Association of a distinctive strain of Epstein-Barr virus with gastric cancer

Epstein-Barr virus (EBV) has been linked to gastric carcinoma (GC) with worldwide geographical variations attributable to types and variants of EBV. Here, we compare EBV strains between EBVaGC and healthy donors in Latin America, a high frequency area for EBVaGC. Tumor samples from 73 EBVaGC cases and throat washings from 329 healthy adults were examined for types 1 and 2 EBV and polymorphism at BamHI-F and BamHI-W1/I1 boundary regions and XhoI restriction site in LMP1 gene. Type 1 and prototype F of BamHI- F polymorphism accounted 59 (81%) and 69 (95%) of EBVaGC cases and 257 (78%) and 267 (81%) of healthy donors, respectively. Types I and "i" of BamHI W1/I1 polymorphism accounted 2 (3%) and 62 (85%) of EBVaGC and 85 (26%) and 170 (52%) of healthy donors, respectively (p<0.001). XhoI+ and - polymorphism accounted 60 (82%) and 4 (5%) of EBVaGC and 142 (43%) and 92 (28%) of healthy donors, respectively (p<0.001). Cosegregation analysis demonstrated that most of the 62 type "i" EBVaGC cases harbor XhoI+ strain (81%). However, among 143 type "i" healthy adults, both XhoI polymorphism were present in relatively similar frequencies (XhoI+ 58% and XhoI- 42%) (OR 9.0; 95% CI 1.2-69). Our findings are against to the proposed hypothesis that EBV strains are geographically but not disease-restricted. We conclude that most of the EBVaGC cases harbor a distinctive EBV strain (type "i"/XhoI +), but in healthy donors, this strain was as common as other strains. This finding is contrary to the proposed hypothesis that EBV strains are geographically but not disease-restricted and identified a healthy population group that share the same strain that predominate in EBVaGC cases.     

Epstein-Barr virus-associated gastric carcinoma in Lima, Peru

We examined 254 gastric carcinomas (GCs) diagnosed in four hospitals in Lima, Peru, and its suburban area during the period between 1994-2001. Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) was identified by the in situ hybridization (ISH) technique to detect EBV-encoded small RNA (EBER) in gastric tissue. EBVaGCs, where EBER ISH staining was observed in all carcinoma cells, accounted for 3.9% (10/254) of gastric adenocarcinomas, the lowest frequency ever reported in Latin American countries. EBVaGC incidence rates in Peru, which we estimated on the basis of the present study and cancer incidence in Lima, were 0.8 per 100,000 among men and 0.5 per 100,000 among women. These estimates are much lower than those reported in our previous studies in Colombia (4.1 and 1.4 per 100,000 among men and women, respectively), a neighboring country, and in Japan (6.4 and 1.1 per 100,000 among men among women, respectively). Interestingly, EBVaGC in Peru showed no evident male predominance, as opposed to the findings reported in a majority of studies. Other clinicopathological features of EBVaGC in Peru were similar to those found in literature: EBVaGC showed no age dependence, a predominance in the non-antrum part of the stomach, and high frequencies in histological subtypes of moderately differentiated tubular adenocarcinoma and solid poorly differentiated adenocarcinoma. There was a case of well-differentiated adenocarcinoma showing a partial EBER-1-positive staining. In this carcinoma, the tumor in the body (middle third of the stomach) was EBER-1 positive but the tumor in the stomach antrum showed no noticeable EBER-1 ISH staining. We suspect this was a case of synchronous double carcinomas. Further studies are needed to identify the cause of the low frequency and lack of male predominance of EBVaGC in Peru.     

Epstein-barr virus detection in tumors of upper gastrointestinal tract. An in situ hybridization study in Pakistan

To examine the potential role of Epstein-Barr virus (EBV) in the carcinogenesis of upper gastrointestinal tract, we conducted an in situ hybridization assay for EBV-encoded small RNA (EBER) expression in the tumors of 56 oral and 50 esophageal squamous cell carcinoma (SCC) cases, and 52 stomach adenocarcinoma cases diagnosed in the King Edward Medical College and Allama Iqbal Medical College Lahore, Pakistan between 1996-2002. There were no malignancies with positive EBER expression in oral and esophageal SCC. Only one out of the 52 gastric adenocarcinoma cases (1.9%) was positive for EBER expression, and this frequency was relatively low as compared to cases reported worldwide. The case was a 42 year-old male patient and histologically classified as moderately differentiated tubular adenocarcinoma. In conclusion, the frequency of EBV-associated gastric carcinoma was relatively low in Pakistan. The present study could not confirm the involvement of EBV in the carcinogenesis of oral and esophageal SCC.     

