Epstein-Barr virus DNA and epithelial markers in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) belongs to the most common malignant tumours in certain parts of the world, e.g. South-East Asia. The undifferentiated type of NPC is associated with genomic Epstein-Barr virus (EBV) DNA. In normal epithelia of the nasopharynx cytokeratins (CK) 4, 5, 6, 13, 14, 15 and 19 are expressed. The aim of this study was to analyse the expression pattern of cytokeratins in NPC in the presence of EBV infection. Twenty primary or metastatic tumours from 13 patients suffering from a NPC were evaluated (formalin-fixed, paraffin-embedded). (35)S-labelled probes were used to detect EBV DNA in the tissue sections. Fourteen specimens (70%) were EBV positive. All positive specimens were undifferentiated NPC. All NPC were identified with broad-spectrum anti-CK antibody. Using a panel of anti-CK antibodies, there was no specific CK-expression pattern in NPC. In summary, undifferentiated NPC are strongly associated with EBV. The cytoskeleton of undifferentiated NPC reveals no specific pattern of CK expression.     

Polymorphism analysis of Epstein-Barr virus isolates of nasopharyngeal carcinoma biopsies from Tunisian patients

Many studies suggest that the focal distribution of nasopharyngeal carcinoma (NPC) may be influenced not only by host genetics, diet and environments but also by interplay with Epstein-Barr virus (EBV) genetics. Specific EBV gene variants (the A and C types, the BamHI f configuration, a C terminal 30 bp deletion and a N terminal loss of an XhoI site in the BNLF1 gene) have been explored in high incidence areas in southern Asian NPC patients. In contrast, in Tunisia where NPC represents the most frequent type of Head and Neck cancer the distribution of these polymorphisms remains poorly investigated. In order to characterize the epidemiology of EBV variants in Tunisian NPC patients, we have investigated the A or B type of the EBV nuclear antigen (EBNA)2 gene, the C or D type of the BamHI W1/I1 region, the F/f variants of the BamHI F region and the presence or the absence of the XhoI site, 30 bp deletion and Taq1 site in the BNLF1 gene in 47 NPC biopsies, 12 being younger than 30 and 35 older than 30. Our results show a unique genetic profile of the tumor EBV strains regarding the A and D types, the prototype F and retention of the XhoI restriction site in the N terminal region of BNLF1 gene. With regard to the C terminal region of this gene, four genetic profiles were detected: (1) the occurrence of the 30 bp deletion in association with the Taq1 site in 39 cases (83%), (2) the presence of the Taq1 site by itself in 5 cases, (3) the occurrence of the 30 bp deletion by itself in 2 cases and (4) the occurrence of a new deletion of 81 bp covering the 30 bp deletion in association with the Taq1 site in one case. With the exception of the 81 bp deletion, which has not been previously described in the literature, the summarized results have shown the same genetic profile in Tunisian NPC tumor isolates as tumor isolates from other North African and Mediterranean countries. Hence, the observed EBV polymorphisms are not fully specific of to the Tunisian NPCs. Nevertheless, the notion of a divergence between North African and Asian tumor EBV isolates is reinforced by this study. Wajdi Ayadi and Lamia Feki wish it to be known that, in their opinion, the first two authors should be regarded as joint first authors.     

Epstein-Barr virus DNA in nasopharyngeal carcinomas from Chinese patients in Hong Kong.

AIMS: To investigate the presence of Epstein-Barr virus (EBV) in cases of nasopharyngeal carcinoma (NPC) in Chinese patients living in Hong Kong. METHODS: Nasopharyngeal biopsy specimens, formalin fixed and paraffin wax embedded, from 24 patients, eight with undifferentiated nasopharyngeal carcinoma, eight with well differentiated squamous carcinoma, and eight showing normal tissue histology, were analysed for the presence of Epstein-Barr virus (EBV) DNA by slot-blot hybridisation on extracted unamplified DNA, and also after amplification of EBV specific sequences by the polymerase chain reaction (PCR). RESULTS: DNA slot-blot analysis showed viral DNA in all the undifferentiated, five of the well differentiated tumours, and none of the normal biopsy specimens. PCR studies confirmed positivity in the eight undifferentiated tumours, but six of the well differentiated tumours and three of the normal biopsy specimens showed viral DNA by this method, illustrating its greater sensitivity. CONCLUSIONS: EBV genome is present in appreciable copy number in most cases of well differentiated NPC in Chinese patients in Hong Kong.     

