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.     

Noninvasive diagnosis of nasopharyngeal carcinoma: nasopharyngeal brushings reveal high Epstein-Barr virus DNA load and carcinoma-specific viral BARF1 mRNA

Nasopharyngeal carcinoma (NPC) is the most prevalent ENT-tumour in Indonesia. We investigated the primary diagnostic value of Epstein-Barr virus (EBV) DNA load and mRNA detection in noninvasive nasopharyngeal (NP) brushings, obtained prospectively from consecutive Indonesian ENT-patients with suspected NPC (N=106) and controls. A subsequent routine NP biopsy was taken for pathological examination and EBER-RISH, yielding 85 confirmed NPC and 21 non-NPC tumour patients. EBV DNA and human DNA load were quantified by real-time PCR. NP brushings from NPC patients contained extremely high EBV DNA loads compared to the 88 non-NPC controls (p<0.0001). Using mean EBV DNA load in controls plus 3 SD as cut-off value, specificity, sensitivity, positive and negative predictive values were 98, 90, 97 and 91%, respectively. Epstein-Barr nuclear antigen 1 (EBNA1) and the carcinoma-specific BARF1 mRNA were detected by nucleic acid sequence based amplification and found in 86 and 74% of NP brushings, confirming NPC tumour cell presence. EBV RNA positivity was even higher in fresh samples stored at -80 degrees C until RNA expression analyses (88% for both EBNA1 and BARF1). EBV RNA-negative NP brushings from proven NPC cases had the lowest EBV DNA loads, indicating erroneous sampling. No EBV mRNA was detected in NP brushings from healthy donors and non-NPC patients. In conclusion, EBV DNA load measurement combined with detection of BARF1 mRNA in simple NP brushings allows noninvasive NPC diagnosis. It reflects carcinoma-specific EBV involvement at the anatomical site of tumour development and reduces the need for invasive biopsies. This procedure may be useful for confirmatory diagnosis in large serological NPC screening programs and has potential as prognostic tool.     

Nasopharyngeal brush biopsies and detection of nasopharyngeal cancer in a high-risk population.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an important tumor in many countries. Ethnic and regional factors strongly influence disease risk. NPC is usually diagnosed late in disease development, and 10-year survival rates are as low as 10%. Epstein-Barr virus (EBV), a possibly causative agent, is present in all cells of essentially all undifferentiated NPCs. We wished to determine the following: 1) whether an ambulatory nasopharyngeal brush biopsy could provide sufficient tumor cell DNA for the detection of EBV and 2) whether the detection of EBV in this locale reflects the presence of tumor cells or simply EBV carrier status. METHODS: We collected nasopharyngeal tissue via ambulatory brush biopsies from 21 patients with newly diagnosed NPC and from 157 subjects with other otolaryngologic complaints. The majority of study subjects were from high-risk populations. Sample DNA was analyzed for the presence of EBV genomic sequences by use of the polymerase chain reaction (PCR). RESULTS: Ninety-six percent of samples yielded sufficient DNA for PCR amplification. Nineteen of 21 patients with NPC brushed positive for EBV DNA, while all but two (1.3%) of 149 informative control subjects were negative for EBV (two-sided P<.0001). One of the EBV-positive control subjects had an EBV-positive inverted sinonasal papilloma; the other EBV-positive control subject exhibited no overt clinical disease. CONCLUSION: Demonstration of EBV DNA in nasopharyngeal brush biopsy specimens detects NPC with a sensitivity of at least 90% (95% confidence interval = 89.63%-91.32%) and a specificity of approximately 99% (95% confidence interval = 98.64%-98.68%). This technique merits further testing as a possible ambulatory screening strategy in high-risk populations.     

Demonstration of Epstein-Barr virus in malignant non-Hodgkin's lymphomas

Lymph nodes or tumor biopsies of 60 persons suspected of having a malignant lymphoma were examined for the presence of Epstein-Barr virus nuclear antigen (EBNA) by anticomplement immunofluorescence. In 8 cases the tissue specimens were also assayed for Epstein-Barr virus (EBV) DNA by nucleic acid hybridization. Serum samples of patients and controls were tested for EBV-related antibodies. The histological tests in 37 cases showed a malignant non-Hodgkin lymphoma, and in 23 cases a reactive lymphadenopathy. A Burkitt lymphoma of a European boy and a polymorphic centroblastoma contained EBNA and approximately 27 or 30 genome equivalents EBV DNA per cell, respectively. EBNA was also demonstrated in about 20% of the cells of a lymph node from a patient with recurrent reactive lymphadenopathy.     

