Direct Detection of Epstein-Barr Virus DNA from a Single Reed-Sternberg Cell of Hodgkin's Disease by Polymerase Chain Reaction

Eleven cases of Hodgkin's disease (HD) were examined for the presence of the Epstein-Barr virus (EBV) genome, using the polymerase chain reaction (PCR) to detect EBV DNA in whole paraffin-embedded tissue specimens and in single cells picked out from the specimens with a micromanipulator. The EBV genome was detected in 5 of the 11 cases by conventional PCR. Single cell PCR demonstrated the EBV genome in Reed-Sternberg cells from all the EBV-positive cases, but not from any of the EBV-negative cases. Background lymphocytes and lysozyme-positive histiocytes from EBV-positive cases did not contain the EBV genome. These results indicate an etiological association of EBV with some cases of HD.     

A Strategy for Precision of Genotyping of Epstein-Barr Virus by Polymerase Chain Reaction: Application for Studying Hodgkin's Lymphoma

Previous studies on the genotyping of Epstein-Barr virus (EBV) have been based on the analysis of a single gene locus. The assignment of genotype of an isolate could easily be overlooked with this assay. Our strategy for precision of EBV genotyping has exploited the existence of two families of EBV strains (type A and B) that can be distinguished at three divergent gene loci (EBNA-2, EBNA-3C, and EBER). To precisely determine the genotype of EBV in Hodgkin's disease (HD), we designed primers and simultaneously analysed these three gene loci that distinguish type A and B viruses by the polymerase chain reaction (PCR) technique. The primers designed to amplify these three gene loci encompass either type-specific deletion sequences (EBNA-2 and EBNA-3C) or type-specific point mutations (EBER) that identify the virus strain based on the sizes of PCR-amplified products or the mobility shifts in single-strand conformation polymorphism (SSCP) analysis. The locations of point mutations were identified by direct sequencing of the PCR-amplified DNA. Fifteen EBV-infected cell lines were analysed and a good correlation between EBNA-2 and EBNA-3C typing results was found. In contrast, approximately 33% of the cell lines analysed maintained type A sequences in EBNA-2 and EBNA-3C genes while carrying type B sequences in the EBER region. Data obtained from analysis of cell lines served as a reference for studying HD samples. EBV DNA was detected in about 70% of HD. Among the EBV-positive samples, 56% were associated with type A virus, 13% with type B. and 31% with dual viral sequences. Thus, type A virus is predominant in HD. Based on the histology, the frequencies of EBV positivity were 83%, 71%, and 33% for mixed cellularity, nodular sclerosis and lymphocyte predominance, respectively. The detection of high frequency of both type A and B sequences in HD may provide a lead in investigating the role of dual viral infection in EBV pathogenesis.     

Detection of Epstein-Barr Virus in Oral Papilloma

Fifty-one cases of malignant and non-malignant oral diseases were investigated for Epstein-Barr virus (EBV). EBV DNA was detected by polymerase chain reaction analysis in 2 of 4 papillomas, but not in other tissues including 36 squamous cell carcinomas and 4 leukoplakias. The copy numbers of EBV DNA in the two positive samples were estimated to be 120 and 36 per cell, respectively. Intense EBV DNA signals were detected on papilloma cells by in situ hybridization. DNAs for the benign and malignant types of human papilloma virus were not detected in papilloma tissues. The present results suggest that EBV is a causative agent of oral papilloma.     

Detection of Epstein-Barr viral genomes in lymph nodes of Hodgkin's disease patients

Previous studies using Southern blot analysis or in situ hybridization have shown that approximately 20% of patients with Hodgkin's disease have Epstein-Barr virus (EBV) in involved tissues. We used the more sensitive polymerase chain reaction (PCR) technique to determine if a higher percentage of EBV could be detected. Of the 16 Hodgkin's disease patients studied, the PCR technique detected EBV in eight (50%). No prognostic significance was associated with the presence of EBV in the eight EBV-positive patients, and the presence of EBV was not associated with B cell monoclonality.     