Detection of Epstein-Barr virus DNA sequences in nasopharyngeal carcinoma cells by enzymatic DNA amplification.

The presence of Epstein-Barr virus (EBV) DNA sequences was examined by the polymerase chain reaction in 50 nasopharyngeal carcinoma (NPC) biopsy specimens and in two primary epithelial tumor cell cultures derived from patients with NPC. The detection limit was a single EBV genome equivalent by agarose gel electrophoresis followed by Southern blot analysis of the amplified products. EBV DNA sequences were detected in all 41 undifferentiated NPC cell specimens, in 2 of 4 moderately differentiated NPC cell specimens, and in 3 of 5 keratinized NPC cell specimens. Undifferentiated NPC cells were also found to contain higher copy numbers of EBV than cells of the other two types of NPC. Our data suggest that EBV replication may be closely associated with the differentiation of NPC tumor cells. The results also demonstrated a sensitive and specific method for the detection of EBV DNA sequences in NPC tumor cells.     

Detection of Epstein-Barr virus DNA in nasopharyngeal carcinoma using a non-radioactive digoxigenin-labelled probe

The presence of Epstein Barr virus (EBV) DNA in biopsies from the post-nasal space (PNS) of patients suspected of nasopharyngeal carcinoma (NPC) was detected by in situ cytohybridization with an EBV DNA probe labelled with the novel labelling compound digoxigenin. The digoxigenin probe was hybridised to cryostat sections of NPC biopsies and subsequently detected by an enzyme immunoassay procedure. It was found that in situ cytohybridization using the digoxigenin probe was much more rapid and sensitive (96 h compared to five weeks) than the current method of using 3H-labelled probe. Using the digoxigenin EBV probe, it was found that in all the eighteen NPC biopsies tested, EBV DNA was detected in malignant epithelial cells and infiltrating lymphocytes. EBV DNA was also detected in some normal epithelial cells in these NPC biopsies. EBV DNA was not detected in epithelial cells of non-malignant biopsies.     

Antibody against Epstein-Barr virus DNA polymerase activity in sera of patients with nasopharyngeal carcinoma

A salt-dependent DNA polymerase activity was demonstrated in the culture of an EBV-producing, lymphoblastoid cell line (NPC-204 cells) treated with 5-iodo-2'-deoxyuridine (IUdR). There was a high frequency of levels of antibody to this enzyme in sera of patients with nasopharyngeal carcinoma (NPC). In contrast, sera from healthy subjects had little or no neutralizing activity. The high antibody level appeared as early as stage 1 of the disease in many NPC patients. The levels of the antibody increased with the progression of the disease and declined in treated patients. The results strongly suggest that tests measuring serum antibody against EBV DNA polymerase activity can be used for early diagnosis and prognosis of NPC.     

Serum Epstein-Barr virus DNA load in primary Epstein-Barr virus infection

Specific viral laboratory diagnosis of primary Epstein-Barr Virus (EBV) infection is usually based on antibody-detection assays. During acute, lytic phase of infection, viral DNA can also be detected in serum. In the present study, the diagnostic utility of EBV DNA detection and quantitation in serum in primary EBV infection was investigated. The level of EBV DNA in the serum of 98 immunocompetent patients aged 1-47 years with symptomatic, antibody-confirmed EBV primary infection was assessed using a quantitative real-time PCR assay. The association between viral load and time after onset of disease, age and clinical and laboratory data was investigated. Quantitative PCR detected EBV DNA in 93 of 98 samples (94.9%), and the measured viral loads ranged from 3.8 x 10(1) to 6.6 x 10(4) copies/ml. EBV DNA detection exhibited a sensitivity of 94.9% and a specificity of 97.4% for primary EBV infection. EBV DNA was always detectable until day 12 after onset of symptoms, whereas no further positive PCR results were found after a period of 22 days after onset of disease. Detection of EBV DNA also showed a clearer association with the clinical manifestation of disease than the presence of EBV specific VCA IgG antibodies of low avidity. EBV DNA load was found to correlate inversely with the time after onset of disease (P < 0.001), and higher viral load levels were detected in younger (P = 0.009) and in hospitalized patients (P = 0.038). The results indicate that real-time PCR is a reliable tool for diagnosis of primary EBV infection early in the course of disease. In addition, EBV DNA detection may serve as a useful diagnostic supplement in serologically indeterminate EBV infections.     