Demonstration of Epstein-Barr virus-associated nuclear antigen in nasopharyngeal carcinoma cells from fresh biopsies

Fresh nasopharyngeal carcinoma (NPC) biopsies were treated in several ways to yield satisfactory cell preparations for detection of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) by anti-complement immunofluorescence tests. If the cells were well dispersed (trypsinization or extensive mechanical dispersal) few or none of them were EBNA-positive. In contrast, if the biopsy preparations contained small to moderately sized tissue fragments (touch preparations or limited mechanical dispersal), nuclear staining was detected in nearly every cell at the margin of the fragments or in cell sheets protruding from them. The stained cells corresponded to the carcinoma cells when compared to histologically stained replicate cell preparations. Nuclear staining was obtaining with anti-EBNA positive sera [healthy donors, NPC patients, convalescents from infectious mononucleosis (IM)], shown to be free of antibodies to other nuclear antigens, but not with anti-EBNA negative sera (healthy donors or patients in the early acute phase of IM). These results confirm and extend previous reports that the carcinoma cells harbor EBV genomes. The implications of these findings are discussed.     

Serum EBV DNA as a biomarker in primary nasopharyngeal carcinoma of Indian origin

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a unique tumor due to its etiology and endemic distribution. Ethnic and regional factors are found to strongly influence the risk of disease; however, there have been no well-conducted studies on Indian patients. The present study assesses the relationship between Epstein-Barr Virus (EBV) and sporadic Indian NPC and the role of serum EBV DNA in NPC detection. METHODS: Primers directed against non-polymorphic Epstein-Barr nuclear antigen-1 (EBNA-1) gene were used to detect the presence of EBV DNA from fresh tissue and serum in NPC, using PCR. RESULTS: EBV DNA was detected in 69% of the biopsies and 58% of the serum of the NPC patients. With respect to histology, WHO Type III NPC, WHO Type II tumors and WHO I tumors showed 100%, 72.2% and 33% EBV positivity, respectively. EBV positivity was also observed in 23% (6/26) of benign samples. All biopsies of patients with positive serum samples were positive for EBV DNA. CONCLUSION: EBV infection was found in sporadic NPC of South Indian origin, which confirms the etiological role of EBV in NPC. Detection of EBNA-1 in the serum and corresponding tissues of NPC patients suggests that the serum EBV DNA originates from NPC and also indicates the benefit of circulating viral DNA as an early marker in the diagnosis of NPC. Serum DNA-PCR methods can be extrapolated to follow-up studies involving tumor regression or to assess the response to various therapies.     

EB病毒与结直肠癌的相关性

背景与目的:研究表明,EB病毒(Epstein-Barrvirus,EBV)除与鼻咽癌密切相关外,可能与其它癌症相关。本研究旨在探讨EBV与中国人结直肠癌的关系。方法:采用PCR方法从90例原发性结直肠癌标本及25例配对的癌旁标本检测EBVLMP1(latentmembraneprotein1)基因的第3外显子及BamHⅠW片段的部分序列;采用免疫组织化学方法检测EBNA1(EBVnuclearantigen1)和LMP1的表达,采用原位杂交检测EBERs(EBV-encodedRNAs)的表达。结果:在90例原发性结直肠癌标本中,EBV基因阳性率为27.7%(LMP1外显子3)和32.2%(W片段),而25例癌旁组织中只有1例(4.0%)EBV基因阳性,癌组织和癌旁组织EBV阳性率差异有显著性(P<0.001)。29例W片段阳性的大肠癌标本中,有23例(79.3%)EBNA1表达阳性,只有1例(3.4%)EBERs阳性,阳性细胞主要为癌细胞,阳性信号集中在核内;而在8例W片段阴性的标本中,未检测到EBNA1的表达,也未检测到EBERs;以上标本中均未检测到LMP1的表达。结论:EBV与部分中国人结直肠癌相关。     

Serum and salivary IgA antibodies against a defined epitope of the Epstein-Barr virus nuclear antigen (EBNA) are elevated in nasopharyngeal carcinoma.

