Liquid biopsy tracking during sequential chemo-radiotherapy identifies distinct prognostic phenotypes in nasopharyngeal carcinoma.
Lv Jiawei,Chen Yupei,Zhou Guanqun,Qi Zhenyu,Tan Kuan Rui Lloyd,Wang Haitao,Lin Li,Chen Foping,Zhang Lulu,Huang Xiaodan,Liu Ruiqi,Xu Sisi,Chen Yue,Ma Jun,Chua Melvin L K,Sun Ying
Liquid biopsies have the utility for detecting minimal residual disease in several cancer types. Here, we investigate if liquid biopsy tracking on-treatment informs on tumour phenotypes by longitudinally quantifying circulating Epstein-barr virus (EBV) DNA copy number in 673 nasopharyngeal carcinoma patients undergoing radical induction chemotherapy (IC) and chemo-radiotherapy (CRT). We observe significant inter-patient heterogeneity in viral copy number clearance that is classifiable into eight distinct patterns based on clearance kinetics and bounce occurrence, including a substantial proportion of complete responders (≈30%) to only one IC cycle. Using a supervised statistical clustering of disease relapse risks, we further bin these eight subgroups into four prognostic phenotypes (early responders, intermediate responders, late responders, and treatment resistant) that are correlated with efficacy of chemotherapy intensity. Taken together, we show that real-time monitoring of liquid biopsy response adds prognostic information, and has the potential utility for risk-adapted treatment de-intensification/intensification in nasopharyngeal carcinoma.
Gemcitabine and Cisplatin Induction Chemotherapy in Nasopharyngeal Carcinoma.
Zhang Yuan,Chen Lei,Hu Guo-Qing,Zhang Ning,Zhu Xiao-Dong,Yang Kun-Yu,Jin Feng,Shi Mei,Chen Yu-Pei,Hu Wei-Han,Cheng Zhi-Bin,Wang Si-Yang,Tian Ye,Wang Xi-Cheng,Sun Yan,Li Jin-Gao,Li Wen-Fei,Li Yu-Hong,Tang Ling-Long,Mao Yan-Ping,Zhou Guan-Qun,Sun Rui,Liu Xu,Guo Rui,Long Guo-Xian,Liang Shao-Qiang,Li Ling,Huang Jing,Long Jin-Hua,Zang Jian,Liu Qiao-Dan,Zou Li,Su Qiong-Fei,Zheng Bao-Min,Xiao Yun,Guo Ying,Han Fei,Mo Hao-Yuan,Lv Jia-Wei,Du Xiao-Jing,Xu Cheng,Liu Na,Li Ying-Qin,Chua Melvin L K,Xie Fang-Yun,Sun Ying,Ma Jun
The New England journal of medicine
BACKGROUND:Platinum-based concurrent chemoradiotherapy is the standard of care for patients with locoregionally advanced nasopharyngeal carcinoma. Additional gemcitabine and cisplatin induction chemotherapy has shown promising efficacy in phase 2 trials. METHODS:In a parallel-group, multicenter, randomized, controlled, phase 3 trial, we compared gemcitabine and cisplatin as induction chemotherapy plus concurrent chemoradiotherapy with concurrent chemoradiotherapy alone. Patients with locoregionally advanced nasopharyngeal carcinoma were randomly assigned in a 1:1 ratio to receive gemcitabine (at a dose of 1 g per square meter of body-surface area on days 1 and 8) plus cisplatin (80 mg per square meter on day 1), administered every 3 weeks for three cycles, plus chemoradiotherapy (concurrent cisplatin at a dose of 100 mg per square meter every 3 weeks for three cycles plus intensity-modulated radiotherapy) or chemoradiotherapy alone. The primary end point was recurrence-free survival (i.e., freedom from disease recurrence [distant metastasis or locoregional recurrence] or death from any cause) in the intention-to-treat population. Secondary end points included overall survival, treatment adherence, and safety. RESULTS:A total of 480 patients were included in the trial (242 patients in the induction chemotherapy group and 238 in the standard-therapy group). At a median follow-up of 42.7 months, the 3-year recurrence-free survival was 85.3% in the induction chemotherapy group and 76.5% in the standard-therapy group (stratified hazard ratio for recurrence or death, 0.51; 95% confidence interval [CI], 0.34 to 0.77; P = 0.001). Overall survival at 3 years was 94.6% and 90.3%, respectively (stratified hazard ratio for death, 0.43; 95% CI, 0.24 to 0.77). A total of 96.7% of the patients completed three cycles of induction chemotherapy. The incidence of acute adverse events of grade 3 or 4 was 75.7% in the induction chemotherapy group and 55.7% in the standard-therapy group, with a higher incidence of neutropenia, thrombocytopenia, anemia, nausea, and vomiting in the induction chemotherapy group. The incidence of grade 3 or 4 late toxic effects was 9.2% in the induction chemotherapy group and 11.4% in the standard-therapy group. CONCLUSIONS:Induction chemotherapy added to chemoradiotherapy significantly improved recurrence-free survival and overall survival, as compared with chemoradiotherapy alone, among patients with locoregionally advanced nasopharyngeal carcinoma. (Funded by the Innovation Team Development Plan of the Ministry of Education and others; ClinicalTrials.gov number, NCT01872962.).
Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling.
Liu Shu-Chen,Hsu Tien,Chang Yu-Sun,Chung An-Ko,Jiang Shih Sheng,OuYang Chun-Nan,Yuh Chiou-Hwa,Hsueh Chuen,Liu Ya-Ping,Tsang Ngan-Ming
Metastasis remains a clinically unsolved issue in nasopharyngeal carcinoma. Here, we report that higher levels of cytoplasmic leukemia inhibitory factor (LIF) and LIF receptor are correlated with poorer metastasis/recurrence-free survival. Further, single nucleotide variations and signal peptide mutation of LIF are identified in NPC. Cytoplasmic LIF reprograms the invasive mode from collective to mesenchymal migration via acquisition of EMT and invadopodia-associated characteristics. Higher cytoplasmic LIF enhances cancer vascular dissemination and local invasion mechanistically through modulation of YAP1-FAK/PXN signaling. Immunohistochemical analyses of NPC biopsies reveal a positive correlation of cytoplasmic LIF expression with focal adhesion kinases. Pharmaceutical intervention with AZD0530 markedly reverses LIF-mediated cancer dissemination and local invasion through promotion of cytoplasmic accumulation of YAP1 and suppression of focal adhesion kinases. Given the significant role of LIF/YAP1-focal adhesion signaling in cancer dissemination, targeting of this pathway presents a promising opportunity to block metastasis.
Antitumor Activity of Nivolumab in Recurrent and Metastatic Nasopharyngeal Carcinoma: An International, Multicenter Study of the Mayo Clinic Phase 2 Consortium (NCI-9742).
Ma Brigette B Y,Lim Wan-Teck,Goh Boon-Cher,Hui Edwin P,Lo Kwok-Wai,Pettinger Adam,Foster Nathan R,Riess Jonathan W,Agulnik Mark,Chang Alex Y C,Chopra Akhil,Kish Julie A,Chung Christine H,Adkins Douglas R,Cullen Kevin J,Gitlitz Barbara J,Lim Dean W,To Ka-Fai,Chan K C Allen,Lo Y M Dennis,King Ann D,Erlichman Charles,Yin Jun,Costello Brian A,Chan Anthony T C
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
Purpose This multinational study evaluated the antitumor activity of nivolumab in nasopharyngeal carcinoma (NPC). Tumor and plasma-based biomarkers were investigated in an exploratory analysis. Patients and Methods Patients with multiply pretreated recurrent or metastatic NPC were treated with nivolumab until disease progression. The primary end point was objective response rate (ORR) and secondary end points included survival and toxicity. The expression of programmed death-ligand 1 (PD-L1) and human leukocyte antigens A and B in archived tumors and plasma clearance of Epstein-Barr virus DNA were correlated with ORR and survival. Results A total of 44 patients were evaluated and the overall ORR was 20.5% (complete response, n = 1; partial response, n = 8). Nine patients received nivolumab for > 12 months (20%). The 1-year overall survival rate was 59% (95% CI, 44.3% to 78.5%) and 1-year progression-free survival (PFS) rate was 19.3% (95% CI, 10.1% to 37.2%). There was no statistical correlation between ORR and the biomarkers; however, a descriptive analysis showed that the proportion of patients who responded was higher among those with PD-L1 positive tumors (> 1% expression) than those with PD-L1-negative tumors. The loss of expression of one or both human leukocyte antigen class 1 proteins was associated with better PFS than when both proteins were expressed (1-year PFS, 30.9% v 5.6%; log-rank P = .01). There was no association between survival and PD-L1 expression or plasma Epstein-Barr virus DNA clearance. There was no unexpected toxicity to nivolumab. Conclusion Nivolumab has promising activity in NPC and the 1-year overall survival rate compares favorably with historic data in similar populations. Additional evaluation in a randomized setting is warranted. The biomarker results were hypothesis generating and validation in larger cohorts is needed.
Analysis of Plasma Epstein-Barr Virus DNA to Screen for Nasopharyngeal Cancer.