Unique variations of Epstein-Barr virus-encoded BARF1 gene in nasopharyngeal carcinoma biopsies

The Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1) gene is frequently expressed in EBV-associated epithelial malignancies and involves in oncogenicity and immunomodulation. To characterize the variations of BARF1 gene in different populations, the sequences of BARF1 gene in Northern Chinese nasopharyngeal carcinoma (NPC), EBV-associated gastric carcinoma (EBVaGC) and healthy donors were analyzed. The correlation of BARF1 variation with polymorphisms of BamHI F fragment (type F and f variants) and EBV-coded viral interleukin-10 (vIL-10) gene (B95-8 and SPM patterns) was also explored. Two major subtypes of BARF1 gene, designated as B95-8 and V29A, were identified. B95-8 subtype had identical amino acid sequence to B95-8 and was the dominant subtype among the EBV isolates from Northern China. V29A subtype, with one consistent amino acid change at residue 29 (V→A) and several nucleotide changes, showed higher frequency in NPC cases (25.3%, 20/79) than in EBVaGC cases (0/45) or healthy donors (4.3%, 2/46) (NPC vs. EBVaGC: P=0.0001; NPC vs. healthy donor: P=0.004). A preferential linkage between BamHI F and BARF1/vIL-10 polymorphisms was found. Type f isolates was specially correlated with the V29A/SPM genotype in NPC isolates and type f/V29A/SPM was preferentially found in NPC. BARF1/c-fms homology domain, transforming domain and cytotoxic T lymphocyte (CTL) epitopes of BARF1 were highly conserved in most isolates, suggesting the important role of BARF1 in virus infection and the potential usefulness in EBV-targeting immunotherapy of EBV-associated tumors. The relatively higher prevalence of type f/V29A/SPM strains in NPC may also suggest the association between these variations in multiple viral genes and NPC.     

Epstein-Barr virus expression within keratinizing nasopharyngeal carcinoma

Three stages of maturation can be seen in keratinizing nasopharyngeal carcinomas. These stages are similar morphologically to basal cells, intermediate and superficial squamous cells seen in normal squamous epithelium. Taking advantage of such a diverse tumour cell population, 10 keratinizing nasopharyngeal carcinoma (NPC) were examined by in situ hybridization for the presence of latent Epstein-Barr Virus (EBV) using EBV encoded RNAs (EBERs) and by immunohistology for the presence of EBV early antigen-diffuse (EA-D) and the 350/220 kd membrane glycoprotein of the EBV. The basal cell-like tumour cells are mainly infected latently with the virus; viral replication was found in isolated intermediate squamous cells, whilst superficial squamous cells are largely depleted of all the viral markers. We used a control series of non-keratinizing nasopharyngeal carcinomas composed of undifferentiated and poorly differentiated tumour cells and EBV latency was present in these tumours. Viral replication was detected by RT-PCR, in the undifferentiated tumours but viral replication was not seen by immunohistology. The possible relationship between EBV life cycle in these tumours and tumour cell differentiation is discussed in the light of these findings. J. Med. Virol. 55:227–233, 1998. © 1998 Wiley-Liss, Inc.     

Epstein-Barr virus serology in the diagnosis of nasopharyngeal carcinoma

The antibody levels to viral capsid antigen (VCA) and early antigen (EA) of Epstein-Barr virus (EBV) in 164 nasopharyngeal carcinoma (NPC) patients from Sarawak, East Malaysia were significantly higher than those in 147 sex, age and ethnically matched healthy controls. As diagnostic markers of NPC, IgG/VCA at reciprocal titers > or =160 was the most sensitive (89%, with 98% specificity), while IgA/EA at > or =5 was the most specific (100%) but the least sensitive (75%). The sensitivity and specificity of IgA/VCA at reciprocal titers > or =10 were 84% and 97%. IgA/VCA has an advantage over IgG/VCA despite the slightly lower sensitivity due to its consistently more distinct fluorescence reaction. The sensitivity and specificity can be marginally improved by a combination of two tests.     

