Molecular Screening of Nasopharyngeal Carcinoma: Detection of LMP-1, LMP-2 Gene Expression in Vietnamese Nasopharyngeal Swab Samples

Objective: This study aimed to characterize the expression of LMP-1, LMP-2 in clinical swab samples in orderto find out the potential molecular based biomarker for NPC diagnosis and screening, which could offer a chance indevelopment of rapid method for NPC diagnosis in Vietnamese population. Materials and Methods: A total of 93nasopharyngeal carcinoma swab samples and 100 healthy nasopharyngeal swab samples were collected to evaluateLMP-1, LMP-2 expression by Real-time reversed PCR. Results: we figured out the significant association betweenthe expression of LMP-1 (counting for 48.39%), LMP-2 (counting for 39.78%) and NPC. No LMP-1 expression wasobserved, and only 1 of 100 specimens was detected with LMP-2 positive in healthy samples. In the combination ofLMP-1 (+) and/or LMP-2 (+), the frequency of positive was 53.76%, greater than each gene expression. Additionally,sensitivity, specificity, positive predictive value, negative predictive value of assay were 99.00%, 98.04%, 69.72%,and 77.02%, respectively. Additionally, the LMP-2 expression level was 5.50 times higher in NPC samples thannon-cancerous samples. Conclusion: Our results indicated the molecular invasive method based on the expression ofLMP-1, LMP-2 in swab samples would be a promising supplement in NPC diagnosis, screening in the near future inVietnam.     

In vitro EBV-infected subline of KMH2, derived from Hodgkin lymphoma, expresses only EBNA-1, while CD40 ligand and IL-4 induce LMP-1 but not EBNA-2

In about 50% of classical Hodgkin lymphomas, the Hodgkin/Reed Sternberg (H/RS) cells carry Epstein-Barr virus (EBV). The viral gene expression in these cells is restricted to EBNA-1, EBERs, LMP-1 and LMP-2 (type II latency). The origin of H/RS cells was defined as crippled germinal center B cells that escaped apoptosis. In spite of numerous attempts, only few typical Hodgkin lymphoma (HL) lines have been established. This suggests that the cells require survival factors that they receive in the in vivo microenvironment. If EBV is expected to drive the cells for growth in culture, the absence of EBNA-2 may explain the incapacity of H/RS cells for in vitro proliferation. In EBV carrying B lymphocytes, functional EBNA-2 and LMP-1 proteins are required for in vitro growth. For analysis of the interaction between EBV and the H/RS cells, we infected the CD21-positive HL line KMH2 with the B958 and Akata viral strains. Only EBNA-1 expression was detected in a few cells in spite of the fact that all cells could be infected. Using a neomycin-resistance-tagged recombinant EBV strain (Akata-Neo) we established an EBV-positive subline that was carried on selective medium. In contrast to the type II EBV expression pattern of H/RS cells in vivo, the KMH2 EBV cells did not express LMP-1. The EBV expression pattern could be modified in this type I subline. LMP-1 could be induced by the histone deacetylase inhibitors TSA and n-butyrate, by 5-AzaC, a demethylating agent, and by phorbol ester. None of these treatments induced EBNA-2. Importantly, exposure to CD40 ligand and IL-4 induced LMP-1 without EBNA-2 expression and lytic replication. The KMH2 EBV cells expressed LMP-2A, but not LMP-2B mRNAs. This result is highly relevant for the type II expression pattern of H/RS cells in vivo, since these stimuli can be provided by the surrounding activated T lymphocytes.     