Chan K C Allen,Woo John K S,King Ann,Zee Benny C Y,Lam W K Jacky,Chan Stephen L,Chu Sam W I,Mak Constance,Tse Irene O L,Leung Samantha Y M,Chan Gloria,Hui Edwin P,Ma Brigette B Y,Chiu Rossa W K,Leung Sing-Fai,van Hasselt Andrew C,Chan Anthony T C,Lo Y M Dennis
The New England journal of medicine
BACKGROUND:Circulating cell-free Epstein-Barr virus (EBV) DNA is a biomarker for nasopharyngeal carcinoma. We conducted a prospective study to investigate whether EBV DNA in plasma samples would be useful to screen for early nasopharyngeal carcinoma in asymptomatic persons. METHODS:We analyzed EBV DNA in plasma specimens to screen participants who did not have symptoms of nasopharyngeal carcinoma. Participants with initially positive results were retested approximately 4 weeks later, and those with persistently positive EBV DNA in plasma underwent nasal endoscopic examination and magnetic resonance imaging (MRI). RESULTS:A total of 20,174 participants underwent screening. EBV DNA was detectable in plasma samples obtained from 1112 participants (5.5%), and 309 (1.5% of all participants and 27.8% of those who initially tested positive) had persistently positive results on the repeated sample. Among these 309 participants, 300 underwent endoscopic examination, and 275 underwent both endoscopic examination and MRI; of these participants, 34 had nasopharyngeal carcinoma. A significantly higher proportion of participants with nasopharyngeal carcinoma that was identified by screening had stage I or II disease than in a historical cohort (71% vs. 20%, P<0.001 by the chi-square test) and had superior 3-year progression-free survival (97% vs. 70%; hazard ratio, 0.10; 95% confidence interval, 0.05 to 0.18). Nine participants declined to undergo further testing, and 1 of them presented with advanced nasopharyngeal carcinoma 32 months after enrollment. Nasopharyngeal carcinoma developed in only 1 participant with negative EBV DNA in plasma samples within 1 year after testing. The sensitivity and specificity of EBV DNA in plasma samples in screening for nasopharyngeal carcinoma were 97.1% and 98.6%, respectively. CONCLUSIONS:Analysis of EBV DNA in plasma samples was useful in screening for early asymptomatic nasopharyngeal carcinoma. Nasopharyngeal carcinoma was detected significantly earlier and outcomes were better in participants who were identified by screening than in those in a historical cohort. (Funded by the Kadoorie Charitable Foundation and the Research Grants Council of the Hong Kong government; ClinicalTrials.gov number, NCT02063399 .).
Evaluation of plasma Epstein-Barr virus DNA load to distinguish nasopharyngeal carcinoma patients from healthy high-risk populations in Southern China.
Ji Ming-Fang,Huang Qi-Hong,Yu Xia,Liu Zhiwei,Li Xinghua,Zhang Li-Fang,Wang Panpan,Xie Shang-Hang,Rao Hui-Lan,Fang Fang,Guo Xiang,Liu Qing,Hong Ming-Huang,Ye Weimin,Zeng Yi-Xin,Cao Su-Mei
BACKGROUND:The utility of circulating Epstein-Barr Virus (EBV) DNA as a tumor marker for nasopharyngeal carcinoma (NPC) detection suggests that it might improve the diagnostic performance of anti-EBV antibody markers in NPC screening. In this study, the authors evaluated whether circulating EBV DNA load is capable of distinguishing NPC patients from high-risk individuals who have positive anti-EBV antibodies. METHODS:In a population-based NPC screening trial in Sihui City and Zhongshan City, Guangdong Province, China, the authors previously identified 862 high-risk participants with 2 screening markers, immunoglobulin A (IgA) antibodies to EBV capsid antigen (VCA/IgA) and nuclear antigen-1 (EBNA1/IgA). In the current study, real-time polymerase chain reaction was used to measure the baseline plasma EBV DNA load among 825 participants (97%). Follow-up was extended to the end of 2011 to evaluate the diagnostic and predictive values of plasma EBV DNA load. RESULTS:By using 0 copies/mL as the cutoff value, plasma EBV DNA had a sensitivity of 86.8% (33 of 38 patients) for NPC detected within the first year of follow-up, yielding a positive predictive value of 30% (33 of 110 participants) and a negative predictive value of 99.3% (696 of 701 participants). The patients who had early stage NPC had lower sensitivity (81.5%; 22 of 27 patients) than those who had advanced NPC (100%; 11 of 11 patients). For the 14 patients who had NPC detected after 1 year of follow-up, only 50% (7 of 14 patients) tested positive for EBV DNA at baseline. CONCLUSIONS:The plasma EBV DNA load may improve the accuracy of diagnosing NPC in high-risk individuals, but it appears to have limited value in screening patients who have early stage NPC and predicting NPC development.
Increase in IkappaB kinase alpha expression suppresses the tumor progression and improves the prognosis for nasopharyngeal carcinoma.
Deng Ling,Li Yan,Ai Ping,Xie Yuxin,Zhu Hong,Chen Nianyong
Recent studies have suggested that the action of IkappaB kinase alpha (IKKα) as a tumor suppressor is crucial in the development of skin carcinoma, but its role in nasopharyngeal carcinoma (NPC) remains unknown. We examined the IKKα expression in specimens from 157 NPC patients by immunohistochemistry and analyzed the effect of IKKα on prognosis. The functional significance of IKKα expression in NPC cell lines was investigated by IKKα overexpression or downregulation in in vitro studies. The in vitro assays revealed that the IKKα expression was negatively correlated with the invasiveness, migration, and angiogenesis of NPC cells. Overexpression or downregulation of IKKα could significantly repress or enhance the above characteristics, respectively, and these effects were independent of IKKα kinase or EBNA1. In 157 NPC cases, IKKα was differentially expressed in NPC tissues. High expression of IKKα was associated significantly with a high disease-free survival (DFS; P = 0.002) or overall survival (OS; P = 0.014). Multivariate analyses showed that the IKKα expression was an independent risk factor for DFS (HR, 2.302; P = 0.011) and OS (HR, 3.578; P = 0.006). Our findings indicated that IKKα plays a crucial role as a tumor suppressor that suppresses the invasion, metastasis, and angiogenesis of NPC cells in vitro and correlates with the survival in NPC patients. Therefore, IKKα is not only a novel independent prognostic indicator in NPC, but also targeting IKKα expression may provide a potential therapeutic strategy for NPC.
Establishment and validation of a two-step screening scheme for improved performance of serological screening of nasopharyngeal carcinoma.
Li Tingdong,Guo Xiaoyi,Ji Mingfang,Li Fugui,Wang Han,Cheng Weimin,Chen Honglin,Ng Munhon,Ge Shengxiang,Yuan Yong,Xia Ningshao
Nasopharyngeal carcinoma (NPC), which is closely associated with Epstein-Barr virus (EBV), is one of the most prevalent cancers in southeast China. Most NPC patients are diagnosed at late stage due to inconspicuous symptoms at the early stage, and the prognosis of these patients is poor. The early diagnosis rate of NPC could be significantly increased by serological screening, but the positive predictive value (PPV) is relatively low. A simple two-step serological screening scheme was established to improve the PPV of the screening strategy and was validated by a prospective cohort. Serum antibodies specific for EBNA1, Zta, Thymidine Kinase (TK), EAD, EAR, and VCA were detected by enzyme-linked immunosorbent assay. The combination of EBNA1/IgA and VCA/IgA was used in the first step of screening, and anti-early antigens (EAs) were used in the second step of screening. EAD/IgA was the most prominent marker in the second step of screening, and other anti-EAs were complementary to EAD/IgA. As validated by a prospective cohort including 4200 participants, using the combination of EAD/IgA and TK/IgA in the second step decreased the number of high-risk participants from 128 to 27, and increased the PPV from 4.69% to 18.52%, with only one very early-stage case missed. The two-step screening scheme provides a standardized approach for NPC screening with an improved PPV and may be used in future field studies. With this two-step serological screening method, more people benefit from the screening program without increasing the need for fiberoptic endoscopy.
Chimerically fused antigen rich of overlapped epitopes from latent membrane protein 2 (LMP2) of Epstein-Barr virus as a potential vaccine and diagnostic agent.
Lin Xiaoyun,Chen Shao,Xue Xiangyang,Lu Lijun,Zhu Shanli,Li Wenshu,Chen Xiangmin,Zhong Xiaozhi,Jiang Pengfei,Sename Torsoo Sophia,Zheng Yi,Zhang Lifang
Cellular & molecular immunology
Epstein-Barr virus (EBV) is prevalent throughout the world and is associated with several malignant diseases in humans. Latent membrane protein 2 (LMP2) of EBV plays a crucial role in the pathogenesis of EBV-associated tumors; therefore, LMP2 has been considered to be a potential immunodiagnostic and immunotherapeutic target. A multi-epitope-based antigen is a promising option for therapeutic vaccines and diagnoses of such malignancies. In this study, we systematically screened cytotoxic T lymphocyte (CTL), helper T cell (Th) and B-cell epitopes within EBV-LMP2 using bioinformatics. Based on the screen, two peptides rich in overlapping epitopes of both T cells and B cells were selected to construct a plasmid containing the sequence for a chimeric multi-epitope protein referred to as EBV-LMP2m, which is composed of LMP2aa195∼232 and LMP2aa419∼436. The EBV-LMP2m protein was expressed in E. coli BL21 (DE3) after prokaryotic codon optimization. Inoculation of the purified chimeric antigen in BALB/c mice induced not only high levels of specific IgG in the serum and secretory IgA in the vaginal mucus but also a specific CTL response. By using purified EBV-LMP2m as an antigen, the presence of specific IgG in the serum specimens of 202 nasopharyngeal carcinoma (NPC) patients was effectively detected with 52.84% sensitivity and 95.40% specificity, which represents an improvement over the traditional detection method based on VCA-IgA (60.53% sensitivity and 76.86% specificity). The above results indicate that EBV-LMP2m may be used not only as a potential target antigen for EBV-associated tumors but also a diagnostic agent for NPC patients.
Translational genomics of nasopharyngeal cancer.