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。     

Epstein-barr virus genotypes in NPC biopsies from North Africa

The genotypes of Epstein-Barr virus (EBV) were investigated in North African nasopharyngeal carcinoma (NPC) biopsies, nasopharyngeal chronic inflammation (NCI) biopsies, and saliva of healthy individuals from Algeria and Tunisia where there is an intermediate incidence of NPC. The prevalence of A-type virus in NPC, NCI biopsies and saliva of healthy individuals was found in these regions by means of a PCR assay. Restriction enzyme polymorphism analysis by Southern blotting revealed that all North African EBV variants have a conserved restriction site on BamHI W'-1′ and XhoI LMP gene. No additional BamHI enzyme site on the BamHI-F fragment was observed; however, the presence of an extra BamHI site on the BamHI-H fragment giving 2 H1 and H2 fragment-like EBV M-ABA strains was found. All EBV strains present in NPC or NCI biopsies at all ages were homogeneous in these polymorphisms and no correlation was observed between the EBV genotypes from NPC patients and clinical stages of the cancer. These characteristics revealed a significant difference between the EBV variants common in Chinese NPC and those in North African NPC.     

Epstein-barr virus-positive gastric carcinoma has a distinct protein expression profile in comparison with epstein-barr virus-negative carcinoma.

PURPOSE: EBV has been detected in 2-16% of gastric carcinomas. However, there is little information available about the gene expression profile of EBV-positive gastric carcinomas. EXPERIMENTAL DESIGN: EBV infection was examined using EBV-encoded small RNAs (EBERs) in situ hybridization, and 63 (5.6%) of 1127 consecutive gastric carcinomas were found to be EBV-positive. The expressions of 27 tumor-associated proteins were evaluated immunohistochemically in 63 EBV-positive gastric carcinomas and 287 EBV-negative carcinomas using the tissue array method. In addition, the genotype of EBV was investigated by PCR amplification of LMP1 (latent membrane protein 1), Epstein-Barr nuclear antigen 2 (EBNA2), and EBNA3B genes. RESULTS: EBV-positive gastric carcinomas are characterized by the presence of lymphoid stroma, proximal location, and predominance in males. In comparison with EBV-negative carcinomas, EBV-positive carcinomas showed frequent loss of expression of p16, smad4, FHIT, and KAI-1 (kangai 1; P < 0.05), but retained the expression of APC (adenomatous polyposis coli), DCC (deleted in colorectal cancer), and some DNA repair proteins (P < 0.05). There was negative association between EBV infection and the expression of MUC1, MUC2, MUC5AC, p53, CEA, C-erbB2, and smad7. Using hierarchical cluster analysis, we divided EBV-positive gastric carcinomas into two clusters. Those patients with cluster 1 (42 cases) carcinomas had a better prognosis than those with cluster 2 (12 cases; P = 0.0002) or those with EBV-negative carcinomas (280 cases; P = 0.0251). Fifty-one (92.7%) of 55 EBV-positive carcinomas demonstrated the 30-bp deletion in LMP1 gene, and 53 (96.4%) of 55 cases were type 1 for EBNA2 and EBNA3B genes. CONCLUSION: EBV-positive gastric carcinomas have a distinct protein expression profile as well as distinct clinicopathological features, as compared with EBV-negative carcinomas. The subclassification of EBV-positive carcinomas, by hierarchical cluster analysis, is significantly associated with patient survival.     

Circulating Epstein-Barr virus DNA in the serum of patients with gastric carcinoma.