EB病毒潜伏膜蛋白LMP-1在鼻咽癌中的表达及临床意义

目的观察EB病毒（EBV）潜伏膜蛋白1（LMP-1）在鼻咽癌（NPC）鼻咽拭子中的表达,探讨LMP-1表达与NPC临床病理特征的关系。方法纳入2007年～2008年福建省肿瘤医院放疗科收治的经病理检查确诊为NPC且同意参加该试验的初治患者129例,均在治疗前应用鼻咽拭子法取鼻咽部黏膜脱落细胞,提取DNA,采用PCR法检测LMP-1基因的表达。结果 129例患者中有114例患者LMP-1呈阳性表达,另15例呈阴性表达,阳性率为88.37%（114/129）;对照组中无1例呈阳性表达。LMP-1阳性表达率有颈部淋巴结转移组明显高于无颈部淋巴结转移组。LMP-1表达与患者年龄、性别、临床分期、T分期及M分期无相关性。结论 PCR法检测鼻咽拭子中LMP-1的检出率高于免疫组化法,LMP-1表达与鼻咽癌患者颈部淋巴结转移密切相关,临床中可预测鼻咽癌患者的转移潜能,LMP-1可能做为抗肿瘤转移靶向治疗的潜在靶点。     

EBV LMP-1 negatively regulates expression and pro-apoptotic activity of Par-4 in nasopharyngeal carcinoma cells

Latent membrane protein-1 (LMP-1) of the Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC), and in this study we sought to determine whether the pro-apoptotic activity of prostate apoptosis response-4 (Par-4) is modulated by LMP-1 in NPC cells. We found that LMP-1 diminished the pro-apoptotic activity of Par-4 and negatively regulated Par-4 protein by de novo synthesis; moreover, although LMP-1 accelerated a Par-4 activator, PKA, we demonstrated that LMP-1 also activated the PI3 K/Akt pathway and increased Bcl-2 expression to suppress the activity of Par-4. Consequently, our results revealed a novel negative action of LMP-1 on the pro-apoptosis protein Par-4 by the coordination of multiple signaling pathways.     

Screening nasopharyngeal carcinoma by detection of the latent membrane protein 1 (LMP-1) gene with nasopharyngeal swabs

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a common head and neck cancer in Taiwan. The goals of the current study were to investigate whether a nasopharyngeal swab technique could provide enough DNA for polymerase chain reaction (PCR) analysis of the Epstein-Barr virus (EBV)-derived latent membrane protein 1 (LMP-1) gene and to determine the feasibility and reliability of diagnosing NPC by detection of LMP-1 in the nasopharynx. METHODS: 320 adults underwent nasopharyngoscopy and nasopharyngeal swab to obtain cells for the LMP-1 PCR assay; some patients also underwent nasopharyngeal biopsy. RESULTS: An amount of DNA that was sufficient for PCR was extracted from 96.3% of the swab samples. By detecting LMP-1 in nasopharyngeal swabs, NPC was diagnosed with a false positive rate of 12.7% (7 of 55 patients), a false negative rate of 1.6% (4 of 253 patients), sensitivity of 87.3% (48 of 55 patients), specificity of 98.4% (249 of 253 patients), a positive predictive value of 92.3% (48 of 52 patients), and a negative predictive value of 97.3% (249/256 patients). NPC was diagnosed by nasopharyngoscopy with a false positive rate of 38% (30 of 79 patients), a false negative rate of 0.4% (1 of 241 patients), sensitivity of 62% (49 of 79 patients), specificity of 99.6% (240 of 241 patients), a positive predictive value of 98% (49 of 50 patients), and a negative predictive value of 88.9% (240 of 270 patients). Only 7 (0.2%) of 256 patients with a diagnosis other than NPC had LMP-1 detected in the nasopharyngeal space. CONCLUSIONS: Detecting EBV genomic LMP-1 by nasopharyngeal swab diagnosed NPC with 87.3% sensitivity and 98.4% specificity. EBV genomic DNA usually is not detected by PCR-based methods in the nasopharyngeal space. Its incidence is estimated to be as low as 0.2% among the general population. The nasopharyngeal swab coupled with PCR-based EBV LMP-1 detection could serve as part of a screening program for high-risk populations.     