Tsang Chi Man,Lui Vivian Wai Yan,Bruce Jeffrey P,Pugh Trevor J,Lo Kwok Wai
Seminars in cancer biology
Nasopharyngeal carcinoma (NPC), also named the Cantonese cancer, is a unique cancer with strong etiological association with infection of the Epstein-Barr virus (EBV). With particularly high prevalence in Southeast Asia, the involvement of EBV and genetic aberrations contributive to NPC tumorigenesis have remained unclear for decades. Recently, genomic analysis of NPC has defined it as a genetically homogeneous cancer, driven largely by NF-κB signaling caused by either somatic aberrations of NF-κB negative regulators or by overexpression of the latent membrane protein 1 (LMP1), an EBV viral oncoprotein. This represents a landmark finding of the NPC genome. Exome and RNA sequencing data from new EBV-positive NPC models also highlight the importance of PI3K pathway aberrations in NPC. We also realize for the first time that NPC mutational burden, mutational signatures, MAPK/PI3K aberrations, and MHC Class I gene aberrations, are prognostic for patient outcome. Together, these multiple genomic discoveries begin to shape the focus of NPC therapy development. Given the challenge of NF-κB targeting in human cancers, more innovative drug discovery approaches should be explored to target the unique atypical NF-κB activation feature of NPC. Our next decade of NPC research should focus on further identification of the -omic landscapes of recurrent and metastatic NPC, development of gene-based precision medicines, as well as large-scale drug screening with the newly developed and well-characterized EBV-positive NPC models. Focused preclinical and clinical investigations on these major directions may identify new and effective targeting strategies to further improve survival of NPC patients.
Current State of PCR-Based Epstein-Barr Virus DNA Testing for Nasopharyngeal Cancer.
Kim Kelly Y,Le Quynh-Thu,Yom Sue S,Pinsky Benjamin A,Bratman Scott V,Ng Raymond H W,El Mubarak Haja S,Chan K C Allen,Sander Miriam,Conley Barbara A
Journal of the National Cancer Institute
Clinical studies have shown plasma Epstein-Barr virus (EBV) DNA level to be an independent prognostic biomarker for nasopharyngeal carcinoma (NPC). However, the proportion of NPC patients whose tumors are associated with EBV vary with geographic location, and there are a variety of assays for plasma EBV. To develop the level of evidence needed to demonstrate the clinical utility of plasma EBV DNA detection for NPC patients and encourage widespread adoption of this biomarker test in clinical laboratories, validated harmonized assays are needed. In 2015, the National Cancer Institute (NCI) convened a Workshop on Harmonization of EBV Testing for Nasopharyngeal Cancer, where experts in head and neck oncology and laboratory medicine addressed the limitations of currently available polymerase chain reaction-based EBV DNA quantitation assays and discussed strategies for advancing the development of harmonized EBV DNA assays and their appropriate clinical use. This article presents the key recommendations to direct future efforts in assay harmonization and validation.
Genome-wide profiling of Epstein-Barr virus integration by targeted sequencing in Epstein-Barr virus associated malignancies.
Xu Miao,Zhang Wei-Long,Zhu Qing,Zhang Shanshan,Yao You-Yuan,Xiang Tong,Feng Qi-Sheng,Zhang Zhe,Peng Rou-Jun,Jia Wei-Hua,He Gui-Ping,Feng Lin,Zeng Zhao-Lei,Luo Bing,Xu Rui-Hua,Zeng Mu-Sheng,Zhao Wei-Li,Chen Sai-Juan,Zeng Yi-Xin,Jiao Yuchen
: Epstein-Barr virus (EBV) is associated with multiple malignancies with expression of viral oncogenic proteins and chronic inflammation as major mechanisms contributing to tumor development. A less well-studied mechanism is the integration of EBV into the human genome possibly at sites which may disrupt gene expression or genome stability. : We sequenced tumor DNA to profile the EBV sequences by hybridization-based enrichment. Bioinformatic analysis was used to detect the breakpoints of EBV integrations in the genome of cancer cells. : We identified 197 breakpoints in nasopharyngeal carcinomas and other EBV-associated malignancies. EBV integrations were enriched at vulnerable regions of the human genome and were close to tumor suppressor and inflammation-related genes. We found that EBV integrations into the introns could decrease the expression of the inflammation-related genes, , , and , in NPC tumors. In the EBV genome, the breakpoints were frequently at or terminal repeats. These breakpoints were surrounded by microhomology sequences, consistent with a mechanism for integration involving viral genome replication and microhomology-mediated recombination. : Our finding provides insight into the potential of EBV integration as an additional mechanism mediating tumorigenesis in EBV associated malignancies.
Sequencing-based counting and size profiling of plasma Epstein-Barr virus DNA enhance population screening of nasopharyngeal carcinoma.
Lam W K Jacky,Jiang Peiyong,Chan K C Allen,Cheng Suk H,Zhang Haiqiang,Peng Wenlei,Tse O Y Olivia,Tong Yu K,Gai Wanxia,Zee Benny C Y,Ma Brigette B Y,Hui Edwin P,Chan Anthony T C,Woo John K S,Chiu Rossa W K,Lo Y M Dennis
Proceedings of the National Academy of Sciences of the United States of America
Circulating tumor-derived DNA testing for cancer screening has recently been demonstrated in a prospective study on identification of nasopharyngeal carcinoma (NPC) among 20,174 asymptomatic individuals. Plasma EBV DNA, a marker for NPC, was detected using real-time PCR. While plasma EBV DNA was persistently detectable in 97.1% of the NPCs identified, ∼5% of the general population had transiently detectable plasma EBV DNA. We hypothesized that EBV DNA in plasma of subjects with or without NPC may have different molecular characteristics. We performed target-capture sequencing of plasma EBV DNA and identified differences in the abundance and size profiles of EBV DNA molecules within plasma of NPC and non-NPC subjects. NPC patients had significantly higher amounts of plasma EBV DNA, which showed longer fragment lengths. Cutoff values were established from an exploratory dataset and tested in a validation sample set. Adopting an algorithm that required a sample to concurrently pass cutoffs for EBV DNA counting and size measurements, NPCs were detected at a positive predictive value (PPV) of 19.6%. This represented superior performance compared with the PPV of 11.0% in the prospective screening study, which required participants with an initially detectable plasma EBV DNA result to be retested within 4 weeks. The observed differences in the molecular nature of EBV DNA molecules in plasma of subjects with or without NPC were successfully translated into a sequencing-based test that had a high PPV for NPC screening and achievable through single time-point testing.
EBV(LMP1)-induced metabolic reprogramming inhibits necroptosis through the hypermethylation of the promoter.
Shi Feng,Zhou Min,Shang Li,Du Qianqian,Li Yueshuo,Xie Longlong,Liu Xiaolan,Tang Min,Luo Xiangjian,Fan Jia,Zhou Jian,Gao Qiang,Qiu ShuangJian,Wu Weizhong,Zhang Xin,Bode Ann M,Cao Ya
EBV infection is a recognized epigenetic driver of carcinogenesis. We previously showed that EBV could protect cancer cells from TNF-induced necroptosis. This study aims to explore the epigenetic mechanisms allowing cancer cells with EBV infection to escape from RIP3-dependent necroptosis. : Data from the TCGA database were used to evaluate the prognostic value of promoter methylation and its expression. Western blotting, real-time PCR, and immunochemistry were conducted to investigate the relationship between LMP1 and RIP3 in cell lines and NPC tissues. BSP, MSP and hMeDIP assays were used to examine the methylation level. Induction of necroptosis was detected by cell viability assay, p-MLKL, and Sytox Green staining. : promoter hypermethylation is an independent prognostic factor of poorer disease-free and overall survival in HNSCC patients, respectively. RIP3 is down-regulated in NPC (a subtype of HNSCC). EBV(LMP1) suppresses RIP3 expression by hypermethylation of the promoter. RIP3 protein expression was inversely correlated with LMP1 expression in NPC tissues. Restoring RIP3 expression in EBV(LMP1)-positive cells inhibits xenograft tumor growth. The accumulation of fumarate and reduction of α-KG in EBV(LMP1)-positive cells led to RIP3 silencing due to the inactivation of TETs. Decreased FH activity caused fumarate accumulation, which might be associated with its acetylation. Incubating cells with fumarate protected NPC cells from TNF-induced necroptosis. : These results demonstrate a pathway by which EBV(LMP1)-associated metabolite changes inhibited necroptosis signaling by DNA methylation, and shed light on the mechanism underlying EBV-related carcinogenesis, which may provide new options for cancer diagnosis and therapy.
The genomic landscape of Epstein-Barr virus-associated pulmonary lymphoepithelioma-like carcinoma.
Hong Shaodong,Liu Dongbing,Luo Shuzhen,Fang Wenfeng,Zhan Jianhua,Fu Sha,Zhang Yaxiong,Wu Xuan,Zhou Huaqiang,Chen Xi,Chen Gang,Zhang Zhonghan,Zheng Qiufan,Li Xiaobo,Chen Jinghao,Liu Xingmin,Lei Mengyue,Ye Chen,Wang Jian,Yang Huanming,Xu Xun,Zhu Shida,Yang Yunpeng,Zhao Yuanyuan,Zhou Ningning,Zhao Hongyun,Huang Yan,Zhang Lanjun,Wu Kui,Zhang Li
Pulmonary lymphoepithelioma-like carcinoma (LELC) is a rare and distinct subtype of primary lung cancer characterized by Epstein-Barr virus (EBV) infection. Herein, we reported the mutational landscape of pulmonary LELC using whole-exome sequencing, targeted deep sequencing and single-nucleotide polymorphism arrays. We identify a low degree of somatic mutation but widespread existence of copy number variations. We reveal predominant signature 2 mutations and frequent loss of type I interferon genes that are involved in the host-virus counteraction. Integrated analysis shows enrichment of genetic lesions affecting several critical pathways, including NF-κB, JAK/STAT, and cell cycle. Notably, multi-dimensional comparison unveils that pulmonary LELC resemble NPC but are clearly different from other lung cancers, natural killer/T-cell lymphoma or EBV-related gastric cancer in terms of genetic features. In all, our study illustrates a distinct genomic landscape of pulmonary LELC and provides a road map to facilitate genome-guided personalized treatment.
