Conserved mutation of Epstein-Barr virus-encoded BamHI-A Rightward Frame-1 (BARF1) gene in Indonesian nasopharyngeal carcinoma

Background  

BamHI-A rightward frame-1 (BARF1) is a carcinoma-specific Epstein-Barr virus (EBV) encoded oncogene. Here we describe the BARF1 sequence diversity in nasopharyngeal carcinoma (NPC), other EBV-related diseases and Indonesian healthy EBV carriers in relation to EBV genotype, viral load and serology markers. Nasopharyngeal brushings from 56 NPC cases, blood or tissue from 15 other EBV-related disorders, spontaneous B cell lines (LCL) from 5 Indonesian healthy individuals and several prototype EBV isolates were analysed by PCR-direct sequencing.     

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

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

EB病毒基因BARF1在鼻咽癌细胞中的表达及意义

目的：探讨EB(Epstein—Barr)病毒新基因BARF1(BamHI A rightward open reading frame1)在人鼻咽癌组织中的表达及意义，为深入阐明EB病毒致癌机制提供实验依据。方法：提取RNA后，采用RT—PCR方法扩增标本中的EBNA1(EB virus associated nuclear antigen 1)和BARF1 mRNA，PCR产物经2％琼脂糖凝胶电泳观察并照相。结果：11例RNA合格的标本均表达EBNA1，提示病例均为EB病毒阳性病例；其中9例表达BARF1，占82％：而且9例中的7例为强阳性。结论：EB病毒新基因BARF1 mRNA在鼻咽癌细胞中高表达，这提示除了已经明确的潜伏性膜蛋白1(LMP1)以外，BARF1可能在鼻咽癌细胞恶性增殖中发挥重要作用，具体机制有待深入研究。     

Laboratory markers of tumor burden in nasopharyngeal carcinoma: a comparison of viral load and serologic tests for Epstein-Barr virus

Epstein-Barr virus (EBV) is present within the tumor cells of most cases of nasopharyngeal carcinoma (NPC). Recent studies suggest that tumor burden is proportional to the level of EBV DNA in blood and that rapid blood testing can be used to guide therapeutic intervention. The relative utility of viral load vs. serology has been insufficiently studied. In our study, EBV viral load was measured by quantitative PCR using either real-time or end-point detection systems in serum samples from 124 NPC patients (93 pretreatment, 13 relapsed, 18 in remission) and 40 controls. Serologic titers against EBV early antigen were measured in the same serum samples. EBV DNA was detectable in 64 of 93 untreated NPC patients (69%; mean viral load 11,211 copies/ml), 11 of 13 relapsed NPC patients (85%; mean 53,039 copies/ml) and 0 of 18 remission patients. EBV DNA was detectable in only 1 of 40 non-NPC controls (3%). In 34 instances where paired plasma and serum samples were available for testing, both were effective sample types, and there was no significant difference between end-point and real-time methods for measuring viral load. Early antigen (EA) IgA and IgG titers were elevated in most NPC patients regardless of whether their disease was active or in remission. EBV viral load was more informative than was EA serology for distinguishing remission from relapsed disease. EBV DNA measurement appears to be a noninvasive way to monitor tumor burden after therapy.     

Epigenetic markers for noninvasive early detection of nasopharyngeal carcinoma by methylation‐sensitive high resolution melting

Nasopharyngeal carcinoma (NPC) is a human malignancy that is closely associated with Epstein‐Barr Virus (EBV). Early diagnosis of NPC will greatly improve the overall survival. However, current EBV DNA marker detection still lacks the predictive value to perform well in high‐risk populations for early detection of NPC. Since aberrant promoter hypermethylation of tumor suppressor genes (TSGs) is widely considered to be an important epigenetic change in early carcinogenesis, this study identified a panel of methylation markers for early detection of NPC and also assessed the clinical usefulness of these markers with noninvasive plasma specimens instead of biopsies. MS‐HRM assays were carried out to assess the methylation status of a selected panel of four TSGs (RASSF1A, WIF1, DAPK1 and RARβ2) in biopsies, NP brushings and cell‐free plasma from NPC patients. High‐risk and cancer‐free groups were used as controls. DNA methylation panel showed higher sensitivity and specificity than EBV DNA marker in cell‐free plasma from NPC patients at early Stages (I and II) and in addition to the EBV DNA marker, MS‐HRM test for plasma and NP brushing DNA methylation significantly increased the detection rate at all NPC stages as well as local recurrence, using this selected four‐gene panel (p < 0.05). MS‐HRM assay on a selected gene panel has great potential to become a noninvasive and complementary test for NPC early and recurrent detection in combination with the EBV DNA test to increase the sensitivity for NPC detection at an early stage.     

Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma

Expression of the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNA 1 to 6) and membrane-associated protein (LMP) was investigated by immunoblotting in 83 nasopharyngeal carcinoma (NPC) biopsies and 25 other tumor and normal tissue specimens from the head and neck region. Fifty-eight of the 83 NPC biopsies were large enough to yield parallel data on virus DNA and viral expression. All 16 cases of clinically diagnosed and histologically confirmed NPCs from North Africa contained EBV DNA and expressed EBNA-1. Of 31 clinically diagnosed NPCs from China, 29 contained EBV DNA and 25 of these expressed EBNA-1. One control tissue biopsy from the oropharynx of NPC patients contained EBV DNA, but none expressed EBNA-1. The latent membrane protein (LMP) was detected in 22/31 of the Chinese and in 10/16 of the North African NPC biopsies. None of the NPC biopsies or control tissues expressed detectable amounts of EBNA 2 or any of the other 4 nuclear antigens which are invariably expressed in EBV-transformed B cells. A smaller number of tumors from Malaysia and East Africa exhibited a similar pattern of expression. EBV was rescued from a nude-mouse-passaged North African NPC tumor by co-cultivation of the tumor cells with umbilical cord blood lymphocytes. The tumor expressed EBNA 1 and LMP, but not EBNA 2 or the other 4 EBNAs. The resulting LCLs expressed all 6 nuclear antigens, EBNA 1 to 6 and LMP. Our data suggest that expression of the EBV genome is regulated in a tissue-specific fashion.     

Quantitative Epstein-Barr virus DNA analysis and detection of gene promoter hypermethylation in nasopharyngeal (NP) brushing samples from patients with NP carcinoma.

PURPOSE: Nasopharyngeal carcinoma (NPC) is highly prevalent in southern China and characterized by a strong association with EBV. We aimed to detect EBV DNA and cancer-related gene promoter hypermethylation in nasopharyngeal (NP) brushing samples and provide a novel noninvasive approach for NPC detection. EXPERIMENTAL DESIGN: Twenty-eight NPC cases and 26 noncancerous subjects were prospectively recruited. NP brushing samples were subjected to quantitative real-time PCR analysis of EBV DNA and methylation-specific PCR analysis of the DAP-kinase, RASSF1A, and p16 genes. RESULTS: EBV DNA quantity in NP brushing samples from NPC patients (median, 8.94 copies/actin) was significantly higher than that of controls (median, 0 copies/actin; P < 0.0001). Twenty-seven of 28 NPC patients had detectable EBV DNA in NP brushes, whereas 25 of 26 controls had undetectable or very low levels of EBV DNA. Elevated EBV DNA level in brushing samples as a tumor marker had a sensitivity of 96.4% and a specificity of 96.2% for NPC detection. Moreover, T(1) disease had a significantly lower EBV DNA level as compared with locally more advanced disease (P = 0.037). In brushing samples of NPC patients, the frequencies of DAP-kinase, RASSF1A, and p16 promoter hypermethylation were 50.0%, 39.3%, and 46.4%, respectively. Seventy-eight percent of cases showed methylation of at least one gene. No aberrant hypermethylation was detected in control samples. CONCLUSIONS: Our study demonstrated the feasibility of detecting multiple molecular tumor markers in NP brushing samples with a high sensitivity and specificity for NPC detection. It offers a powerful yet noninvasive approach for the diagnosis of NPC in high-risk populations.     

Quantification of Epstein–Barr virus DNA load in nasopharyngeal brushing samples in the diagnosis of nasopharyngeal carcinoma in southern China

Nasopharyngeal carcinoma (NPC) is highly incident in southern China, where 40% of world's new cases arise each year. Detection of Epstein–Barr virus (EBV) DNA load in nasopharyngeal (NP) brush/swab samples has gradually been established as a method for diagnosis of NPC. However, its applicable value in NPC diagnosis has never been investigated in southern China. It is important to explore whether such a test could be applicable to our local population. A total of 245 consecutive participants undergoing NP brushing examination were recruited to obtain the NP brushing samples in this study. Quantitative PCR assays were used to obtain the EBV DNA load. Mann–Whitney, ANOVA and receiver operating characteristic tests were used to analyze its diagnostic value. NP brushing samples from NPC patients showed extremely high levels of EBV DNA load (mean = 46360 copy/ng DNA) compared to its expression from non‐NPC control (mean = 28 copy/ng DNA) and high‐risk control (mean = 50 copy/ng DNA) groups. It produced 96% sensitivity and 97% specificity, at the COV = 225 copy/ng DNA. Furthermore, EBV DNA load could reflect disease progress. Our data showed a better performance of EBV DNA load in NP brushing samples compared with an initial biopsy, immunoglobulin A (IgA) antibody titers to viral capsid antigen in serum and EBV DNA load in plasma. Detection of EBV DNA load in NP brushing samples could be an effective supplement for NPC diagnosis. Being minimally invasive and low cost, NP brush sampling combined with EBV DNA detection demonstrates great potential for screening high‐risk populations for NPC.     