Detection of LMP-1 gene in middle ear effusion of NPC

Nasopharyngeal carcinoma (NPC) is closely related with Epstein-Barr virus (EBV). Almost every NPC tumor cells carries clonal EBV genomes. Detection of EBV derived latent membrane protein-1 gene (LMP-1) indicate the presence of NPC. Middle ear effusion (MEE) is a frequent sign of NPC. There have been no reports on LMP-1 in MEE. Tympanocentesis of 88 ears with MEE of 66 patients were done in three groups of patients, group (I) NPC, 31 patients, 50 ears, (II) other head and neck cancers, five patients, six ears and (III) no cancer history, 30 patients, 32 ears. The middle ear aspirate and nasopharyngeal swab specimen were collected to detect LMP-1 with a PCR-based method. Sixty aspirates (68%) out of 88 ears with MEE had enough DNA for PCR amplification. LMP-1 was detected in six middle ear aspirate specimen from three patients in group I who had petrous apex invasion. LMP-1 was detected in 30 swab specimen (93.8%) out of 32 nasopharyngeal swabs in group I. LMP-1 was not detected in middle ear aspirates or nasopharyngeal swab in group II and III patients. LMP-1 was not detected in MEE in patients without NPC. In NPC patients, the detection of LMP-1 may indicate petrous apex invasion.     

DNA载体介导RNA干扰抑制LMP-1基因对鼻咽癌细胞转移能力的影响

目的探讨应用DNA载体介导RNA干扰技术,抑制鼻咽癌细胞中EB病毒潜伏膜蛋白1（LMP-1）表达的可能性,观察LMP-1基因沉默对鼻咽癌细胞转移能力的影响。方法将能转录出干扰RNA的DNA模板插入pAVU6＋27质粒,构建能够介导RNA干扰的发夹状干扰RNA（shRNA）表达载体。采用磷酸钙共沉淀法,将质粒导入EB病毒（＋）的鼻咽癌细胞C611,筛选LMP-1基因沉默的克隆,通过观察其贴壁生长能力、基底膜穿透能力和移动能力的变化,分析LMP-1基因沉默对鼻咽癌细胞转移能力的影响。结果在shRNA表达载体有效转染的C611细胞中,LMP-1基因的表达受到稳定抑制。肿瘤细胞转移实验表明,LMP-1基因沉默的C611细胞,贴壁生长能力提高,基底膜穿透能力和细胞移动能力均明显下降。结论shRNA表达载体能够在鼻咽癌细胞内介导RNA干扰,并获得对LMP-1基因稳定的抑制。LMP-1基因可能通过贴壁生长能力、基底膜穿透能力和细胞移动能力等,影响鼻咽癌细胞的转移。     

Molecular Grading of Oral Squamous Cell Carcinomas Infected with EBV

Background: Squamous Cell Carcinoma (SCC) is a type of cancer that is often found in oral cavity and areas ofthe head and neck. Viruses are major etiological factors through production of factors that can disturb proliferationand apoptosis regulators such as p53i, c-myc and bcl-2. This study aimed to determine the molecular grading of oralsquamous cell carcinoma (OSCCs) infected with the Epstein-Barr Virus (EBV). Methods: Twenty-seven OSCC patientsunderwent biopsy to detect EBV infection through in situ hybridization for RNA EBV (EBER) and immunohistochemicalanalysis of latent membrane protein-1 (LMP-1) and EBV nuclear antigen-1 (EBNA-1). To assess molecular grades, cellproliferation and apoptosis regulator expression i.e. inactive p53 (p53i), c-myc and bcl-2, were immunohistochemicallyanalysed. Results: The cases were divided into two groups; infected and non-infected by EBV. Regression analysisshowed that only EBNA-1 expression could affect p53i expression. Based on regression equations molecular grading ofOSCCs infected by EBV was divided into three: Grade I (low), EBNA-1 expression was 7.60, and p53i expressionwas 9.74-17.5; Grade II (medium), EBNA-1 expression was 7.61-19.7, and p53i 17.5-30.1; Grade III (high), EBNA-1expression was 19.71, and p53i ≥ 30.1. Conclusion: In OSCC infected with EBV, only EBNA-1 expression caninfluence p53i expression.     