Complementary roles of MRI and endoscopic examination in the early detection of nasopharyngeal carcinoma.
King A D,Woo J K S,Ai Q Y,Chan J S M,Lam W K J,Tse I O L,Bhatia K S,Zee B C Y,Hui E P,Ma B B Y,Chiu R W K,van Hasselt A C,Chan A T C,Lo Y M D,Chan K C A
Annals of oncology : official journal of the European Society for Medical Oncology
BACKGROUND:Early-stage nasopharyngeal carcinoma (NPC) evades detection when the primary tumor is hidden from view on endoscopic examination. Therefore, in a prospective study of subjects being screened for NPC using plasma Epstein-Barr virus (EBV) DNA, we conducted a study to investigate whether magnetic resonance imaging (MRI) could detect endoscopically occult NPC. PATIENTS AND METHODS:Participants with persistently positive EBV DNA underwent endoscopic examination and biopsy when suspicious for NPC, followed by MRI blinded to the endoscopic findings. Participants with a negative endoscopic examination and positive MRI were recalled for biopsy or surveillance. Diagnostic performance was assessed by calculating sensitivity, specificity and accuracy, based on the histologic confirmation of NPC in the initial study or in a follow-up period of at least two years. RESULTS:Endoscopic examination and MRI were performed on 275 participants, 34 had NPC, 2 had other cancers and 239 without cancer were followed-up for a median of 36 months (24-60 months). Sensitivity, specificity and accuracy were 76.5%, 97.5% and 94.9%, respectively, for endoscopic examination and 91.2%, 97.5% and 96.7%, respectively, for MRI. NPC was detected only by endoscopic examination in 1/34 (2.9%) participants (a participant with stage I disease), and only by MRI in 6/34 (17.6%) participants (stage I = 4, II = 1, III = 1), two of whom had stage I disease and follow-up showing slow growth on MRI but no change on endoscopic examination for 36 months. CONCLUSION:MRI has a complementary role to play in NPC detection and can enable the earlier detection of endoscopically occult NPC.
Analysis of Plasma Epstein-Barr Virus DNA in Nasopharyngeal Cancer After Chemoradiation to Identify High-Risk Patients for Adjuvant Chemotherapy: A Randomized Controlled Trial.
Chan Anthony T C,Hui Edwin P,Ngan Roger K C,Tung Stewart Y,Cheng Ashley C K,Ng Wai T,Lee Victor H F,Ma Brigette B Y,Cheng Hoi C,Wong Frank C S,Loong Herbert H F,Tong Macy,Poon Darren M C,Ahuja Anil T,King Ann D,Wang Ki,Mo Frankie,Zee Benny C Y,Chan K C Allen,Lo Y M Dennis
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
Purpose The contribution of adjuvant chemotherapy after chemoradiation therapy (CRT) in nasopharyngeal cancer (NPC) remains controversial. Plasma Epstein-Barr virus (EBV) DNA is a potential biomarker of subclinical residual disease in NPC. In this prospective, multicenter, randomized controlled trial, we used plasma EBV DNA to identify patients with NPC at a higher risk of relapse for adjuvant chemotherapy. Patients and Methods Eligible patients with histologically confirmed NPC of Union for International Cancer Control stage IIB to IVB, adequate organ function, and no locoregional disease or distant metastasis were screened by plasma EBV DNA at 6 to 8 weeks after radiotherapy (RT). Patients with undetectable plasma EBV DNA underwent standard surveillance. Patients with detectable plasma EBV DNA were randomly assigned to either adjuvant chemotherapy with cisplatin and gemcitabine for six cycles (arm 1) or observation (arm 2). Patients were stratified for primary treatment (RT v CRT) and stage (II/III v IV). The primary end point was relapse-free survival (RFS). Results Seven hundred eighty-nine patients underwent EBV DNA screening. Plasma EBV DNA was undetectable in 573 (72.6%) and detectable in 216 (27.4%); 104 (13.2%) with detectable EBV DNA were randomly assigned to arms 1 (n = 52) and 2 (n = 52). After a median follow-up of 6.6 years, no significant difference was found in 5-year RFS rate between arms 1 and 2 (49.3% v 54.7%; P = .75; hazard ratio for relapse or death, 1.09; 95% CI, 0.63 to 1.89). The level of post-RT plasma EBV DNA correlated significantly with the hazards of locoregional failure, distant metastasis, and death. Conclusion In patients with NPC with detectable post-RT plasma EBV DNA, adjuvant chemotherapy with cisplatin and gemcitabine did not improve RFS. Post-RT plasma EBV DNA level should be incorporated as the selection factor in future clinical trials of adjuvant therapy in NPC.
Exome and genome sequencing of nasopharynx cancer identifies NF-κB pathway activating mutations.
Li Yvonne Y,Chung Grace T Y,Lui Vivian W Y,To Ka-Fai,Ma Brigette B Y,Chow Chit,Woo John K S,Yip Kevin Y,Seo Jeongsun,Hui Edwin P,Mak Michael K F,Rusan Maria,Chau Nicole G,Or Yvonne Y Y,Law Marcus H N,Law Peggy P Y,Liu Zoey W Y,Ngan Hoi-Lam,Hau Pok-Man,Verhoeft Krista R,Poon Peony H Y,Yoo Seong-Keun,Shin Jong-Yeon,Lee Sau-Dan,Lun Samantha W M,Jia Lin,Chan Anthony W H,Chan Jason Y K,Lai Paul B S,Fung Choi-Yi,Hung Suet-Ting,Wang Lin,Chang Ann Margaret V,Chiosea Simion I,Hedberg Matthew L,Tsao Sai-Wah,van Hasselt Andrew C,Chan Anthony T C,Grandis Jennifer R,Hammerman Peter S,Lo Kwok-Wai
Nasopharyngeal carcinoma (NPC) is an aggressive head and neck cancer characterized by Epstein-Barr virus (EBV) infection and dense lymphocyte infiltration. The scarcity of NPC genomic data hinders the understanding of NPC biology, disease progression and rational therapy design. Here we performed whole-exome sequencing (WES) on 111 micro-dissected EBV-positive NPCs, with 15 cases subjected to further whole-genome sequencing (WGS), to determine its mutational landscape. We identified enrichment for genomic aberrations of multiple negative regulators of the NF-κB pathway, including CYLD, TRAF3, NFKBIA and NLRC5, in a total of 41% of cases. Functional analysis confirmed inactivating CYLD mutations as drivers for NPC cell growth. The EBV oncoprotein latent membrane protein 1 (LMP1) functions to constitutively activate NF-κB signalling, and we observed mutual exclusivity among tumours with somatic NF-κB pathway aberrations and LMP1-overexpression, suggesting that NF-κB activation is selected for by both somatic and viral events during NPC pathogenesis.
Structure-based design of small-molecule inhibitors of EBNA1 DNA binding blocks Epstein-Barr virus latent infection and tumor growth.
Messick Troy E,Smith Garry R,Soldan Samantha S,McDonnell Mark E,Deakyne Julianna S,Malecka Kimberly A,Tolvinski Lois,van den Heuvel A Pieter J,Gu Bai-Wei,Cassel Joel A,Tran Donna H,Wassermann Benjamin R,Zhang Yan,Velvadapu Venkata,Zartler Edward R,Busson Pierre,Reitz Allen B,Lieberman Paul M
Science translational medicine
Epstein-Barr virus (EBV) is a DNA tumor virus responsible for 1 to 2% of human cancers including subtypes of Burkitt's lymphoma, Hodgkin's lymphoma, gastric carcinoma, and nasopharyngeal carcinoma (NPC). Persistent latent infection drives EBV-associated tumorigenesis. Epstein-Barr nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all EBV-associated tumors and is therefore an attractive target for therapeutic intervention. It is a multifunctional DNA binding protein critical for viral replication, genome maintenance, viral gene expression, and host cell survival. Using a fragment-based approach and x-ray crystallography, we identify a 2,3-disubstituted benzoic acid series that selectively inhibits the DNA binding activity of EBNA1. We characterize these inhibitors biochemically and in cell-based assays, including chromatin immunoprecipitation and DNA replication assays. In addition, we demonstrate the potency of EBNA1 inhibitors to suppress tumor growth in several EBV-dependent xenograft models, including patient-derived xenografts for NPC. These inhibitors selectively block EBV gene transcription and alter the cellular transforming growth factor-β (TGF-β) signaling pathway in NPC tumor xenografts. These EBNA1-specific inhibitors show favorable pharmacological properties and have the potential to be further developed for the treatment of EBV-associated malignancies.
Genome sequencing analysis identifies Epstein-Barr virus subtypes associated with high risk of nasopharyngeal carcinoma.