Association of Epstein-Barr virus with nasopharyngeal carcinoma in Alaskan native patients: serum antibodies and tissue EBNA and DNA

Biopsy specimens from Alaskan Native patients with nasopharyngeal carcinoma (NPC) and from other patients seen on the otolaryngology service were tested for Epstein-Barr virus-specific DNA and nuclear antigen (EBNA). Serum samples from both groups were tested for various EBV-related antibodies. EBV DNA and EBNA results were in agreement in 29 of 31 tissue specimens tested by the two methods. Ten of 11 biopsies containing NPC cells were positive for EBV DNA. Two NPC patients had biopsies that showed only atypical epithelium but were also positive for EBV DNA or EBNA. The other tissue specimens were negative except for biopsies from two patients: one with a parotid gland lymphoepithelial lesion; another with undifferentiated carcinoma of salivary gland origin.     

Reconstitution of nasopharyngeal carcinoma-type EBV infection induces tumorigenicity

Several reports have shown that the EBV-encoded BARF1 gene has oncogenic activity. We have recently reported that BARF1 is expressed as a latent gene in most nasopharyngeal carcinomas (NPC), suggesting that BARF1 may have an important role in NPC oncogenesis. However, we found that when the NPC-derived EBV-negative cell lines, HONE-1 and CNE-1, were infected with EBV in vitro, BARF1 was not expressed, although the expression of other latent genes was identical to that of NPC tumors. Therefore, we generated a recombinant EBV (rEBV) carrying the BARF1 gene (BARF1-rEBV) under the SV40 promoter to reconstitute the NPC-type EBV infection. NPC-derived EBV-negative cell lines were stably infected with either a wild-type rEBV (wild-rEBV) or BARF1-rEBV. The resultant BARF1-rEBV-infected NPC cell clones represented NPC-type EBV expression, and BARF1 expression was similar to that observed in NPC tissues. BARF1-rEBV-infected cell clones grew to a higher cell density and were more resistant to apoptosis than wild-rEBV-infected counterparts. BARF1 protein was quickly secreted into the culture medium, and secreted BARF1 contributed to the increase of cell densities in NPC cells, but it had no effect on resistance to apoptosis. Furthermore, BARF1-rEBV-infected cell clones became tumorigenic in nude mice. These results suggest that BARF1 plays an important role in NPC development.     

EB病毒相关胃癌组织中病毒基因的表达

背景与目的：EB病毒(Epstein—Barr virus,EBV)与多种恶性肿瘤的发生有关,在鼻咽癌及淋巴瘤等组织中EBV的存在形式和表达已有报道,研究表明不同肿瘤组织中EBV的存在形式和表达不同,在胃癌组织中EBV某些基因尤其是裂解性基因的表达情况报道较少。为明确胃癌组织中EBV潜伏性基因和裂解性基因的表达情况,本研究从mRNA水平检测胃癌组织中EBV潜伏感染和裂解感染相关基因的表达,从分子水平探讨EBV编码基因与胃癌发生发展的关系。方法：应用PCR—Southern杂交检测185例胃癌及其相应癌旁组织中特异性EBVDNA片段,进一步用原位杂交(ISH)技术检测PCR阳性标本EBV编码小RNA1(EBERl)的表达,癌细胞EBERl阳性者确定为EBV相关胃癌(EBV—associated gastric carcinoma,EBVaGC)。采用RT-PCR和Southern杂交技术检测EBVaGCs组织中EBV核抗原(EBNAs)基因转录启动子(Qp、Cp和Wp)、潜伏性基因(EBNA1、EBNA2基因,潜伏膜蛋白LMP1、LMP2A和LMP2B基因)和裂解性基因(即刻早期基因BZLF1和BRLF1,早期基因BARF1和BHRF1,晚期基因BcLF1和BLLF1)的表达。结果：185例胃癌标本EBV阳性率为7．03％(13／185),癌旁组织均为阴性。13例EBVaGCs组织中均检测到启动子Qp mRNA,而Wp和Cp mRNA则为阴性。潜伏性基因中EBNA1 mRNA均阳性(13／13),而EBNA2、LMP1和LMP2B均未见表达,5例标本(5／13)检测到LMP2A mRNA。裂解性基因中BcLF1有7例(7／13)表达阳性,2例(2／13)BHRF1表达阳性,6例标本(6／13)检测到BZLF1 mRNA,BARF1亦有6例(6／13)表达阳性,而BRLF1和BLLF1mRNA均为阴性。结论：EBVaGC中EBV潜伏类型为Ⅰ型或介于Ⅰ和Ⅱ型之间的独特类型;EBVaGC组织中部分裂解性基因表达阳性,部分胃癌存在BARF1和BHRF1表达,其在胃癌中所起的作用有待进一步研究。     