EB病毒诱发淋巴瘤中LMP的表达

背景与目的:EB病毒潜伏膜蛋白-1(latent membrane protein-1,LMP-1)是一种病毒癌蛋白,可能在淋巴瘤发生中起重要的作用.本文旨在了解EB病毒诱发的淋巴瘤细胞与正常人淋巴细胞中LMP的差异表达情况.方法:应用实时定量PCR方法检测EB病毒诱发淋巴瘤细胞和正常人淋巴细胞中LMP (LMP-1、LMP-2A及LMP-2B)的差异表达情况.采用Western blot检测LMP-1蛋白表达.结果:建立了EBV诱发淋巴瘤的动物模型获得诱发淋巴瘤,检测LMP-1、LMP-2A、LMP-2B在诱发淋巴瘤细胞及正常人淋巴细胞中的表达情况.实时定量PCR结果表明,LMP-1在诱发淋巴瘤细胞中的表达比在正常细胞中的表达上调255.7倍,表达差异有统计学意义(P＜0.05),LMP-2A在诱发淋巴瘤细胞中的表达比在正常细胞中的表达上调37.74倍,表达差异有统计学意义(P＜0.01),LMP-2B在诱发淋巴瘤细胞中的表达比在正常细胞中的表达上调330.63倍,表达差异有统计学意义(P＜0.05).Western blot结果表明LMP-1在诱发的淋巴瘤细胞中的表达比在正常淋巴细胞中的表达上调.结论:在EB病毒诱发的淋巴瘤发生过程中LMP可能起重要作用.     

EB病毒膜潜伏蛋白LMP-1筛选鼻咽癌的初步结果

Objective  

Early diagnosis of nasopharyngeal carcinoma (NPC) is an important method to improve the survival rate. However, the sensitivity and specificity of the screening protocols which was widely used in clinic now are considered to be unsatisfactory. Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP-1) is one of the proteins that have been suggested to be a classic oncogene with transformation properties. The current study set out to discuss the clinical significance of LMP-1 on the screening of NPC.     

韩国淋巴瘤与中国延边地区淋巴瘤和鼻咽癌中EB病毒类型和30bp缺失型潜伏膜蛋白-1基因表达的比较研究

目的 比较韩国淋巴瘤与中国延边地区淋巴瘤和鼻咽癌中。EB病毒类型的分布特点。方法 采用聚合酶链反应(PCR),巢式PCR,限制性酶切分析和Southern印迹杂交技术检测。EB病毒的类型分布。结果 在韩国非霍奇金淋巴瘤中,57％的B细胞淋巴瘤和75％的T细胞淋巴瘤表现为EBNA-1阳性。“C”变异型为44％,“f”变异型为9％。而在15例霍奇金淋巴瘤中,4例为“CF”变异型,11例为“DF”型。在延边地区,22％的淋巴瘤和81％的鼻咽癌表现为EBNA-1阳性。除1例“f”变异型以外,均为“F”型,“C”变异型占鼻咽癌的63％。此外,30bp缺失型潜伏膜蛋白-1基因在韩国淋巴瘤病例中占80％,未缺失型占14％,两者兼有者占6％。而在延边地区,缺失型在淋巴瘤中仅占43％,在鼻咽癌中占40％。结论 EB病毒-1型为韩国和延边地区的主要EB病毒株。。BamHI“f”变异型在两地区均罕见。但在韩国淋巴瘤病例中,30bp缺失型LMP-1基因的检出率则明显高于我国延边地区。     

Epstein-Barr Virus-encoded Latent Membrane Protein 1 Co-expresses with Epidermal Growth Factor Receptor in Nasopharyngeal Carcinoma

Latent membrane protein 1 (LMP-1) is the only Epstein-Barr virus (EBV)-encoded oncogenic protein that has been detected in nasopharyngeal carcinoma (NPC), a cancer that is closely associated with EBV. Previous in-vitro studies have demonstrated that LMP-1 can upregulate epidermal growth factor receptor (EGFR) in epithelial cells. It was not established whether this cellular effect exists in NPC. To assess the association between LMP-1 and EGFR in NPC tissues, 60 NPC specimens were examined by immunohistochemistry using anti-LMP-1 antibody (CS 1–4) and anti-EGFR antibodies (EGFR 1, EGFR 1005). The results revealed that 41 (68.3%) specimens were immunopositive for LMP-1 and 44 (73.3%) specimens over-expressed EGFR. Morphologically, the expressions of LMP-1 and EGFR were homogeneously distributed in the tumor nests. In addition, the correlation between LMP-1 and EGFR was statistically significant (P<0.001, χ² test, d.f.=1). To elucidate further the correlation between LMP-1 and EGFR in vivo and in situ , an indirect dual immunofluorescence assay was conducted, using secondary antibodies conjugated with fluorescein isothiocyanate (FITC) or indocarbocyanine (Cy3). The results disclosed an intimate co-expression of LMP-1 and EGFR. In summary, the data indicate that over-expression of EGFR is a common phenomenon in NPC, and that EGFR is co-expressed with LMP-1 in NPC. Thus, EBV may play a role in the tumorigenesis of NPC through the effects of LMP-1 and EGFR.     