Detection of Epstein-Barr Virus DNA in a Japanese Case of Lymphoepithelioma-like Thymic Carcinoma

Epstein-Barr virus DNA was detected in a case of lymphoepithelioma-like thymic carcinoma. A homogeneous terminal structure of the viral DNA was demonstrated in this case, indicating the presence of the viral genome in clonally expanded tumor cells. Since all of 26 other thymic epithelial tumors (eight non-invasive, 13 invasive thymomas and four non-lymphoepithelioma-like thymic carcinomas) in Japanese were negative by polymerase chain reaction, it is suggested that lymphoepithelioma-like thymic carcinoma may represent a unique pathological entity distinct from Epstein-Barr virus-negative thymic epithelial tumors, which are in the majority in Japan.     

Rearrangement of bcl-2 Is Detectable in Hodgkin's Disease by Polymerase Chain Reaction

The authors examined the occurrence of the t(14;18) chromosomal translocation in 44 cases of Hodgkin's disease (HD) using the polymerase chain reaction and Southern blot hybridization with non-radioactive oligonucleotide probes. DNAs were extracted from unfixed, fresh-frozen and formalin-fixed, paraffin-embedded biopsy specimens. Southern blot hybridization of the amplification product showed that, of 44 HD DNAs, three had a detectable t(14;18) breakpoint at the mbr (major breakpoint region), while none had a detectable t(14;18) breakpoint at the mcr (minor cluster region). Of the three cases positive for a t(14;18) breakpoint at the mbr, two were of lymphocyte predominance type, and the other was of mixed cellularity type.     

Sporadic Activation of Epstein-Barr Virus in Thyroid Lymphoma

The causal role of Epstein-Barr virus (EBV) in the development of B-cell lymphoma, especially in immunocompromised individuals, has been suggested. The purpose of the present study was to evaluate an association of EBV with thyroid lymphoma (TL) and chronic lymphocytic thyroiditis (CLTH) which is known to play an important role in the development of TL. Thirty cases with TL and 28 with CLTH were studied for presence or absence of EBV genome in the lesions using the polymerase chain reaction (PCR) and the in situ hybridization method. EBV genomes were detected by PCR in one and two cases with CLTH and TL. respectively. Subtyping of EBV genome was possible in one TL case showing B-type in EBNA-2 coding region. In situ hybridization revealed positive signals in the nucleus of lymphoma cells, which also expressed latent membrane protein-I. The present findings indicate that activation of EBV in TL is not common.     

中线T细胞淋巴瘤与EB病毒关系的研究

为了解ＥＢ病毒在中线Ｔ细胞淋巴瘤中的感染情况，作者对遵义地区４６例中线Ｔ细胞淋巴瘤进行ＥＢＶ检测，用聚合酶链反应检测ＥＢＶ特征性的ＤＮＡ序列（ＥＢＶＤＮＡ），用ＲＮＡ原位杂交法检测ＥＢＶ编码的ＲＮＡ（ＥＢＥＲ１／２）。结果：ＥＢＶＤＮＡ阳性率为７６．１％（３５／４６）；ＥＢＥＲ１／２阳性率为６７．４％（３１／４６）。鼻腔、鼻咽、口咽Ｔ淋巴瘤ＥＢＥＲ１／２阳性率分别为８８．９％（１６／１８）、７１．４％（５／７）、４７．６％（１０／２１）。在多形细胞性淋巴瘤中，中多形和大多形细胞性ＥＢＥＲ１／２阳性率为８０．０％（１６／２０），小多形细胞性为７３．３％（１１／１５）。结果表明ＥＢＶ感染与中线Ｔ淋巴瘤关系密切，它在该肿瘤的发病中可能起重要作用，中线Ｔ淋巴瘤的ＥＢＶ感染率与解剖学部位有关，多形细胞性淋巴瘤ＥＢＶ感染率较高，特别是中多形和大多形细胞性     