Serologic diagnosis of nasopharyngeal carcinoma. A double-blind study of four EB virus antibodies with evaluation by sequential discrimination

It is generally known that a close relationship exists between Epstein-Barr Virus (EBV) and nasopharyngeal carcinoma (NPC). Recently, patients with early lesions of NPC have been detected in the general population by use of serologic mass survey. Using the double-blind method, we have studied the diagnostic value of the four EBV antibody titers, VCA-IgA, VCA-IgG, EA-IgA and EA-IgG, in four groups of subjects, each consisting of 50 persons: patients with nasopharyngeal carcinoma (NPC group), patients with cancers other than NPC in the head and neck regions (HNC group), patients with cancers outside of head and neck regions (OC group) and normal individuals (NS group). The results of these four antibodies were evaluated both singularly and together by multivariate sequential discrimination. Taking 1:10 as the criterion of being positive, in the NPC group, the positive rate of VCA-IgA is 88%, the VCA-IgG rate is 100%, the EA-IgA rate is 48% and the EA-IgG rate is 74%. In the non-NPC group, the positive rates of VCA-IgA are as high as 86%-92%, but those of the other antibodies are as low as 0-42%. The positive rates and the geometric mean titers of these four antibodies were all elevated as compared with those in the three non-NPC groups. These differences are statistically significant. VCA-IgG is unimportant in the diagnosis of NPC because of its low specificity. By treating the antibody titers of VCA-IgA, VCA-IgG, EA-IgA and EA-IgG with sequential discrimination, the correlation rate between the serology and pathology of NPC is 88% and the false positive rate is 7.3%.     

Epstein-Barr virus DNA loads in the peripheral blood cells predict the survival of locoregionally-advanced nasopharyngeal carcinoma patients

Objective:Circulating cell-free Epstein-Barr virus (EBV) DNA has been shown to be a valuable biomarker for population screening and prognostic surveillance for nasopharyngeal carcinoma (NPC). Despite important insights into the biology of persistence, few studies have addressed the clinical significance of cell-based EBV-DNA loads in peripheral blood cells (PBCs).Methods:A prospective observational cohort study was conducted involving 1,063 newly diagnosed, locoregionally-advanced NPC patients at Sun Yat-sen University Cancer Center from 2005 to 2007. Cox regression analysis was conducted to identify the association of PBC EBV DNA loads to overall survival (OS) and other prognostic outcomes. Prognostic nomograms were developed based on PBC EBV DNA loads to predict survival outcomes for NPC patients.Results:After a median follow-up of 108 months, patients with higher PBC EBV-DNA loads had significantly worse OS [hazard ratio (HR) of medium, medium-high, and high vs. low were 1.50, 1.52, and 1.85 respectively; P_trend < 0.001]. Similar results were found for progression-free survival and distant metastasis-free survival. The concordance index of the prognostic nomogram for predicting OS in the training set and validation set were 0.70 and 0.66, respectively. Our data showed that the PBC EBV DNA load was an independent and robust survival biomarker, which remained significant even after adjusting for plasma EBV DNA loads in a subset of 205 patients of the cohort (HR: 1.88; P = 0.025). Importantly, a combination of PBC EBV DNA load and plasma EBV DNA load improved the predicted OS.Conclusions:The EBV-DNA load in PBCs may be an independent prognosis marker for NPC patients.     