EB病毒的潜伏感染及其编码的癌基因在鼻咽非霍奇金恶性淋巴瘤中的表达及意义

目的：探讨鼻咽非霍奇金恶性淋巴瘤患者中EB病毒的感染情况以及在EB病毒潜伏感染状态下编码的病毒癌基因产物潜伏膜蛋白-1(LMP-1)与鼻咽部非霍奇金恶性淋巴瘤的发生及预后的关系，另外还对该组鼻咽淋巴瘤的组织细胞起源进行了分析。方法：我们选用EBER-1／2 mRNA探针，经原位杂交方法检测了70例鼻咽恶性淋巴瘤和10例鼻咽慢性炎症病例中的EB病毒感染，同时使用免疫组化染色方法，分别标记了LMP-1、CD45R0、CD20及CD56阳性细胞。结果：EB病毒mRNA在鼻咽恶性淋巴瘤患者中的阳性表达率(62、9％)明显高于慢性炎症患者(0％)。LMP-1蛋白在上述两者间也有差异表达，肿瘤组(68．6％)高于炎症组(20．0％)(P=0．003)。本组有12例NK／T细胞淋巴瘤，特征性表达CD56，与LMP-1相关，但不具有特殊的生物学行为．LMP-1阳性高表达率除与鼻咽淋巴瘤患者出现临床B症状相关(P=0．043)外，与临床分期、出现区域淋巴结肿大均不相关，但LMP-1的表达与患者的预后密切相关，在死亡患者组中有LMP-1的高表达。结论：在鼻咽恶性淋巴瘤的发生、发展过程中，EB病毒的感染及其编码的癌蛋白LMP-1的产生起到了致关重要的作用。此外，LMP-1与不良临床特征和预后相关，同时还与CD56( )的NK/T细胞淋巴瘤相关。     

中国南方鼻咽鳞状细胞癌与EB病毒感染的关系

目的：探讨中国南方鼻咽癌高发区鼻咽鳞状细胞癌或称角化性鳞状细胞癌（Ｋｅｒａｔｉｎｉｚｉｎｇ ｓｑｕａｍｏｕｓ ｃｅｌｌ ｃａｒｃｉｎｏｍａ,ＫＳＣＣ）是否也如非角化性癌那样与ＥＢ病毒感染有密切的关系。方法：应用ＰＣＲ Ｓｏｕｔｈｅｒｎ ｂｌｏｔｔｉｎｇ、原 位分子杂交和免疫组化法检测３８例鼻咽鳞状细胞癌细胞中有ＥＢ病毒ＤＮＡ及其产物ＥＢＮＡ－１,ＥＢＮＡ－２,ＥＢＥＲＳ,ＬＭＰ－１,ＺＥＢＲＡ,Ｅ     

Complementary serum test of antibodies to Epstein-Barr virus nuclear antigen-1 and early antigen: a possible alternative for primary screening of nasopharyngeal carcinoma

