of NPC with 5-year follow-up. The SVM1 displayed highly predictive sensitivity (sensitivity, specificity were 88.0% and 81.9%, respectively) by integrating the expression of 7 molecular biomarkers. The SVM2 model showed highly predictive specificity (sensitivity, specificity were 84.0% and 94.5%, respectively) by grouping the expression level of 12 molecular biomarkers and 3 EBV-related serological biomarkers. The SVM3 model, constructed by combination SVM1 with SVM2, displayed a high predictive capacity (sensitivity, specificity were 88.0% and 90.3%, respectively). We found that 3 SVM models had strong power in classification of prognosis. Moreover, Cox multivariate regression analysis confirmed these 3 SVM models were all the significant independent prognostic model for overall survival in testing set and overall patients. CONCLUSIONS/SIGNIFICANCE:Our SVM prognostic models designed in the RCT displayed strong power in refining patient prognosis for locally advanced NPC, potentially directing future target therapy against the related signaling pathways.

BACKGROUND:Lifestyle behaviors have been widely reported to influence the survival of patients with head and neck cancer. However, the relationship between pretreatment lifestyle behaviors and survival among patients with nasopharyngeal carcinoma (NPC) is unclear. METHODS:A prospective cohort study was designed to determine the relationship between lifestyle behaviors and survival in 1,533 NPC patients recruited from October 2005 to October 2007. Pretreatment lifestyle behaviors (such as body-mass index [BMI], smoking, alcohol, diet) of the patients were investigated. Univariate and multivariate proportional-hazards models were used to assess the impact of lifestyle behaviors on patient survival. RESULTS:Smoking was a predictor of survival; both current smokers (hazard ratio [HR] = 1.88; 95% CI, 1.33 to 2.65) and heavy smokers (≥ 25 Pack-years; HR = 1.84; 95% CI, 1.30 to 2.60) showed associations with poor survival. Higher BMI was significantly associated with a lower risk of death (P(trend) = 0.002). Compared with under/normal-weight patients (BMI less than 22.99 kg/m(2)), the multivariate HR for survival was 0.66 (95% CI, 0.48 to 0.90) and 0.47 (95% CI, 0.23 to 0.97) for overweight and obese patients, respectively. No alcohol intake and high fruit intake were associated with favorable survival in the univariate analysis but lost significance in the multivariate model. CONCLUSION:Our findings indicate that pretreatment lifestyle behaviors, especially smoking status and BMI, as easily available data, provide prognostic value for survival in NPC patients.

BACKGROUND: Substantial evidence suggests that the presence of inflammatory cells plays a critical role in the development and/or progression of human tumors. Neutrophils are the common inflammatory cells in tumors; however, the infiltration of intratumoral neutrophils in colorectal carcinoma (CRC) and its effect on CRC patients' prognosis are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the methods of tissue microarray and immunohistochemistry (IHC) were used to investigate the prognostic significance of intratumoral CD66b+ neutrophil in CRC. According to receiver operating characteristic curve analysis, the cutoff score for high intratumoral CD66b+ neutrophil in CRC was defined when the mean counts were more than 60 per TMA spot. In our study, high intratumoral CD66b+ neutrophil was observed in 104/229 (45.4%) of CRCs and in 29/229 (12.7%) of adjacent mucosal tissues. Further correlation analysis showed that high intratumoral neutrophil was positively correlated with pT status, pM status and clinical stage (P

BACKGROUND: Topotecan produces DNA damage that induces autophagy in cancer cells. In this study, sensitising topotecan to colon cancer cells with different P53 status via modulation of autophagy was examined. METHODOLOGY/PRINCIPAL FINDINGS: The DNA damage induced by topotecan treatment resulted in cytoprotective autophagy in colon cancer cells with wild-type p53. However, in cells with mutant p53 or p53 knockout, treatment with topotecan induced autophagy-associated cell death. In wild-type p53 colon cancer cells, topotecan treatment activated p53, upregulated the expression of sestrin 2, induced the phosphorylation of the AMPKα subunit at Thr172, and inhibited the mTORC1 pathway. Furthermore, the inhibition of autophagy enhanced the anti-tumour effect of topotecan treatment in wild-type p53 colon cancer cells but alleviated the anti-tumour effect of topotecan treatment in p53 knockout cells in vivo. CONCLUSIONS/SIGNIFICANCE: These results imply that the wild-type p53-dependent induction of cytoprotective autophagy is one of the cellular responses that determines the cellular sensitivity to the DNA-damaging drug topotecan. Therefore, our study provides a potential therapeutic strategy that utilises a combination of DNA-damaging agents and autophagy inhibitors for the treatment of colon cancer with wild-type p53.

The role of microRNA-155 (miR-155) has been associated with oncogenesis of several human tumors. However the expression pattern of miR-155 has not been investigated in nasopharyngeal carcinoma (NPC). The present study was to assess miR-155 expression pattern and its possible function in NPC, to identify its targets and evaluate their clinical applications in NPC. MiR-155 was found to be upregulated in two Epstein-Barr virus (EBV) negative NPC derived cell lines CNE1 and TW03, as well as in NPC clinical samples by quantitative Real-time PCR and in situ hybridization detection. EBV encoded LMP1 and LMP2A could further enhance the expression of miR-155 in NPC CNE1 and TW03 cells. JMJD1A and BACH1 were identified as putative targets of miR-155 in a bioinformatics screen. Overexpression of miR-155 downregulated a luciferase transcript fused to the 3'UTR of JMJD1A and BACH1. MiR-155 mimic could downregulate the expression of JMJD1A and BACH1, while miR-155 inhibitor could upregulate JMJD1A expression in NPC cell lines. Moreover, downregulation of JMJD1A was significantly correlated with N stage in TNM classification (p = 0.023), a lower five-year survival rate (p = 0.021), and a lower five-year disease-free survival rate (p = 0.049) of NPC patients. Taken together, up-regulation of miR-155 in NPC is partly driven by LMP1 and LMP2A, and results in downregulation of JMJD1A, which is associated with N stage and poor prognosis of NPC patients. The potential of miR-155 and JMJD1A as therapeutic targets in NPC should be further investigated.