PURPOSE: We investigated the detectability of EBV DNA in the serum of gastric carcinoma patients in Hong Kong. Previous data have shown that approximately 10% of gastric carcinomas in Hong Kong are associated with EBV. EXPERIMENTAL DESIGN: We recruited 51 patients with gastric carcinoma, 30 patients with gastritis, and 197 apparently healthy controls. For gastric carcinoma patients, blood samples were obtained before surgery. After surgery, the resected tumor samples from the cancer cases were subjected to in situ hybridization for small EBV-encoded RNA (EBER). Serum EBV DNA in all cases was measured by real-time quantitative PCR. RESULTS: Serum EBV DNA was detectable in 5 of 5 (100%) EBER-positive gastric carcinoma cases (median concentration, 1063 copies/ml), in 13 of 14 (93%) EBER-negative gastric carcinoma cases with EBER-positive infiltrating lymphocytes (median concentration, 50 copies/ml), and in 0 of 32 (0%) EBER-negative cases. In the nontumor controls, serum EBV DNA was detectable in 7 of 30 (23%) gastritis cases (median concentration, 0 copies/ml) and in 7 of 197 (3.6%) apparently healthy individuals (median concentration, 0 copy/ml). CONCLUSIONS: Our data indicate that serum EBV DNA reflects tumoral EBER status and opens up the possibility that circulating EBV DNA may be used as a tumor marker for the EBER-positive gastric carcinomas. The biological and clinical significance of the presence of low levels of circulating EBV DNA in the minority of gastritis patients and healthy individuals remains to be elucidated.     

中线T细胞淋巴瘤与EB病毒关系的观察

为研究中线Ｔ细胞淋巴瘤（ＭＴＬ）与ＥＢ病毒的关系，运用原位杂交和免疫组化技术检测２０例中线Ｔ细胞淋巴瘤，观察其ＥＢ病毒的存在情况，结果２０例ＭＴＬ均显示Ｔ细胞标记阳性，Ｂ细胞标记阴性；原位杂交ＥＢ病毒（ＥＢＥＲ－１）阳性率７０％（１４／２０），ＬＭＰ－１检出率５０％（１０／２０），结果表明ＥＢ病毒感染与ＭＴＬ存在密切关系，可能在其发病中起着重要作用     

Detection of Epstein-Barr virus in gastrectomy specimens

Epstein Barr virus (EBV) has been reported to be present in a minority of gastric carcinomas and may be implicated in its pathogenesis. This study was aimed at determining the occurrence of EBV in 43 consecutive gastrectomy specimens with a variety of benign and malignant lesions. In situ hybridisation was used for detection of EBER RNA, the marker for latent EBV infection. Only 1/20 (5%) gastric cancers was EBER positive; a moderately differentiated adenocarcinoma with a heavy lymphocytic infiltration. The interstitial lymphocytic infiltrate was predominantly of B cell type, but the majority of lymphocytes overlying the tumour cells were CD8+ T cells. The other gastric lesions examined, which included 15 peptic ulcers, 6 stromal tumours and 2 lymphomas, were all EBER negative. Using a biotin detection system, scattered EBER positive cells were seen in adjacent normal gastric and/or duodenal mucosa in 9 sections from 8 cases (i.e., in 19% of all 43 cases examined). However, on using a digoxygenin detection system, no reactivity was found in these normal cells. An immunoperoxidase stain for chromogranin A showed that these apparently 'EBER positive' cells corresponded to normal chromogranin positive neuroendocrine cells within the gastric and duodenal mucosa. We conclude that EBV infection occurred only in the lymphoepithelioma type of gastric carcinoma and was absent from the other adenocarcinomas and from normal and benign tissues. The occasional EBER positive reaction encountered in normal cells was probably the result of a false signal arising from neuroendocrine cells as a consequence of the biotin-containing detection system.     

EB病毒阳性的胃弥漫大B 细胞淋巴瘤特点及预后

目的:探讨EB病毒阳性患者的胃弥漫大B 细胞淋巴瘤（diffuse large B-celllymphoma,DLBCL ）病理学特点及预后。方法:回顾性分析北京大学基础医学院病理学系2009年1 月至2015年1 月75例胃弥漫大B 细胞淋巴瘤患者的临床资料,15例EB病毒（Epstein-Barr virus,EBV ）阳性者为病例组,60例EBV 阴性者为对照组,采用免疫组织化学法和EB病毒RNA 探针原位杂交法检测Bcl- 2、c-myc 蛋白表达及EBV-EBER 情况,分析EBV 阳性的胃DLBCL 患者的病理学特点及预后。结果:EBV 阳性组在临床表现、年龄、性别、起源、细胞形态等方面与EBV 阴性组相比,差异无统计学意义（P > 0.05）;在Bcl- 2、c-myc 蛋白表达方面,EBV 阳性组与EBV 阴性组相比,差异无统计学意义（P > 0.05）;R-CHOP 方案治疗下,EBV 阳性组与EBV 阴性组相比,中位总生存期（median overallsurvival,OS）分别为15.1 个月和31.4 个月,差异具有统计学意义（P = 0.01）。 结论:发生于胃DLBCL 患者中,EB病毒感染对临床表现、瘤细胞的起源、形态、蛋白表达等方面无明显影响;EB病毒阳性的DLBCL 患者并不局限于老年人;R-CHOP 治疗下EB病毒阳性患者的预后比EB病毒阴性的患者预后差。      