This hospital-based cohort study evaluated the efficacy of three Epstein-Barr virus (EBV) - associated assays for nasopharyngeal carcinoma (NPC) primary screening and monitoring treatment outcome. Five hundred and seventeen consecutive subjects, including 156 NPC patients, 264 healthy volunteers and 97 patients with head and neck squamous cell carcinoma (HNSCC) were enrolled. The sensitivity and specificity of EBV IgAs to viral capsid antigen (VCA), complementary EBV IgAs to early antigen and nuclear antigen-1 (EA+EBNA-1), and EBV DNA load were examined by immunofluorescent assays, enzyme-linked immunosorbent assays, and quantitative real-time PCR, respectively. After constructing the receiver operating characteristics to demonstrate screening efficacy, EBV EA+EBNA-1 IgA (AUC: 0.952; 95% CI, 0.930-0.974) was proved superior to EBV VCA IgA (AUC: 0.888; 95% CI, 0.854-0.922) or EBV DNA load (AUC: 0.893; 95% CI, 0.854-0.932) in differentiating NPC patients from controls. Comparison of screening efficacy between NPC patients and HNSCC patients revealed EBV EA+EBNA-1 IgA (AUC: 0.964; 95% CI, 0.943-0.985) still outperformed EBV VCA IgA (AUC: 0.884; 95% CI, 0.845-0.923). In subjects with higher serum titer or level equal to or above 1:80 and 6 EU/ml for EBV VCA IgA and EA+EBNA-1 IgA, the specificity reached as high as 99.2% and 95.1%, respectively, in the control groups. However, correlation of these three assays with clinicopathological manifestations of NPC, revealed only EBV DNA load significantly associated with N stage and overall stage in NPC patients. Additionally, EBV DNA load could be used to further raise the specificity of EBV EA+EBNA-1 IgA assays and was also the only assay to be consistently predictive of tumor relapse in post-treatment patients according to serial test results by time frame. Consequently, an EBV EA+EBNA-1 IgA-based protocol is recommended for mass screening, but EBV DNA load should be used solely for post-treatment monitoring for NPC in endemic areas.     

Identification of a novel variant of LMP-1 of EBV in patients with endemic Burkitt lymphoma in western Kenya

Background

Epstein Barr virus (EBV) is a gammaherpesvirus that is associated with nasopharyngeal carcinoma (NPC) and endemic Burkitt lymphoma (eBL). EBV carries several latent genes that contribute to oncogenesis including the latent membrane protein 1 (LMP-1), a known oncogene and constitutively active CD40 homolog. Variation in the C terminal region of LMP-1 has been linked to NPC pathogenesis, but little is known regarding LMP-1 variation and eBL.

Results

In the present study, peripheral blood samples were obtained from 38 eBL patients and 22 healthy controls in western Kenya, where the disease is endemic. The LMP-1 C-terminal region from these samples was sequenced and analyzed. The frequency of a 30 base pair deletion of LMP-1 previously linked to NPC was not associated with eBL compared to healthy controls. However a novel LMP-1 variant was identified, called K for Kenya and for the G318K mutation that characterizes it. The K variant LMP-1 was found in 40.5% of eBL sequences and 25.0% of healthy controls. All K variant sequences contained mutations in both of the previously described minimal T cell epitopes in the C terminal end of LMP-1. These mutations occurred in the anchor residue at the C-terminal binding groove of both epitopes, a pocket necessary for MHC loading.

Conclusions

Overall, our results suggest that there is a novel K variant of LMP-1 in Kenya that may be associated with eBL. Further studies are necessary to determine the functional implications of the LMP-1 variant on early events in eBL genesis.

     

Prognostic impact of EBV-related LMP-1, histologic type, and environmental factors in nasopharyngeal carcinoma in a German population

INTRODUCTION: This retrospective study addressed the possible involvement of latent Epstein-Barr virus (EBV) infection, in particular LMP-1 expression, and further exogenous factors, i.e. tobacco, alcohol and occupational hazardous substances, in nasopharyngeal carcinoma (NPC) in a German population. PATIENTS AND METHODS: From 1980 to 2000, 44 patients suffering from histologically confirmed NPC were entered into the study. 33 specimens were available for immunostaining (IHC) to analyze LMP-1 expression. Information about environmental exposures were obtained employing a detailed standardized questionnaire. RESULTS: Outcome of patients with squamous cell NPC (SC-NPC) was significant worse than that of those with non-keratinizing NPC (NK-NPC). Age and tumor size correlated with response to therapy. The group with negative conventional LMP-1 staining showed better overall survival after 5 years compared to the group with positive or marginally positive LMP-1 detection (not significant). Nevertheless, after staining by tyramid-augmented IHC (TSA-IHC), nearly all specimens with negative LMP-1-staining in conventional IHC were found to be clearly positive. All patients with SC-NPC were smokers. The distribution of smokers and non-smokers in the group of NK-NPC was balanced. Comparable to the tobacco observation, there was also a correlation between high alcohol consumption and SC-NPC. CONCLUSION: Prognosis of NPC is mainly dependent on histologic type. Prognostic impact of LMP-1 is still unclear since LMP-1 was detected in all specimens using TSA-IHC. Therefore, TSA-IHC-LMP-1 detection might be interesting for diagnostic specification and development of new therapeutic strategies in NPC.     