The Epstein-Barr virus nuclear antigen I (EBNA I) is the only latent EBV antigen consistently expressed in malignant tissues of the nasopharynx. A 20-amino-acid synthetic peptide, p107 contains a major epitope of EBNA I. We tested sera from 210 patients with nasopharyngeal carcinoma (NPC) and from 128 normal individuals (NHS) for IgA antibodies to p107 using an enzyme-linked immunosorbent assay (ELISA). Whereas 191/210 (91%) of NPC patients had IgA antibodies to p107, only 17/128 (13.3%) of NHS had such antibodies and only 6/57 (10.5%) of sera from patients with malignancies other than NPC had IgA-p107 reactivity. Thirty-nine salivary samples from 46 NPC patients (84.8%) also contained IgA-p107 antibodies whereas only 3/42 (7.1%) of normal saliva samples were IgA-p107 positive. The results suggest that IgA antibodies to EBNA I may become a useful, easily measurable, marker for NPC.     

抗重组EB病毒抗原双重抗体检测血清学诊断鼻咽癌的研究

背景与目的：在评估4种EB病毒抗原酶联免疫吸附法的基础上,探讨优化抗重组：EB病毒抗原双重抗体检测应用于血清学诊断鼻咽癌。方法：收集广州地区57例治疗前鼻咽癌患者和58例健康成人的血清。应用：EB病毒特异抗原(谷胱甘肽转移酶重组融合蛋白)为基础的4种免疫酶联吸附法,即：EBNA1-IgA,EBNA1-Igg,Zta-IgA和Zta-IgG检测血清中抗EB病毒的抗体水平。结果：EBNA1-IgA的灵敏度(O．9123)和阴性预测值(0．9074)是单独使用4种ELISA实验中最高的。Zta-IgA具有最高的正确率(π,0．8870)和Youden指数(J,0．7738)。当评估配对的ELISA时,EBNA1-IgA和Zta-IgA双重阳性的所有指标是4种双重阳性实验中最高的。5例：EBNA1-IgA阴性的鼻咽癌患者呈Zta-IgA阳性,而7例Zta-IgA阴性的鼻咽癌患者呈EBNA1-IgA阳性。结论：EBNA1-IgA酶联免疫吸附的单独检测在血清学诊断鼻咽癌时优于其他3项(EBNA1-IgG、Zta-IgA和Zta-IgG)单独酶联免疫吸附检测。EBNA1-IgA和Zta-IgA两项的组合应用在血清学诊断鼻咽癌时有互补作用,是血清学检测的合适组合。     

Epstein-Barr virus in nasopharyngeal and salivary gland carcinomas of Greenland Eskimoes

Biopsy specimens from nasopharyngeal carcinomas (NPC) or salivary-gland carcinomas (SGC) in Greenland Eskimoes were examined for the presence of Epstein-Barr virus (EBV) DNA and sera from the patients were tested for EBV-specific antibody titres. Six out of 7 NPCs and one from an undifferentiated SGG were positive for EBV DNA. The EBV-specific antibody spectra and titres of the patients with NPC or undifferentiated SGG conformed to the results of earlier studies in other high-incidence areas.     

Detection of the EBV-determined nuclear antigen (EBNA) in Burkitt's lymphoma and nasopharyngeal carcinoma biopsies by the acid fixed nuclear binding (AFNB) technique.

Epstein-Barr virus (EBV) carrying biopsies of Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC) were used to examine the question whether the EBV-associated nuclear antigen (EBNA) can be demonstrated by the acid fixed nuclear binding (AFNB) technique, developed previously for the demonstration of EBNA in cultured cell lines [11]. Extracts of 5 BL and 5 NPC biopsies gave a brilliant, EBNA specific fluorescence after binding to acid fixed chicken red cells. Similar extracts of 3 other African tumors that are not known to carry the EBV-genome were negative, in spite of the fact that they were derived from EBV-seropositive patients with relatively high anti-EBV (VCA) antibody titers. Crude extraction and DNA-cellulose purification gave equally active extracts, provided that incubation was carried out at 4 degrees C. These results show that the acid fixed nuclear binding technique can be applied to biopsy material. This may be helpful in searching for EBNA carrying cells in heterogeneous normal and tumor tissues in vivo where the direct in situ ACIF staining for EBNA is known to meet great difficulties.     