胃癌组织幽门螺杆菌感染与EB病毒潜伏膜蛋白表达的相关性

目的：观察胃癌组织中幽门螺杆菌（H．pylori）感染和EB病毒潜伏膜蛋白（EBV—LMP）的同步表达情况，探讨两者在胃癌中的相互关系。方法：80例正常胃黏膜组织和97例胃癌组织，利用HE染色对所取标本进行组织病理学诊断，EBV—LMP测定采用免疫组化sP法，H．pylori感染判定采用HE，H．pylofi—DNA PCR和血清ELISA法测定IgG抗体三种方法。结果：正常胃黏膜组织未见EBV—IMP的表达，胃癌黏膜组织EBV—LMP表达阳性率为7．2％（7／97），胃癌组织EBV—LMP表达明显高于正常胃黏膜组织，差异有统计学意义（P〈0．05）。本组病例中H．pylofi感染的胃癌黏膜组织中EBV—LMP的阳性表达率为13．5％（7／52），无H．pylori感染的胃癌组织EBV—LMP的阳性表达率为0，H．pylori感染的胃癌组织EBV—LMP表达明显高于无H．pylofi感染的胃癌组织，差异有统计学意义（P〈0．05）。结论：正常胃黏膜组织无EBV—LMP的表达，胃癌组织中EBV—LMP表达明显高于正常胃黏膜组织，H．pylofi感染胃癌组织比无H．pylori感染胃癌组织EBV—LMP呈现高表达，H．pylofi感染和EB病毒感染在胃癌的发生和发展过程中可能具有更加深在的相互关系。     

胃肠道B细胞淋巴瘤中bcl-2的表达及与EB病毒的相关性研究

 目的　研究胃肠道B细胞淋巴瘤与bcl 2蛋白的表达及与EB病毒感染的关系。方法　采用免疫组织化学、原位杂交方法分别检测 16例胃肠道B细胞淋巴瘤中bcl 2、LMP 1蛋白及EB病毒编码的E BER1/2表达水平。结果　bcl 2蛋白阳性表达 13例 (81.3% ) ,均为高表达 ,其中 7例低度恶性MALT型淋巴瘤均为阳性。EB病毒编码的EBER1/2及LMP 1均为阴性。结论　胃肠道B细胞淋巴瘤特别是低度恶性MALT型B细胞淋巴瘤 ,与bcl 2蛋白的高表达密切相关 ,而与EB病毒的感染似乎无相关性     

唐山地区胃癌患者EB病毒感染与p53基因表达的关系

 目的 探讨胃癌患者癌组织中EB病毒感染与p53表达的关系。方法 原位杂交法检测胃癌患者石蜡标本中EB病毒编码的小RNA（EBER1）； 免疫组化法检测EB病毒阳性胃癌及EB病毒阴性胃癌患者标本中抑癌基因p53的表达状况。结果 217例胃癌患者癌组织标本中检测到23例呈EBER1阳性； EB病毒阳性胃癌中P53蛋白表达的平均面积、平均吸光度、积分吸光度值均显著高于EB病毒阴性胃癌（P＜0.05）。结论 EB病毒感染与癌组织中抑癌基因p53的异常表达密切相关。     

Detection of Epstein-Barr virus in gastric carcinoma cells and surrounding lymphocytes