Nasopharyngeal Epstein-Barr virus DNA loads in high-risk nasopharyngeal carcinoma families: Familial aggregation and host heritability

Nasopharyngeal carcinoma (NPC), the most common head and neck cancer, is characterized by distinct geographic distribution and familial aggregation. Multiple risk factors, including host genetics, environmental factor, and EBV infection, have been linked to the development of NPC, particularly in the familial clustering cases. However, the cause of NPC endemicity remains enigmatic due possibly to the complicated interplay between these risk factors. Recently, positive Epstein-Barr virus (EBV) DNA loads at nasopharyngeal (NP) cavity has been found to reflect NPC development and applied in NPC screening. To examine whether the increased NP EBV loads could aggregate in the families and be affected by host genetics and environmental factor, EBV loads were obtained by 510 NP brushing samples from eligible unaffected individuals, who have two or more relatives affected with NPC, in 116 high-risk NPC families. The correlation of relative pairs was estimated using S.A.G.E. (version 6.4, 2016), and host heritability of NP EBV loads was calculated with variance component models using SOLAR (version 8.4.2, 2019). In result, significant correlations of EBV loads were observed between parent-offspring pairs and sibling-sibling pairs (P < .001), but not in distant kin relationship pairs. Interestingly, after excluding the shared environmental factor within families, host genetics contributes significantly to NP EBV loads with a heritability of 56.41% (P = 1.00 × 10⁻⁷), and its effect was slightly elevated (68.86%, P = 3.40 × 10⁻⁶) in families with more NPC cases (≥3). These findings indicate that additional host-genetic variants involved in the EBV local NP mucosal behavior may be especially important for the development of NPC.     

Epstein-Barr virus genotypes associated with nasopharyngeal carcinoma in southern China

We have used probes representing approximately two-thirds of the Epstein-Barr virus (EBV) genome to examine genetic polymorphism of wild-type EBV directly detected in 28 nasopharyngeal carcinoma (NPC) tissues from Southern China. In the hypervariable regions containing reiterated sequences, this analysis showed that while naturally occurring viruses are genetically diverse, only one genotype was detectable in each tumor specimen. Unique variants in NPC were detected in four regions of the EBV genome. Two of these appeared significant in their distribution. The type C variant resulting from loss of a BamHl site between the BamHl W1* and l1* regions was prevalent among isolates from Southern China, but not so common in the United States. An "f" variant having an extra BamHl site in the BamHl F region was almost exclusively detected in only Asian NPC biopsies.     

Epstein-Barr virus infection in the pathogenesis of nasopharyngeal carcinoma.

The association of nasopharyngeal carcinoma (NPC) with the Epstein-Barr virus (EBV) was firmly established as early as 1973. Nevertheless, the role for the virus in the pathogenesis of NPC is still controversial. In this article, the evidence implicating EBV in the development of NPC is reviewed, focusing on the cellular site of EBV persistence, the association of the virus with different NPC histotypes, the tumour cell phenotype in the context of viral latent gene expression, and the possible role of the lymphoid stroma.     