Xu Miao,Yao Youyuan,Chen Hui,Zhang Shanshan,Cao Su-Mei,Zhang Zhe,Luo Bing,Liu Zhiwei,Li Zilin,Xiang Tong,He Guiping,Feng Qi-Sheng,Chen Li-Zhen,Guo Xiang,Jia Wei-Hua,Chen Ming-Yuan,Zhang Xiao,Xie Shang-Hang,Peng Roujun,Chang Ellen T,Pedergnana Vincent,Feng Lin,Bei Jin-Xin,Xu Rui-Hua,Zeng Mu-Sheng,Ye Weimin,Adami Hans-Olov,Lin Xihong,Zhai Weiwei,Zeng Yi-Xin,Liu Jianjun
Epstein-Barr virus (EBV) infection is ubiquitous worldwide and is associated with multiple cancers, including nasopharyngeal carcinoma (NPC). The importance of EBV viral genomic variation in NPC development and its striking epidemic in southern China has been poorly explored. Through large-scale genome sequencing of 270 EBV isolates and two-stage association study of EBV isolates from China, we identify two non-synonymous EBV variants within BALF2 that are strongly associated with the risk of NPC (odds ratio (OR) = 8.69, P = 9.69 × 10 for SNP 162476_C; OR = 6.14, P = 2.40 × 10 for SNP 163364_T). The cumulative effects of these variants contribute to 83% of the overall risk of NPC in southern China. Phylogenetic analysis of the risk variants reveals a unique origin in Asia, followed by clonal expansion in NPC-endemic regions. Our results provide novel insights into the NPC endemic in southern China and also enable the identification of high-risk individuals for NPC prevention.
Methylation analysis of plasma DNA informs etiologies of Epstein-Barr virus-associated diseases.
Lam W K Jacky,Jiang Peiyong,Chan K C Allen,Peng Wenlei,Shang Huimin,Heung Macy M S,Cheng Suk Hang,Zhang Haiqiang,Tse O Y Olivia,Raghupathy Radha,Ma Brigette B Y,Hui Edwin P,Chan Anthony T C,Woo John K S,Chiu Rossa W K,Lo Y M Dennis
Epstein-Barr virus (EBV) is associated with a number of diseases, including malignancies. Currently, it is not known whether patients with different EBV-associated diseases have different methylation profiles of circulating EBV DNA. Through whole-genome methylation analysis of plasma samples from patients with nasopharyngeal carcinoma (NPC), EBV-associated lymphoma and infectious mononucleosis, we demonstrate that EBV DNA methylation profiles exhibit a disease-associated pattern. This observation implies a significant potential for the development of methylation analysis of plasma EBV DNA for NPC diagnostics. We further analyse the plasma EBV DNA methylome of NPC and non-NPC subjects from a prospective screening cohort. Plasma EBV DNA fragments demonstrate differential methylation patterns between NPC and non-NPC subjects. Combining such differential methylation patterns with the fractional concentration (count) and size of plasma EBV DNA, population screening of NPC is performed with an improved positive predictive value of 35.1%, compared to a count- and size-based only protocol.
Viral Carcinogenesis Beyond Malignant Transformation: EBV in the Progression of Human Cancers.
Elgui de Oliveira Deilson,Müller-Coan Bárbara G,Pagano Joseph S
Trends in microbiology
Cancer progression begins when malignant cells colonize adjacent sites, and it is characterized by increasing tumor heterogeneity, invasion and dissemination of cancer cells. Clinically, progression is the most relevant stage in the natural history of cancers. A given virus is usually regarded as oncogenic because of its ability to induce malignant transformation of cells. Nonetheless, oncogenic viruses may also be important for the progression of infection-associated cancers. Recently this hypothesis has been addressed because of studies on the contribution of the Epstein-Barr virus (EBV) to the aggressiveness of nasopharyngeal carcinoma (NPC). Several EBV products modulate cancer progression phenomena, such as the epithelial-mesenchymal transition, cell motility, invasiveness, angiogenesis, and metastasis. In this regard, there are compelling data about the effects of EBV latent membrane proteins (LMPs) and EBV nuclear antigens (EBNAs), as well as nontranslated viral RNAs, such as the EBV-encoded small nonpolyadenylated RNAs (EBERs) and viral microRNAs, notably EBV miR-BARTs. The available data on the mechanisms and players involved in the contribution of EBV infection to the aggressiveness of NPC are discussed in this review. Overall, this conceptual framework may be valuable for the understanding of the contribution of some infectious agents in the progression of cancers.
Incidence and mortality of nasopharyngeal carcinoma: interim analysis of a cluster randomized controlled screening trial (PRO-NPC-001) in southern China.
Ji M F,Sheng W,Cheng W M,Ng M H,Wu B H,Yu X,Wei K R,Li F G,Lian S F,Wang P P,Quan W,Deng L,Li X H,Liu X D,Xie Y L,Huang S J,Ge S X,Huang S L,Liang X J,He S M,Huang H W,Xia S L,Ng P S,Chen H L,Xie S H,Liu Q,Hong M H,Ma J,Yuan Y,Xia N S,Zhang J,Cao S M
Annals of oncology : official journal of the European Society for Medical Oncology
BACKGROUND:Previous mass screening studies have shown that IgA antibodies against Epstein-Barr Virus (EBV) can facilitate early detection of nasopharyngeal carcinoma (NPC), but the impact of EBV-antibody screening for NPC-specific mortality remains unknown. PATIENTS AND METHODS:A prospective, cluster randomized, controlled trial for NPC screening (PRO-NPC-001) was conducted in 3 selected towns of Zhongshan City and 13 selected towns of Sihui City in southern China beginning in 2008. Serum samples of the screening group were tested for two previously selected anti-EBV antibodies. Subjects with serological medium risk were subsequently retested annually for 3 years, and those with serological high risk were referred to otorhinolaryngologists for diagnostic check-up. An interim analysis was carried out to evaluate the primary end points of the NPC-specific mortality and the early diagnostic rate, and the secondary end point of the NPC incidence, through linkage with the database of Zhongshan City. RESULTS:Among 70 296 total subjects, 29 413 screened participants (41.8% of the total subjects) in the screening group and 50 636 in the control group, 153 (43.3 per 100 000 person-year), 62 (55.3 per 100 000 person-year) and 99 (33.1 per 100 000 person-year) NPC cases were identified. The early diagnostic rates of NPC were significantly higher in the participants (79.0%, P < 0.0001) and the screening group (45.9%, P < 0.0001) compared with the control group (20.6%). Although no differences were found between NPC-specific mortality of the screening group and the control group [relative risk (RR)= 0.82, 95% confidence interval (CI) 0.37-1.79], lower NPC-specific mortality was noticed among participants from the screening group versus the control group (RR = 0.22, 95% CI 0.09-0.49). CONCLUSION:IgA antibodies against EBV can identify high-risk population and was effective in screening for early asymptomatic NPC. Although the mortality reduction was not significant in the primary end point, we noted encouraging evidence of a mortality reduction in screening participants in this interim analysis. CLINICAL TRIAL NUMBER:NCT00941538.
Vasculogenic mimicry formation in EBV-associated epithelial malignancies.
Xiang Tong,Lin Yu-Xin,Ma Wenlong,Zhang Hao-Jiong,Chen Ke-Ming,He Gui-Ping,Zhang Xiao,Xu Miao,Feng Qi-Sheng,Chen Ming-Yuan,Zeng Mu-Sheng,Zeng Yi-Xin,Feng Lin
Epstein-Barr virus (EBV)-associated epithelial cancers, including nasopharyngeal carcinoma (NPC) and approximately 10% of gastric cancers, termed EBVaGC, represent 80% of all EBV-related malignancies. However, the exact role of EBV in epithelial cancers remains elusive. Here, we report that EBV functions in vasculogenic mimicry (VM). Epithelial cancer cells infected with EBV develop tumor vascular networks that correlate with tumor growth, which is different from endothelial-derived angiogenic vessels and is VEGF-independent. Mechanistically, activation of the PI3K/AKT/mTOR/HIF-1α signaling cascade, which is partly mediated by LMP2A, is responsible for EBV-induced VM formation. Both xenografts and clinical samples of NPC and EBVaGC exhibit VM histologically, which are correlated with AKT and HIF-1α activation. Furthermore, although anti-VEGF monotherapy shows limited effects, potent synergistic antitumor activities are achieved by combination therapy with VEGF and HIF-1α-targeted agents. Our findings suggest that EBV creates plasticity in epithelial cells to express endothelial phenotype and provides a novel EBV-targeted antitumor strategy.
Down-regulation of LPA receptor 5 contributes to aberrant LPA signalling in EBV-associated nasopharyngeal carcinoma.
Yap Lee Fah,Velapasamy Sharmila,Lee Hui Min,Thavaraj Selvam,Rajadurai Pathmanathan,Wei Wenbin,Vrzalikova Katerina,Ibrahim Maha Hafez,Khoo Alan Soo-Beng,Tsao Sai Wah,Paterson Ian C,Taylor Graham S,Dawson Christopher W,Murray Paul G
The Journal of pathology
Undifferentiated nasopharyngeal carcinoma (NPC) is a highly metastatic disease that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the contribution of lysophosphatidic acid (LPA) signalling to the pathogenesis of NPC. Here we demonstrate two distinct functional roles for LPA in NPC. First, we show that LPA enhances the migration of NPC cells and second, that it can inhibit the activity of EBV-specific cytotoxic T cells. Focusing on the first of these phenotypes, we show that one of the LPA receptors, LPA receptor 5 (LPAR5), is down-regulated in primary NPC tissues and that this down-regulation promotes the LPA-induced migration of NPC cell lines. Furthermore, we found that EBV infection or ectopic expression of the EBV-encoded LMP2A was sufficient to down-regulate LPAR5 in NPC cell lines. Our data point to a central role for EBV in mediating the oncogenic effects of LPA in NPC and identify LPA signalling as a potential therapeutic target in this disease.
Downregulation of ABI2 expression by EBV-miR-BART13-3p induces epithelial-mesenchymal transition of nasopharyngeal carcinoma cells through upregulation of c-JUN/SLUG signaling.