EBV 抗体和EBV-DNA 在鼻咽癌诊断及分期的研究*

目的：评估EBNA1/IgA 、Zta/IgA 、VCA/IgA 和EBV-DNA对不同分期鼻咽癌的诊断效能,探讨各指标阳性率与鼻咽癌分期的关系。方法：收集2010年3 月至2015年9 月中山大学附属中山医院收治的初诊鼻咽癌患者152 例,健康体检者675 例。采用酶联免疫吸附法（ELISA）检测血清EBNA1/IgA 、Zta/IgA 和VCA/IgA 抗体ROD 值,荧光定量PCR （fluorescence quantitative PCR,FQ-PCR ）检测血浆EBV-DNA水平。比较单独和联合应用EBV 标记物对各期鼻咽癌的诊断效能,同时分析各指标阳性率与鼻咽癌分期的关系。结果：鼻咽癌患者EBNA1/IgA 、Zta/IgA 、VCA/IgA 和EBV-DNA阳性率显著高于健康体检者（P < 0.01）。 EBNA1/IgA 在早期鼻咽癌表达相对较高,灵敏度为77.8% ,而EBV-DNA在晚期鼻咽癌的灵敏度最高为88.8% ,两者特异度均在96% 以上。联合检测中EBNA1/IgA 并联EBV-DNA检测的灵敏度为92.1%（早期为82.5% 、晚期为98.9%）,特异度为96.9% 。EBV-DNA阳性率与鼻咽癌临床分期和N 分期呈正相关,Zta/IgA 阳性率与N 分期呈正相关（P < 0.01）。 结论：在无症状人群中进行鼻咽癌筛查,单项指标首选EBNA1/IgA 。晚期患者的辅助诊断则推荐EBV-DNA。两者并联检测可进一步提高鼻咽癌诊断效能。EBV-DNA是鼻咽癌分期和病情监测的重要指标,Zta/IgA 可间接反映淋巴结转移情况,有望对患者病情评估起到参考作用。     

Detection of distinct Epstein-Barr virus genotypes in NPC biopsies from southern Chinese and Caucasians.

Using the polymerase chain reaction (PCR) to analyze paraffin sections from 12 Caucasian patients, we detected only the prototype F Epstein-Barr virus (EBV) in 10 specimens from patients with nasopharyngeal carcinoma (NPC). This is in contrast to the higher frequency of association of "f" variants in NPC biopsies from Southern Chinese. The results of EBV genotyping support evidence that the EBV strains associated with NPC in the Southern Chinese population differ from those found in Caucasians. DNA sequencing confirmed that a simple point mutation is responsible for the restriction-fragment-length polymorphism which distinguishes the prototype F virus from the "f" variant.     

Relationship between EB virus and nasopharyngeal carcinoma. I. Detection of EBV DNA-related sequences in biopsy specimens of nasopharyngeal carcinoma

Assay of biopsy materials of nasopharyngeal carcinoma (NPC) by nucleic hybridization (dot blot) technique with 32P labelled EBVDNA BamHIW fragment probe (specific radioactivity: 1-3 x 10(7) cpm/micrograms DNA) are described. 57 biopsies of different pathologic types were taken from NPC patients from Beijing and Qing Dao, Shandong province, and 54 control biopsies were taken from patients with other tumors and inflammatory tissues from the nasopharynx and head-neck regions. It was shown that EBVDNA positive reaction was found in 52 out of 57 NPC biopsies (91%), while only 10 positive reaction were observed in 54 control samples (18.5%). There was a significant statistical difference therein (P less than 0.001), but no significant difference of EBV DNA positive rates was found among the different pathological types (P greater than 0.05). Also no significant difference was found in biopsies taken from various geographical regions in both groups.     

Frequency of Epstein–Barr Virus DNA in Formalin-fixed Paraffin-embedded Tissue of Patients with Ductal Breast Carcinoma

Background: Ductal carcinoma is one of the most common breast cancer (BrC) among the women in the world.Several factors may involve in establishment of breast cancer. The role of viral infections have been investigated inBrC, Among them the association of Epstein Barr virus have been reported in the patients with breast cancer typeductal carcinoma. Thus this study was conducted to evaluate the rate of Epstein Barr virus in women with breast cancertype ductal carcinoma. Material and methods: A total of 72 formalin-fixed paraffin-embedded tissue blocks sampleswere collected from 37 (51.38%) women with breast cancer type ductal carcinoma and 35 (48.61%) samples of breastwith fibro adenoma as control group. The DNA was extracted for all the samples. The detection of EBNA 3C EBVDNA was done by nested PCR. The results of positive were sequenced to confirm PCR product and determine EBVgenotypes. Results: About 10/37 (27.02%) samples of ductal breast carcinoma were showed positive for EBNA 3CEBV DNA while 4/35 (11.42%) of fibro adenoma were positive for EBNA 3C EBV DNA (p= 0.095). Randomly 7PCR products were sequenced and the results of sequencing EBNA 3C shows, the detected EBVDNA were type 1EBV type. Conclusion: This study shows high prevalence of 27.02% EBV DNA type 1 was found in formalin-fixedparaffin-embedded tissue of Patients with ductal breast carcinoma. The outcomes of this study suggesting that EBVmight have a significant role in breast cancer in Ahvaz city, south west region of Iran. However the expression of EBVoncoproteins ,EBNA1, LMP1, and LMP2 require to be determined with ductal carcinoma cells. About 72.97% breastsamples showed negative for EBVDNA. The role other viruses including Human cytomegalovirus, papilloma virusesand Merkel viruses are required to be investigated in further studies.     