鼻咽癌组织中EB病毒潜伏相关基因的表达

目的： 通过检测鼻咽癌组织中EB病毒的潜伏膜蛋白LMP1的序列以及LMP1、EBNA1、EBNA2的mRNA表达来探讨EB病毒的感染状态及其表达产物与鼻咽癌的关系。方法: 应用PCR法检测鼻咽癌组织中LMP1 DNA的存在，并对鼻咽癌来源的LMP1和EB病毒永生化狨猴B淋巴细胞系B95-8来源的LMP1进行测序，比较序列的差异。利用巢式RT-PCR检测鼻咽癌组织中LMP1、EBNA1、EBNA2的mRNA表达。结果: 47例鼻咽癌组织均含有LMP1 DNA，所有鼻咽癌来源的LMP1 DNA与B95-8来源的LMP1 DNA序列比较均存在着多个单核苷酸变异，最明显的是XhoⅠ酶切位点的丢失。测序后显示鼻咽癌来源的LMP1 DNA有30个核苷酸的丢失。巢式RT-PCR显示LMP1、EBNA1、EBNA2在鼻咽癌中的mRNA表达率分别为76.6％、80.0％和74.5％。其中EBNA1的表达是由Qp启动的，而B95-8细胞中EBNA1的表达是由Cp启动的。结论: 鼻咽癌中EB病毒的作用途径比较复杂，LMP1、EBNA1、EBNA2等潜伏期基因还有早期裂解基因BARF1均可能参与鼻咽癌的发生发展过程。     

Epstein-Barr virus immune response in high-risk nasopharyngeal carcinoma families in Greenland

Undifferentiated nasopharyngeal carcinoma is associated with Epstein-Barr virus (EBV) infection. Presence of EBV IgA antibodies is rare among healthy individuals and is used as a marker of nasopharyngeal carcinoma in high-incidence populations. Reasons for EBV IgA seropositivity are unknown, but high EBV IgA levels have been found among unaffected close family members and spouses to nasopharyngeal carcinoma patients in Chinese populations. In Greenland, a nasopharyngeal carcinoma-high-incidence area, we compared EBV serology and viral load in high-risk nasopharyngeal carcinoma family members (N = 20) and controls without nasopharyngeal carcinoma-affected relatives (N = 90). There was no significant difference in EBV viral loads between relatives and controls, and EBV was detected in plasma in 5.0% of relatives and 11.4% of controls. There was no significant difference in EBV serology, but the seroprevalence of EBV viral capsid antigen (VCA) IgA was high in both relatives (25.0%) and controls (20.5%). Compared with anti-VCA IgA-negative, anti-VCA IgA-positive individuals had significantly higher EBV viral loads in peripheral blood mononuclear cells (PBMCs) (P < 0.01). The very high prevalence of anti-VCA IgA indicates that this antibody is unsuitable for nasopharyngeal carcinoma screening among Inuits.     

Response of nasopharyngeal carcinoma cells to Epstein-Barr virus infection in vitro

Many nasopharyngeal carcinoma (NPC) biopsy specimens contain Epstein-Barr virus (EBV). However, the response of NPC cells to EBV infection in vitro and in vivo is not well characterized. In this experiment we infected NPC cells with EBV particles through endocytosis of a complex of EBV immunoglobulin A (IgA) secretory component (SC) protein to observe the response of host cells to the foreign viral infection in vitro. We found that EBV particles were endocytosed and stabilized in NPC nuclei 24 hours after infection; the EBV genomes were then gradually decreased after serial passages within 3 to 4 weeks by the following pathway: the EBV genomes first moved toward the nuclear envelope from the center of the nucleus; after crossing the nuclear envelope, they moved into the cytoplasm and toward the plasma membrane and were discharged by exocytosis. At the 10th day of EBV infection, EBV-latent membrane protein-1 and Epstein-Barr nuclear antigen (EBNA)-1 protein expressions could be detected, but not EBV-viral capsid antigen. Observation of EBNA-1 protein and host growth factor and cytokine gene expressions in the weeks after incubation revealed that the EBNA-1 protein expression was decreased proportionally with decrease of EBV genome. The mRNA expression of epithelial growth factor receptor, transforming growth factor (TGF)-alpha, interleukin (IL)-1beta, IL-6, and granulocyte-macrophage colony-stimulating factor increased within 1 to 2 weeks after infection, and gradually recovered to the original level at 3 to 4 weeks, whereas the mRNAs of TGFbeta1, TGFbeta receptor type I (TGFbetaRI), TGFbetaR type II, IL-8, and tumor necrosis factor-alpha remained unchanged. It is concluded that in vitro EBV infection in NPC cells results in increase of certain growth factor and cytokine gene expressions in host cells. The change in gene expression returns to the original level approximately 3 to 4 weeks after infection because of exocytosis of EBV DNA by the infected cells through an unidentified mechanism.