Huang Jing,Qin You,Yang Chensu,Wan Chao,Dai Xiaomeng,Sun Yajie,Meng Jingshu,Lu Yanwei,Li Yan,Zhang Zhanjie,Wu Bian,Xu Shuangbing,Jin Honglin,Yang Kunyu
Existing evidence has shown that circulating Epstein-Barr virus (EBV)-miR-BART13-3p is highly expressed in plasma of nasopharyngeal carcinoma (NPC) patients, especially among patients with advanced diseases. However, the exact role that EBV-miR-BART13-3p plays in the development of NPC remains poorly understood. Here we show that up-regulated expression of EBV-miR-BART13-3p leads to increased capacity in migration and invasion of NPC cells and causes tumor metastasis . Furthermore, we find that EBV-miR-BART13-3p directly targets ABI2, known as a tumor suppressor and a cell migration inhibitor, drives epithelial-mesenchymal transition (EMT) by activating c-JUN/SLUG signaling pathway. Silencing ABI2 shows similar effects to overexpression of EBV-miR-BART13-3p, whereas reconstitution of ABI2 resulted in a phenotypic reversion, highlighting the role of ABI2 in EBV-miR-BART13-3p-driven metastasis in NPC. Besides, expression levels of ABI2 in NPC tissue samples correlate with N stages of NPC patients. Taken together, these results suggest a novel mechanism by which ABI2 downregulation by EBV-miR-BART13-3p promotes EMT and metastasis of NPC via upregulating c-JUN/SLUG signaling pathway.
5-hydroxymethylation of the EBV genome regulates the latent to lytic switch.
Wille Coral K,Nawandar Dhananjay M,Henning Amanda N,Ma Shidong,Oetting Kayla M,Lee Dennis,Lambert Paul,Johannsen Eric C,Kenney Shannon C
Proceedings of the National Academy of Sciences of the United States of America
Latent Epstein-Barr virus (EBV) infection and cellular hypermethylation are hallmarks of undifferentiated nasopharyngeal carcinoma (NPC). However, EBV infection of normal oral epithelial cells is confined to differentiated cells and is lytic. Here we demonstrate that the EBV genome can become 5-hydroxymethylated and that this DNA modification affects EBV lytic reactivation. We show that global 5-hydroxymethylcytosine (5hmC)-modified DNA accumulates during normal epithelial-cell differentiation, whereas EBV+ NPCs have little if any 5hmC-modified DNA. Furthermore, we find that increasing cellular ten-eleven translocation (TET) activity [which converts methylated cytosine (5mC) to 5hmC] decreases methylation, and increases 5hmC modification, of lytic EBV promoters in EBV-infected cell lines containing highly methylated viral genomes. Conversely, inhibition of endogenous TET activity increases lytic EBV promoter methylation in an EBV-infected telomerase-immortalized normal oral keratinocyte (NOKs) cell line where lytic viral promoters are largely unmethylated. We demonstrate that these cytosine modifications differentially affect the ability of the two EBV immediate-early proteins, BZLF1 (Z) and BRLF1 (R), to induce the lytic form of viral infection. Although methylation of lytic EBV promoters increases Z-mediated and inhibits R-mediated lytic reactivation, 5hmC modification of lytic EBV promoters has the opposite effect. We also identify a specific CpG-containing Z-binding site on the BRLF1 promoter that must be methylated for Z-mediated viral reactivation and show that TET-mediated 5hmC modification of this site in NOKs prevents Z-mediated viral reactivation. Decreased 5-hydroxymethylation of cellular and viral genes may contribute to NPC formation.
EBV-miR-BART7-3p promotes the EMT and metastasis of nasopharyngeal carcinoma cells by suppressing the tumor suppressor PTEN.
Cai L-M,Lyu X-M,Luo W-R,Cui X-F,Ye Y-F,Yuan C-C,Peng Q-X,Wu D-H,Liu T-F,Wang E,Marincola F-M,Yao K-T,Fang W-Y,Cai H-B,Li X
The epithelial-mesenchymal transition (EMT) is crucial to cancer progression and metastasis. Although multiple cellular miRNAs have been identified to regulate the EMT and metastasis in cancers, the role of viral miRNAs in cancer progression remains largely unknown. Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy typically characterized by its early metastasis. In the present study, we have discovered the involvement of a viral miRNA, EBV-miR-BART7-3p, in the EMT and metastasis of NPC cells. Initially, we observed that EBV-miR-BART7-3p was highly expressed in NPC and positively correlated with lymph node metastasis and clinical stage of NPC. Subsequently, we demonstrated that EBV-miR-BART7-3p enhanced cell migration/invasion in vitro, cancer metastasis in vivo, and particularly the EMT characterized by loss of epithelial markers and gain of mesenchymal features in NPC cells. Furthermore, mechanistic studies disclosed that EBV-miR-BART7-3p targeted a major human tumor suppressor PTEN, modulating PI3K/Akt/GSK-3β signaling and eventually leading to the high expression and nuclear accumulation of Snail and β-catenin, which favor EMT. Knockdown of PTEN could phenocopy the effect of EBV-miR-BART7-3p, whereas re-expression of PTEN resulted in a phenotypic reversion. Moreover, these findings were supported by an observation of an EBV-positive cell model in which silencing of endogenous EBV-miR-BART7-3p partially attenuated cell migration/invasion and altered EMT protein expression pattern via reverting PI3K/Akt, Snail and β-catenin expression. Thus, this study suggests a novel mechanism by which EBV-miR-BART7-3p modulates the EMT and metastasis of NPC cells, and a clinical implication of EBV-miR-BART7-3p as a potential biomarker or therapeutic target.
The role of the EBV-encoded latent membrane proteins LMP1 and LMP2 in the pathogenesis of nasopharyngeal carcinoma (NPC).
Dawson Christopher W,Port Rebecca J,Young Lawrence S
Seminars in cancer biology
Although frequently expressed in EBV-positive malignancies, the contribution of the oncogenic latent membrane proteins, LMP1 and LMP2, to the pathogenesis of nasopharyngeal carcinoma (NPC) is not fully defined. As a key effector in EBV-driven B cell transformation and an established "transforming" gene, LMP1 displays oncogenic properties in rodent fibroblasts and induces profound morphological and phenotypic effects in epithelial cells. LMP1 functions as a viral mimic of the TNFR family member, CD40, engaging a number of signalling pathways that induce morphological and phenotypic alterations in epithelial cells. Although LMP2A plays an essential role in maintaining viral latency in EBV infected B cells, its role in epithelial cells is less clear. Unlike LMP1, LMP2A does not display "classical" transforming functions in rodent fibroblasts but its ability to engage a number of potentially oncogenic cell signalling pathways suggests that LMP2A can also participate in EBV-induced epithelial cell growth transformation. Here we review the effects of LMP1 and LMP2 on various aspects of epithelial cell behaviour highlighting key aspects that may contribute to the pathogenesis of NPC.
Dual-function nanostructured platform for isolation of nasopharyngeal carcinoma circulating tumor cells and EBV DNA detection.
Lee Sheng-Wei,Chen Yi-Wei,Kuan Edward C,Lan Ming-Ying
Biosensors & bioelectronics
Circulating tumor cells (CTCs) and plasma levels of Epstein-Barr virus (EBV) DNA are sensitive prognostic tools for monitoring disease status in nasopharyngeal carcinoma (NPC) patients. Herein, we introduce a novel and low-cost platform for capturing CTCs, the Si nanowires/microscale pyramids (NWs/MPs) hierarchical substrate, which could capture NPC cells in vitro and also detect EBV DNA at very low concentrations. In this study, Si NWs/MPs hierarchical substrates with varying wire length were fabricated using a metal-assisted chemical etching method. Anti-EpCAM antibodies were further conjugated on the substrate for capturing NPC CTCs in vitro. Capture efficiency was evaluated using immunofluorescence and scanning electronic microscopy (SEM) was utilized to understand cell morphology. The Si NWs/MPs substrate was also transformed into a Surface enhanced Raman scattering (SERS) substrate by coating with Ag nanoparticles (AgNPs) for detection of EBV DNA by Raman spectroscopy. The results demonstrated that Si NWs/MPs with 20 min of etch time had the best capturing performance. Additionally, SEM observations revealed good contact of CTCs with Si NWs/MPs substrates. Moreover, the AgNPs-coated NWs/MPs substrate was shown to be a sensitive EBV DNA detector, by which the DNA detection limit can reach up to 10M. In conclusion, the Si NWs/MPs platform not only exhibits superior cell capturing ability, but also can sensitively detect EBV DNA at very low concentrations. This platform has great potential to become a promising diagnostic tool for monitoring disease status and prognostication of NPC patients.
Cinobufotalin powerfully reversed EBV-miR-BART22-induced cisplatin resistance via stimulating MAP2K4 to antagonize non-muscle myosin heavy chain IIA/glycogen synthase 3β/β-catenin signaling pathway.