Epstein-Barr virus-encoded latent membrane protein 1 co-expresses with epidermal growth factor receptor in nasopharyngeal carcinoma.

Latent membrane protein 1 (LMP-1) is the only Epstein-Barr virus (EBV)-encoded oncogenic protein that has been detected in nasopharyngeal carcinoma (NPC), a cancer that is closely associated with EBV. Previous in-vitro studies have demonstrated that LMP-1 can upregulate epidermal growth factor receptor (EGFR) in epithelial cells. It was not established whether this cellular effect exists in NPC. To assess the association between LMP-1 and EGFR in NPC tissues, 60 NPC specimens were examined by immunohistochemistry using anti-LMP-1 antibody (CS 1-4) and anti-EGFR antibodies (EGFR 1, EGFR 1005). The results revealed that 41 (68.3%) specimens were immunopositive for LMP-1 and 44 (73.3%) specimens over-expressed EGFR. Morphologically, the expressions of LMP-1 and EGFR were homogeneously distributed in the tumor nests. In addition, the correlation between LMP-1 and EGFR was statistically significant (P<0.001, chi2 test, d.f. = 1). To elucidate further the correlation between LMP-1 and EGFR in vivo and in situ, an indirect dual immunofluorescence assay was conducted, using secondary antibodies conjugated with fluorescein isothiocyanate (FITC) or indocarbocyanine (Cy3). The results disclosed an intimate co-expression of LMP-1 and EGFR. In summary, the data indicate that over-expression of EGFR is a common phenomenon in NPC, and that EGFR is co-expressed with LMP-1 in NPC. Thus, EBV may play a role in the tumorigenesis of NPC through the effects of LMP-1 and EGFR.     

Expression of Etk/Bmx tyrosine kinase in the tumorigenicity of nasopharyngeal epithelium and its relation with EB virus infection and the apoptosis-related protein Bcl-2

We compared Etk/Bmx expression in nasopharyngeal carcinoma (NPC) and non-neoplastic nasopharyngeal lesions in order to learn whether the expression of this non-receptor protein tyrosine kinase is associated with the development of NPC. We also related Etk/Bmx expression to factors resulting from Epstein-Barr virus (EBV) infection. We used immunohistochemistry (IHC) and in situ hybridization to examine 20 non-neoplastic nasopharyngeal lesions and 49 cases of NPC to assess Etk/Bmx, EB virus latent membrane protein-1 (LMP-1), Bcl-2 and EBV-encoded small RNA-1 expression in these samples. Etk/Bmx expression was present in the basal cell nuclei of the nasopharyngeal epithelium in 1/9 (11.1%) cases of chronic nasopharyngitis and 2/11 cases (18.2%) of dysplasia. While 13/49 (26.5%) NPC cases expressed Etk/Bmx, the difference in frequency between the expression of Etk/Bmx in the non-neoplastic and NPC cases was not significant. Etk/Bmx expression was correlated with the presence of EBER-1 immunopositivity in dysplasia and in NPC but not in chronic nasopharyngitis. The presence of Etk/Bmx immunopositivity was independent of the expression of either LMP-1 or Bcl-2 in either the nasopharyngeal carcinoma or the non-neoplastic lesions. This suggests that in some cases of non-neoplastic and neoplastic nasopharyngeal lesions, Etk/Bmx may participate in regulating epithelial differentiation. While EBV-related small RNA-1 may participate in this regulation, neither LMP-1 or Bcl-2 expression appears to be related to Etk/Bmx expression.