鼻咽癌患者血浆EBV DNA水平的研究进展

EB病毒与鼻咽癌的发生有密切关系。近年来,随着PCR技术的发展,在鼻咽癌患者血浆中可定量检测EBVDNA水平。大量研究表明,鼻咽癌患者血浆EBVDNA的检出率及拷贝数明显高于正常人群。放疗后转移、复发患者其EBVDNA的阳性率及拷贝数高于持续缓解患者。血浆EBVDNA水平在鼻咽癌的早期诊断、临床分期、预后判断及监测治疗后转移、复发中均有重要临床意义。     

EB病毒VCA/IgA、Rta/IgG及EBNA1/IgA抗体水平在鼻咽癌高发区不同人群中的分布

目的 观察分析鼻咽癌高发区中的鼻咽癌患者、非鼻咽癌头颈部相似疾病患者和健康体检人群中EB病毒VCA/IgA、Rta/IgG及EBNA1/IgA的抗体水平分布情况。方法 收集211例未经治疗的鼻咽癌患者、203例头颈部相似症状患者和210例健康体检者的血清,采用免疫酶法检测VCA/IgA,采用酶联免疫吸附法（ELISA）检测Rta/IgG和EBNA1/IgA。应用秩和检验、受试者工作特征(ROC)曲线、多分类logistic回归模型等方法对结果进行分析评价。结果 鼻咽癌组的VCA/IgA、Rta/IgG及EBNA1/IgA抗体水平均显著高于头颈部相似疾病组和健康对照组(P<0.001)。头颈部相似疾病组的Rta/IgG及VCA/IgA抗体水平也明显高于健康对照组(P<0.001)。以头颈部相似疾病组和健康体检组为分析人群,分别作相关抗体的ROC曲线,VCA/IgA 的ROC曲线下面积为0.565,Rta/IgG抗体的ROC曲线下面积为0.604,具有统计学意义(P<0.05)。综合年龄、性别和3种EB病毒抗体等因素的多分类logistic回归分析显示,鼻咽癌、头颈部相似疾病和健康体检者的预测准确率分别为95.3％、70.9％和55.2％。结论 在鼻咽癌高发区EB病毒VCA/IgA及Rta/IgG抗体水平在头颈部相似疾病人群和健康人群中存在一定差异,在鼻咽癌的人群筛查和临床诊断中可根据具体情况设定不同的抗体阳性临界值。     

The 30-bp deletion variant: a polymorphism of latent membrane protein 1 prevalent in endemic and non-endemic areas of nasopharyngeal carcinomas in China.

Development of nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. However, NPC occurs with a marked geographic and racial distribution, whereas EBV infection is ubiquitous in the world. This leads to a question whether certain subtypes of EBV have a greater potential to induce cell transformation. Latent membrane protein 1 (LMP1) is an EBV-encoded oncogenic protein and its 30-bp deleted variant (del-LMP1) has been reported to be predominant in biopsies of NPC. We have assessed the polymorphism of LMP1 in 47 biopsies of NPC, 107 cases of throat washings (TWs) from NPC patients, and 106 cases of TWs from non-NPC patients in Guangzhou, an endemic area of NPC in southern China, as well as 103 cases of TWs from healthy donors in Haerbin, a non-endemic area of NPC in northern China. Our results found a similar extent of the LMP1 polymorphism between NPC patients and non-NPC patients in Guangzhou, with the del-LMP1 being predominant in both Guangzhou and Haerbin. Sequence analyses showed identical substitutions in other coding regions of the del-LMP1 isolated from Guangzhou and Haerbin. These results indicate that del-LMP1 represents a geographic or race-associated polymorphism rather than an NPC disease phenotype-associated polymorphism.     

Immunity to antigens associated with a cell line derived from nasopharyngeal cancer (NPC) in non-Chinese NPC patients.

Delayed hypersensibility to antigens derived from four lymphoid cell lines was measured in 27 non-Chinese patients with nasopharyngeal cancer (NPC) and 63 non-NPC cancer patients. Of the NPC patients, 17/27 (63%) had a positive skin test response to antigens derived from HKLY-28, a lymphoid cell line which was developed from an NPC biopsy. Only 10/51 (20%) and 1/13 (8%) patients with solid tumors and hematopoietic malignancies, respectively, had positive skin test responses to HKLY-28. Positive skin tests were found less frequently when extracts from cell lines derived from normal individuals or lymphoma patients were utilized, although NPC patients were more reactive to two of the non-NPC derived cell lines than the controls. The NPC patients in this study also had significantly elevated antibody titers to the Epstein-Barr virus (EBV), capsid antigen (VCA) and early antigen (EA). Titers were highest in the patients with more anaplastic nasopharyngeal carcinomas. The skin test and serologic data are consistent with studies in Chinese patients, indicating that NPC in non-Chinese and Chinese patients is biologically similar.