Liu Yiyi,Jiang Qingping,Liu Xiong,Lin Xian,Tang ZiBo,Liu Chen,Zhou Jin,Zhao Mengyang,Li Xin,Cheng Zhao,Li Libo,Xie Yingying,Liu Zhen,Fang Weiyi
BACKGROUND:Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-related tumor. The role of EBV-encoding miR-BART22 is still unclear in NPC. This study aimed to identify the detailed mechanisms by which EBV-miR-BART22 functions as a tumor-promoting factor and evaluate the action of cinobufotalin in treating EBV-miR-BART22-overexpressing NPC cells. METHODS:Using real-time PCR, western blotting, immunohistochemistry, and In situ hybridization, we detected the expression of miR-BART22 and MAP2K4 in tissues and cells, as well as evaluated their clinical relevance in NPC patients. The effects of miR-BART22 on cell metastasis, stemness and DDP chemoresistance were examined by sphere formation assay, side population analysis, transwell, boyden, in vivo xenograft tumor mouse model et al. Western blotting, immunofluorescence staining, luciferase reporter assay, ChIP, EMSA and Co-IP assay et al. were performed to explore the detailed molecular mechanism of EBV-miR-BART22 in NPC. Finally, we estimated the effects and molecular basis of Cinobufotalin on EBV-miR-BART22-overexpressing NPC cells in vitro and in vivo assays. FINDINGS:We observed that EBV-miR-BART22 not only promoted tumor stemness and metastasis, but also enhanced the resistance to Cisplatin (DDP) in vitro and in vivo. Mechanistic analysis indicated that EBV-miR-BART22 directly targeted the MAP2K4 and upregulated non-muscle myosin heavy chain IIA (MYH9) expression by PI3K/AKT/c-Jun-induced transcription. Further, MYH9 interacted with glycogen synthase 3β(GSK3β) protein and induced its ubiquitin degradation by activating PI3K/AKT/c-Jun-induced ubiquitin transcription and the latter combined with increased TRAF6 E3 ligase, which further bound to GSK3β protein. Reductions in the GSK3β protein thus promoted β-catenin expression and nuclear translocation, which induced tumor stemness and the epithelial-to-mesenchymal transition (EMT) signals. Furthermore, we observed that cinobufotalin, a new chemically synthesized compound, significantly suppressed EBV-miR-BART22-induced DDP chemoresistance by upregulating MAP2K4 to suppress MYH9/GSK3β/β-catenin and its downstream tumor stemness and EMT signals in NPC. Finally, clinical data revealed that increased miR-BART22 and reduced MAP2K4 expression caused the poor prognoses of NPC patients. INTERPRETATION:Our study provides a novel mechanism that cinobufotalin reversed the DDP chemoresistance and EMT induced by EBV-miR-BART22 in NPC.
High Levels of Antibody that Neutralize B-cell Infection of Epstein-Barr Virus and that Bind EBV gp350 Are Associated with a Lower Risk of Nasopharyngeal Carcinoma.
Coghill Anna E,Bu Wei,Nguyen Hanh,Hsu Wan-Lun,Yu Kelly J,Lou Pei-Jen,Wang Cheng-Ping,Chen Chien-Jen,Hildesheim Allan,Cohen Jeffrey I
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:Elevated IgA antibodies indicative of ongoing exposure to Epstein-Barr virus (EBV) are high-risk biomarkers for nasopharyngeal carcinoma (NPC), an EBV-related epithelial tumor. However, protective biomarkers that limit exposure to the virus have not been defined. We evaluated whether antibodies that can neutralize EBV infection by targeting glycoproteins involved in viral cell entry, including EBV vaccine candidate glycoprotein 350 (gp350), were associated with lower NPC risk. EXPERIMENTAL DESIGN:In a prospective cohort of 2,557 individuals from 358 high-risk NPC multiplex families in Taiwan, we identified 21 incident NPC cases and 50 disease-free controls. To complement data from high-risk families, we further identified 30 prevalent NPC cases and 50 healthy controls from the general Taiwanese population. We quantified EBV-neutralizing antibody, antibodies against EBV glycoproteins involved in B-cell and epithelial cell entry, and anti-EBNA1 IgA, a high-risk NPC biomarker. RESULTS:EBV-neutralizing antibodies blocking B-cell infection and anti-gp350 antibodies were present at significantly higher levels in disease-free controls compared with incident NPC cases (P < 0.03). Family members with both low EBV-neutralizing potential and elevated EBNA1 IgA had a 7-fold increased risk of NPC (95% CI, 1.9-28.7). Neutralizing antibodies against epithelial cell infection did not differ between incident cases and disease-free controls. Anti-glycoprotein antibody levels measured at diagnosis (prevalent NPC) were significantly higher than levels measured prior to diagnosis (P < 0.01). CONCLUSIONS:Elevated titers of EBV-neutralizing antibody and anti-gp350 antibody were low-risk biomarkers for NPC. These data suggest that a vaccine that induces potent EBV gp350 and B-cell-neutralizing antibodies could reduce the risk of EBV-related cancers such as NPC. Clin Cancer Res; 22(14); 3451-7. ©2016 AACR.
Establishment and characterization of new tumor xenografts and cancer cell lines from EBV-positive nasopharyngeal carcinoma.
Lin Weitao,Yip Yim Ling,Jia Lin,Deng Wen,Zheng Hong,Dai Wei,Ko Josephine Mun Yee,Lo Kwok Wai,Chung Grace Tin Yun,Yip Kevin Y,Lee Sau-Dan,Kwan Johnny Sheung-Him,Zhang Jun,Liu Tengfei,Chan Jimmy Yu-Wai,Kwong Dora Lai-Wan,Lee Victor Ho-Fun,Nicholls John Malcolm,Busson Pierre,Liu Xuefeng,Chiang Alan Kwok Shing,Hui Kwai Fung,Kwok Hin,Cheung Siu Tim,Cheung Yuk Chun,Chan Chi Keung,Li Bin,Cheung Annie Lai-Man,Hau Pok Man,Zhou Yuan,Tsang Chi Man,Middeldorp Jaap,Chen Honglin,Lung Maria Li,Tsao Sai Wah
The lack of representative nasopharyngeal carcinoma (NPC) models has seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. Here we report the successful growth of five NPC patient-derived xenografts (PDXs) from fifty-eight attempts of transplantation of NPC specimens into NOD/SCID mice. The take rates for primary and recurrent NPC are 4.9% and 17.6%, respectively. Successful establishment of a new EBV-positive NPC cell line, NPC43, is achieved directly from patient NPC tissues by including Rho-associated coiled-coil containing kinases inhibitor (Y-27632) in culture medium. Spontaneous lytic reactivation of EBV can be observed in NPC43 upon withdrawal of Y-27632. Whole-exome sequencing (WES) reveals a close similarity in mutational profiles of these NPC PDXs with their corresponding patient NPC. Whole-genome sequencing (WGS) further delineates the genomic landscape and sequences of EBV genomes in these newly established NPC models, which supports their potential use in future studies of NPC.
Prognostic utility of anti-EBV antibody testing for defining NPC risk among individuals from high-risk NPC families.
Yu Kelly J,Hsu Wan-Lun,Pfeiffer Ruth M,Chiang Chun-Ju,Wang Cheng-Ping,Lou Pei-Jen,Cheng Yu-Juen,Gravitt Patti,Diehl Scott R,Goldstein Alisa M,Chen Chien-Jen,Hildesheim Allan
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:Epstein-Barr virus (EBV) infection and a family history of nasopharyngeal carcinoma (NPC) are associated with NPC risk. We examined the risk associated with EBV markers and their clinical utility to identify NPC susceptibles within high-risk NPC families. EXPERIMENTAL DESIGN:We evaluated antibody titers against viral capsid antigen (VCA) IgA, EBV nuclear antigen-1 (EBNA1) IgA, and DNase among unaffected relatives of NPC cases from 358 multiplex families in Taiwan. Incident NPC cases were identified via linkage to the National Cancer Registry. Clinical examinations of 924 individuals were also done to identify occult, asymptomatic NPC. Baseline EBV serology was used to estimate NPC risk using rate ratios with 95% CI. Associated sensitivity/specificity and receiver operating characteristic (ROC) curves were calculated. RESULTS:A total of 2,444 unaffected individuals with 15,519 person-years (6.5 years median follow-up) yielded 14 incident NPC cases (nearly 11 times the general population rate). The absolute rate of NPC among anti-EBV EBNA1 IgA seropositives using a standard positivity cutoff versus an optimized cutoff point defined by ROC analyses was 265/100,000 person-years with a 4.7-fold increased risk of NPC (95% CI: 1.4-16) and 166/100,000 person-years with a 6.6-fold increase (95% CI: 1.5-61), respectively. Sensitivity and specificity using the optimized positivity cutoff points were 85.7% and 51.2%, respectively. It is estimated that active evaluation of 49% of individuals from high-risk NPC families seropositive for this marker could lead to earlier detection of up to 86% of NPC cases. Risks associated with the other three EBV markers were weaker. CONCLUSIONS:Future efforts are needed to identify susceptibility markers among high-risk NPC families that maximize both sensitivity and specificity.
Identification of a Novel, EBV-Based Antibody Risk Stratification Signature for Early Detection of Nasopharyngeal Carcinoma in Taiwan.
Coghill Anna E,Pfeiffer Ruth M,Proietti Carla,Hsu Wan-Lun,Chien Yin-Chu,Lekieffre Lea,Krause Lutz,Teng Andy,Pablo Jocelyn,Yu Kelly J,Lou Pei-Jen,Wang Cheng-Ping,Liu Zhiwei,Chen Chien-Jen,Middeldorp Jaap,Mulvenna Jason,Bethony Jeff,Hildesheim Allan,Doolan Denise L
Clinical cancer research : an official journal of the American Association for Cancer Research
Epstein-Barr virus (EBV) is necessary for the development of nasopharyngeal carcinoma (NPC). By adulthood, approximately 90% of individuals test EBV-positive, but only a fraction develop cancer. Factors that identify which individuals are most likely to develop disease, including differential antibody response to the virus, could facilitate detection at early stages when treatment is most effective. We measured anti-EBV IgG and IgA antibody responses in 607 Taiwanese individuals. Antibodies were measured using a custom protein microarray targeting 199 sequences from 86 EBV proteins. Variation in response patterns between NPC cases and controls was used to develop an antibody-based risk score for predicting NPC. The overall accuracy [area under the curve (AUC)] of this risk score, and its performance relative to currently used biomarkers, was evaluated in two independent Taiwanese cohorts. Levels of 60 IgA and 73 IgG anti-EBV antibodies differed between stage I/IIa NPC cases and controls ( < 0.0002). Risk prediction analyses identified antibody targets that best discriminated NPC status-BXLF1, LF2,BZLF1, BRLF1, EAd, BGLF2, BPLF1, BFRF1, and BORF1. When combined with currently used VCA/EBNA1 IgA biomarkers, the resulting risk score predicted NPC with 93% accuracy (95% CI, 87%-98%) in the general Taiwanese population, a significant improvement beyond current biomarkers alone (82%; 95% CI, 75%-90%, ≤ 0.01). This EBV-based risk score also improved NPC prediction in genetically high-risk families (89%; 95% CI, 82%-96%) compared with current biomarkers (78%; 95% CI, 66%-90%, ≤ 0.03). We identified NPC-related differences in 133 anti-EBV antibodies and developed a risk score using this microarray dataset that targeted immune responses against EBV proteins from all stages of the viral life cycle, significantly improving the ability to predict NPC. .