Background. Epstein-Barr virus (EBV), the etiological agent of infectious mononucleosis, has an important role in the oncogenesis of EBV-related malignant diseases. The association of EBV with gastric carcinoma cells has become well known recently, but there are only a few reports concerning its association with surrounding epithelia and infiltrating lymphocytes. In this study, we investigated the association of EBV with gastric carcinoma and surrounding cells. Methods. One hundred and two cases of gastric carcinoma were studied. The specimens were studied for the presence of the EBV genome by polymerase chain reaction (PCR), and then by in situ hybridization (ISH) technique to determine the localization of EBV. Results. Of 97 informative cases, EBV was detected in 21 cases (21.6%) by the PCR method. ISH studies showed that EBV RNA was expressed in 5 of the 97 cases (5.2%) and was localized to the nuclei of carcinoma cells. All these 5 lesions were found in male patients. In these 5 cases, 3 were diffuse type and 2 were intestinal type, and all cases arose in the proximal region of the stomach. EBV RNA was not detected in non-neoplastic epithelia, but it was detected in 24 of the 97 cases (24.7%) in small lymphocytes. Conclusion. EBV was detected in 5.2% of gastric carcinomas and in 24.7% of infiltrating lymphocytes by the ISH method. The high positive rate (21.6%) by the PCR method corresponds to the presence of the EBV genome in surrounding lymphocytes.     

中线恶网中的EB病毒感染

目的　观察一组中线恶网病变组织中 EB病毒感染的存在情况及其异形淋巴样细胞( atypic al lymphoid cells,AL Cs)的免疫表型。方法　 37例病变组织切片作 DNA- RNA原位杂交 ,检测 EB病毒编码的小分子 RNA( EBER) ,作免疫组化染色看 EB病毒隐伏膜蛋白 ( L MP)抗原的表达及 ALCs的免疫表型 ;其中 15例用 PCR方法检测了 EBV- DNA。结果　 ( 1) 31/ 37例 ,占 83.8%之ALCs呈 EBER 1/ 2阳性反应 ;12 / 15例 ,占 80 %检出 EBV- DNA;5/ 19例 ,占 2 6.3%之少部分 AL Cs呈 L MP阳性反应。 ( 2 ) 2 0 / 37例 ,占 54.1%之 ALCs同时表达 CD3及 CD56抗原 ;5/ 19例 ,占 2 6.3%之少部分 ALCs呈 CD30 反应。结论　该组中线恶网病例中存在着较高比率的 EB病毒隐性感染 ,其ALCs既表达 T细胞分化抗原 ,同时也高水平地表达自然杀伤细胞抗原 ,CD56。作者还复习讨论了多种淋巴增生性疾病中 EBV感染的存在情况。     

EBV-positive gastric adenocarcinomas: a distinct clinicopathologic entity with a low frequency of lymph node involvement.

PURPOSE: Epstein-Barr virus (EBV) is detected in a substantial subgroup of gastric adenocarcinomas worldwide. We have previously reported that these EBV-positive gastric carcinomas carry distinct genomic aberrations. In the present study, we analyzed a large cohort of EBV-positive and EBV-negative gastric adenocarcinomas for their clinicopathologic features to determine whether they constitute a different clinical entity. PATIENTS AND METHODS: Using a validated polymerase chain reaction/enzyme immunoassay-based prescreening method in combination with EBER1/2-RNA in situ hybridization, EBV was detected in the tumor cells of 7.2% (n = 41) of the gastric carcinomas from the Dutch D1D2 trial (N = 566; mean follow-up, 9 years). EBV status was correlated with clinicopathologic features collected for the Dutch D1D2 trial. RESULTS: EBV-positive gastric carcinomas occurred significantly more frequently in males (P <.0001) and in younger patients (P =.012). Most were of the intestinal type according to the Laurén classification (P =.047) or tubular according to the WHO classification (P =.006) and located in the proximal part of the stomach (P <.0001). A significantly lower tumor-node-metastasis system-stage (P =.026) was observed in the patients with EBV-carrying carcinomas, which was solely explained by less lymph node (LN) involvement (P =.034) in these cases. In addition, a better prognosis, as reflected by a longer disease-free period (P =.04) and a significant better cancer-related survival (P =.02), was observed for these patients, which could be explained by less LN involvement, less residual disease, and younger patient age. CONCLUSION: EBV-carrying gastric adenocarcinomas are a distinct entity of carcinomas, characterized not only by unique genomic aberrations, but also by distinct clinicopathologic features associated with significantly better prognosis.