EBV specific antibody-based and DNA-based assays in serologic diagnosis of nasopharyngeal carcinoma

We assessed 5 EBV specific assays for their capacity to effect serologic diagnosis of suspected NPC. The assays were the immunofluorescent assays, VCA IgA and EA IgA, the enzyme-linked immunosorbent assays specific for EBNA 1 IgA or zta IgG and an EBV DNA assay. Serum samples were taken from 218 symptomatic NPC patients presenting consecutively at a public hospital in Hong Kong, 51 of whom were subsequently diagnosed as having NPC; 4 had EBV-associated lung cancer with similar serology as NPC. The remaining patients included 23 who had other cancers and 140 who had other diseases. Objectives of serodiagnosis under such clinical settings, therefore, are to both exclude and predict a diagnosis of NPC. None of the assays individually can meet both requirements adequately, however. The difficulty was best overcome by combining EBNA 1 IgA and zta IgG. It was shown that 68.3% of the patients gave a confirmed test results, negative or positive, by both tests. A confirmed negative result was associated with a negative predictive value of 99.1%, providing a clear indication to exclude a diagnosis of NPC; a confirmed positive result was associated with a positive predictive value of 86.8%, providing a clear indication to proceed with diagnostic work-up of NPC. The remaining patients gave equivocal test results, being positive for one or the other test, which were associated with a positive predictive value of 43.3% and 24.2%, respectively.     

定量分析鼻咽癌患者外周血游离EBV

目的:分析外周血游离Epstein-Barr病毒(EBV)是否特异地出现于鼻咽癌患者。方法:收集鼻咽癌患者和非鼻咽癌肿瘤患者血浆各50例,正常人血浆30例,提取DNA,荧光定量PCR检测EBV拷贝数,比较游离EBV在上述人群的差异。结果:在25例(50%)鼻咽癌患者血浆中检测到游离EBV,其拷贝数波动于1×103拷贝/ml血浆～2.6×106拷贝/ml血浆,中位数为5.3×105拷贝/ml血浆。在50例非鼻咽癌肿瘤患者和30例正常人血浆中均未检测到游离EBV。结论:游离EBV出现于鼻咽癌患者外周血,可能是一个较特异的鼻咽癌标志物。     

Rearranged Epstein-Barr virus genomes and clonal origin in nasopharyngeal carcinoma

Epstein-Barr virus (EBV) is known to be associated with two malignant diseases, nasopharyngeal carcinoma (NPC) and endemic Burkitt's lymphoma. In this study, the genomes of EBV in biopsy specimens from 4 NPC patients in Japan were analyzed using Southern blot hybridization. The NPC tissues of all examined cases contained rearranged EBV genomes whose BamHI H fragments were larger than those of prototype EBV genomes. One of them had a BamHI fragment containing contiguous sequences of BamHI Y and H. A single-sized EBV DNA terminus was observed in these NPC tissues, implying the evolution of the carcinoma from a single EBV-infected cell.     

The differentiated form of nasopharyngeal carcinoma contains Epstein-Barr virus DNA

Immunologic studies of Epstein-Barr virus (EBV) have implicated EBV in undifferentiated and partially differentiated, non-keratinizing nasopharyngeal carcinoma (NPC). Patients with the well-differentiated, keratinizing form of NPC have EBV serologic patterns similar to those of control populations. In addition, viral DNA has not been detected in the differentiated tumors using viral cRNA probes to DNA immobilized on filters. In this study we have tested for EBV DNA using recombinant DNA probes to Southern blots of DNA from 33 NPC specimens. The 24 undifferentiated and 4 partially differentiated specimens generally contained a relatively high number of EBV genome equivalents, while the 5 well-differentiated NPC all contained detectable EBV, but at low copy number. The viral DNA from one of the well-differentiated specimens was cloned into a cosmid vector. Five recombinant clones representing the fused viral termini were obtained, indicating the presence of episomal, intracellular DNA in the tumor. These findings indicate that all histologic subsets of NPC contain EBV DNA.