IgE, Reed-Sternberg Cells, and Eosinophilia in Hodgkin's Disease

Tissues containing Hodgkin's disease tumors of the nodular sclerosis and mixed cellularity subtypes are frequently infiltrated by numerous degranulating eosinophils that release granule proteins such as eosinophil peroxidase and major basic protein. Until recently, the causes of the eosinophil infiltration and degranulation in Hodgkin's disease tumors were unknown. Analysis of Hodgkin's disease tissues by a sensitive and specific immunoperoxidase technique has now demonstrated the extensive presence of IgE in the Reed-Sternberg cells and adjacent cells of Hodgkin's disease tumors. Because eosinophils express a cell-surface receptor (CD23) for IgE and degranulate in the presence of IgE deposits, the extensive eosinophilia that is frequently present in Hodgkin's disease tumors is, at least in part, attributable to the IgE deposits within the tumor. In this review, we discuss the possible mechanisms and biological significance of IgE-related eosinophilia in Hodgkin's disease.     

淋巴组织增生性疾病组织中EB病毒的原位杂交检测

目的探讨ＥＢ病毒与我国各类淋巴组织增生性疾病的关系。方法以ＥＢ病毒ＬＭＰ基因为探针,对２１４例淋巴组织增生性疾病组织中ＥＢ病毒进行原位杂交检测,并用ＳＹＳＴＡＴ软件对实验结果进行分析。结果霍奇金淋巴瘤（ＨＤ）、非霍奇金淋巴瘤（ＮＨＬ）、淋巴组织良性增生（ＢＬＰ）组织中ＥＢ病毒阳性率分别为３０．０％（１５／５０）,１４．０％（１８／１２９）及２．９％（１／３５）。在ＮＨＬ中,高度恶性（ＨＮＨＬ）、中度恶性（ＭＮＨＬ）及低度恶性（ＬＮＨＬ）ＥＢ病毒阳性率分别为２８．１％（９／３２）、１０．５％（９／８４）及０％（０／９）。ＨＤ与ＨＮＨＬ间ＥＢ病毒阳性率均显著高于ＢＬＰ（Ｐ＜０．０１,Ｐ＜０．０５）,ＭＮＨＬ和ＬＮＨＬ与ＢＬＰ间ＥＢ病毒阳性率差异无显著性（Ｐ＞０．０５）。结论ＥＢ病毒与ＨＤ及ＨＮＨＬ的发生有关,而与ＭＮＨＬ及ＬＮＨＬ的发生关系不大。     

Specific Detection of Metastasized Human Tumor Cells in Embryonic Chicks by the Polymerase Chain Reaction

We have established a highly sensitive method for specific detection of metastasized human tumor cells in embryonic chicks using the polymerase chain reaction (PCR). The cells (HT-1080 or KMST-6) were inoculated into the chorioallantoic membrane vein of the chick embryo and DNA of each embryonic organ was extracted. Then, a human β-globin-related sequence (576 bp) in the DNA from the embryonic liver and lung was specifically amplified and detected by gel electrophoresis and by a specific oligonucleotide probe. The amplified fragments from the liver DNA samples increased gradually from 2 h to 7 days after HT-1080 inoculation. On the other hand, with inoculation of non-tumorigenic human embryonal fibroblast KMST-6 cells, the DNA from the embryonic liver 7 days after inoculation did not show the PCR-amplified product. This detection technique can contribute significantly to the precise detection of microscopic metastasis.     

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。     

Detection of HTLV-I Genome in Seronegative Infants Born to HTLV-I Seropositive Mothers by Polymerase Chain Reaction

We applied the polymerase chain reaction (PCR) method to detect gag, env and pX sequences of human T cell leukemia virus type I (HTLV-I) provirus in peripheral blood lymphocytes of seronega-tive infants born to HTLV-I seropositive mothers. Out of 22, five subjects were found to contain the HTLV-I provirus genome. Two of the five cases were judged to be negative for not only anti-HTLV-I antibodies but also the viral antigens on cultivated lymphocytes by the conventional antibody/antigen detection methods. These results indicate that PCR is of great use as a simple and highly sensitive method detect HTLV-I infection.     