EBV-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma.
Lung Raymond W-M,Hau Pok-Man,Yu Ken H-O,Yip Kevin Y,Tong Joanna H-M,Chak Wing-Po,Chan Anthony W-H,Lam Ka-Hei,Lo Angela Kwok-Fung,Tin Edith K-Y,Chau Shuk-Ling,Pang Jesse C-S,Kwan Johnny S-H,Busson Pierre,Young Lawrence S,Yap Lee-Fah,Tsao Sai-Wah,To Ka-Fai,Lo Kwok-Wai
The Journal of pathology
Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein-Barr virus (EBV) infection. In NPC, miR-BARTs, the EBV-encoded miRNAs derived from BamH1-A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV-encoded miRNAs in a panel of NPC patient-derived xenografts and an EBV-positive NPC cell line by small RNA sequencing. Among the 40 miR-BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV-miRNAs, BART5-5p, BART7-3p, BART9-3p, and BART14-3p could negatively regulate the expression of a key DNA double-strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'-UTR. Notably, the expression of these four miR-BARTs represented more than 10% of all EBV-encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT-PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5-5p, BART7-3p, BART9-3p, and BART14-3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR-BARTs in EBV-positive NPC cells, we further demonstrated the novel function of miR-BARTs in inhibiting Zta-induced lytic reactivation. These findings imply that the four viral miRNAs work co-operatively to modulate ATM activity in response to DNA damage and to maintain viral latency, contributing to the tumorigenesis of NPC. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Vesicle-bound EBV-BART13-3p miRNA in circulation distinguishes nasopharyngeal from other head and neck cancer and asymptomatic EBV-infections.
Ramayanti Octavia,Verkuijlen Sandra A W M,Novianti Putri,Scheepbouwer Chantal,Misovic Branislav,Koppers-Lalic Danijela,van Weering Jan,Beckers Lisa,Adham Marlinda,Martorelli Debora,Middeldorp Jaap M,Pegtel Dirk Michiel
International journal of cancer
Cell-free microRNA (miRNA) in biofluids released by tumors in either protein or vesicle-bound form, represent promising minimally-invasive cancer biomarkers. However, a highly abundant non-tumor background in human plasma and serum complicates the discovery and detection of tumor-selective circulating miRNAs. We performed small RNA sequencing on serum and plasma RNA from Nasopharyngeal Carcinoma (NPC) patients. Collectively, Epstein Barr virus-encoded miRNAs, more so than endogenous miRNAs, signify presence of NPC. However, RNAseq-based EBV miRNA profiles differ between NPC patients, suggesting inter-tumor heterogeneity or divergent secretory characteristics. We determined with sensitive qRT-PCR assays that EBV miRNAs BART7-3p, BART9-3p and BART13-3p are actively secreted by C666.1 NPC cells bound to extracellular vesicles (EVs) and soluble ribonucleoprotein complexes. Importantly, these miRNAs are expressed in all primary NPC tumor biopsies and readily detected in nasopharyngeal brushings from both early and late-stage NPC patients. Increased levels of BART7-3p, BART9-3p and particularly BART13-3p, distinguish NPC patient sera from healthy controls. Receiver operating characteristic curve analysis using sera from endemic NPC patients, other head and neck cancers and individuals with asymptomatic EBV-infections reveals a superior diagnostic performance of EBV miRNAs over anti-EBNA1 IgA serology and EBV-DNA load (AUC 0.87-0.96 vs 0.86 and 0.66 respectively). The high specificity of circulating EBV-BART13-3p (97%) for NPC detection is in agreement with active secretion from NPC tumor cells. We conclude EV-bound BART13-3p in circulation is a promising, NPC-selective, biomarker that should be considered as part of a screening strategy to identify NPC in endemic regions.
Emodin Inhibits EBV Reactivation and Represses NPC Tumorigenesis.
Wu Chung-Chun,Chen Mei-Shu,Cheng Yu-Jhen,Ko Ying-Chieh,Lin Su-Fang,Chiu Ing-Ming,Chen Jen-Yang
Nasopharyngeal carcinoma (NPC) is a unique malignancy derived from the epithelium of the nasopharynx. Despite great advances in the development of radiotherapy and chemotherapy, relapse and metastasis in NPC patients remain major causes of mortality. Evidence accumulated over recent years indicates that Epstein-Barr virus (EBV) lytic replication plays an important role in the pathogenesis of NPC and inhibition of EBV reactivation is now being considered as a goal for the therapy of EBV-associated cancers. With this in mind, a panel of dietary compounds was screened and emodin was found to have potential anti-EBV activity. Through Western blotting, immunofluorescence, and flow cytometric analysis, we show that emodin inhibits the expression of EBV lytic proteins and blocks virion production in EBV- positive epithelial cell lines. In investigating the underlying mechanism, reporter assays indicated that emodin represses Zta promoter (Zp) and Rta promoter (Rp) activities, triggered by various inducers. Mapping of the Zp construct reveals that the SP1 binding region is important for emodin-triggered repression and emodin is shown to be able to inhibit SP1 expression, suggesting that it likely inhibits EBV reactivation by suppression of SP1 expression. Moreover, we also show that emodin inhibits the tumorigenic properties induced by repeated EBV reactivation, including micronucleus formation, cell proliferation, migration, and matrigel invasiveness. Emodin administration also represses the tumor growth in mice which is induced by EBV activation. Taken together, our results provide a potential chemopreventive agent in restricting EBV reactivation and NPC recurrence.
Targeting Exosomal EBV-LMP1 Transfer and miR-203 Expression via the NF-κB Pathway: The Therapeutic Role of Aspirin in NPC.
Zuo Lielian,Xie Yan,Tang Jinyong,Xin Shuyu,Liu Lingzhi,Zhang Siwei,Yan Qijia,Zhu Fanxiu,Lu Jianhong
Molecular therapy. Nucleic acids
Nasopharyngeal carcinoma (NPC) is an invasive head-and-neck tumor with Epstein-Barr virus (EBV) as an important etiological cause. The EBV oncoprotein Latent membrane protein 1 (LMP1) can be trafficked into exosomes with unclear roles, and this trafficking is a potential problem in NPC control. MicroRNA-203 (miR-203) was found by us to be downregulated by LMP1, and it functions as a tumor suppressor in NPC. In this study, aspirin reversed the epithelial-mesenchymal transition (EMT) by promoting miR-203 expression in cells, and, remarkably, it repressed exosomal LMP1 (exo-LMP1) secretion from EBV-positive cells. Nuclear factor κB (NF-κB) activation was required for the exo-LMP1 production. The exo-LMP1 uptake influenced the EMT potential of EBV-negative recipient NPC cells. The exo-LMP1 level was upregulated in clinical NPC plasma samples. Aspirin treatment observably inhibited NPC lung metastasis in nude mice. The study revealed that aspirin is a promising drug for NPC therapy via its targeting of exo-LMP1 transfer and the regulatory effect of LMP1 on miR-203 expression. EBV can regulate its own tumorigenesis via the LMP1/NF-κB/exo-LMP1 axis, opening a new avenue for understanding the pathogenesis of this tumor virus. Our study also provides a rationale for the use of exo-LMP1 or exosomal miR-203 (exo-miR203) in EBV-targeted therapy by aspirin in invasive NPC.
A comparison of EBV serology and serum cell-free DNA as screening tools for nasopharyngeal cancer: Results of the Singapore NPC screening cohort.
Tay Joshua K,Siow Chor H,Goh Han L,Lim Chwee M,Hsu Pon P,Chan Soh H,Loh Kwok S
International journal of cancer
We aimed to evaluate the effectiveness of nasopharyngeal cancer (NPC) screening by comprehensive clinical follow-up and adjunctive Epstein-Barr virus (EBV) testing. In a prospective cohort study, 524 individuals with a first-degree family history of NPC were recruited at a university clinical center in Singapore. The cohort was evaluated at baseline and at 6 monthly intervals, with a complete head and neck examination including nasopharyngeal endoscopy. Blood was taken at baseline and at yearly intervals for EBV Viral Capsid Antigen (VCA) IgA, EBV Early Antigen (EA) IgA serology and serum cell-free EBV DNA. Nasopharyngeal biopsy was performed when any irregularity in the nasopharynx was observed, or when EBV markers were elevated. The mean duration of follow-up was 57.7 months, with an average of 8.6 clinical visits per participant. Five participants (0.96%) were identified to have NPC, giving a prevalence of 199 per 100,000 person-years of screening. Four of the five NPC cases identified had asymptomatic T1 disease, at an earlier stage compared to NPC patients diagnosed in the clinic during the same time period (p = 0.0297). All NPC cases identified had elevated EBV-EA IgA titers ≥1:10, with a specificity of 94.6% and a positive predictive value of 15.2%, outperforming EBV-VCA IgA and serum EBV DNA. Two NPC cases were biopsied only because of elevated EBV serology titers, with increasing EBV-EA IgA titers preceding the diagnosis of NPC. In conclusion, screening for NPC is effective in identifying early-stage disease. Adjunctive EBV-EA IgA testing improved the effectiveness of screening.