EB 病毒与非霍奇金淋巴瘤——附180 例报道

 目的　探讨 NHL与 EBV感染的相关性。方法　用 ISH方法检测 1 80例 NHL中EBV编码的 RNA( EBER1 /2 )。结果　 ( 1 ) EBER1 /2感染率为 42 .8% ( 77/1 80 ) ,感染率依次为TCL64.8%、NK/T46.2 %、BCL9.5 %。其中 TCL、NK/T与 BCL相比 ,P≤ 0 .0 0 5 ;( 2 )结内、结外EBV感染率依次为 2 2 .2 %、5 3.8% ,有显著差异 ,P<0 .0 0 5 ;( 3)面中线 (鼻腔 /口咽环 ) EBER1 /2感染率 72 .4% ( 5 5 /76) ,多形细胞性 EBER1 /2感染率 70 .1 % ( 5 4 /77)。结论　本地区 NHL与 EBV感染关系密切 ,TCL、NK/T淋巴瘤感染率明显高于 BCL,而且有部位限制性和亚型倾向性.     

Hodgkin's Disease Involving Waldeyer's Ring: A Study of Four Cases

We report four cases of Hodgkin's disease who presented with involvement of Waldeyer's ring. Their clinical, morphological and immunohistochemical features are discussed. The patients were immuno-competent, were between 17 and 65 years of age and presented with symptoms related to swelling in the nasopharynx or oropharynx and cervical lymphadenopathy. The nasopharyngeal biopsy and the cervical lymph node in all four cases showed features of classical Hodgkin's disease. The Reed Sternberg cells expressed CD15 and CD30, and in three cases, CD20 and Epstein-Barr virus-latent membrane protein-1 (EBV-LMP-1). Extranodal involvement by Hodgkin's disease which is not in continuity with nodal disease is rare in immunocompetent patients. Morphologically, such extranodal lesions, especially in locations like the oropharynx and nasopharynx, should be differentiated from EBV-associated lymphoproliferations.     

鼻咽癌组织中EB病毒的不同原位检测方法比较

目的：探讨在鼻咽癌组织石蜡切片标本中原位检测ＥＢ病毒的更快速、更灵敏的方法。方法：用地高辛标记的寡核苷酸ＥＢＥＲ－１和ＥＢＶ ＤＮＡ的ＢａｍＨＩ－Ｗ片段为探针，分别用原位杂交、原位ＰＣＲ检测鼻咽癌组织切片中ＥＢＶ的分布情况。结果：用寡核苷酸ＥＢＥＲ－１作探针原位杂交检测ＥＢ病毒的方法比用ＢａｍＨＩ－Ｗ片段作探鱼原位杂交及原位ＰＣＲ的方法更灵敏、更简单。结论：用ＥＢＥＲ－１作探鱼原位杂交检测鼻咽癌组     

定量和定位检测EB病毒在鼻咽癌组织中的感染状态

背景与目的EB病毒(Epstein-Barrvirus,EBV)与鼻咽癌密切相关,但EBV是鼻咽癌的致瘤因素还是仅仅是伴随关系有待进一步研究,而且EBV在鼻咽癌发生过程中的潜伏状态也不明确,本研究通过检测EBV在不同鼻咽组织中的表达变化,为进一步阐明EBV与鼻咽癌发生的关系提供线索。方法应用荧光定量PCR(real-timePCR)技术定量检测47例鼻咽低分化鳞癌患者癌、癌旁及相对正常鼻咽粘膜组织中的平均EBV拷贝数;并采用原位杂交技术(地高辛标记的EBER-1探针)对鼻咽癌、癌旁、对侧正常鼻咽粘膜中的EBV进行定位检测。以10例正常人鼻咽粘膜组织作对照。结果正常人鼻咽粘膜EBV检出率为80%(8/10),平均拷贝数为6.7×102/滋gDNA;100%(47/47)鼻咽癌和癌旁组织以及72.3%(34/47)的对侧正常鼻咽粘膜组织检出EBV,平均拷贝数分别为6.9×105/滋gDNA、1.5×105/滋gDNA、9.8×103/滋gDNA。正常人鼻咽粘膜、鼻咽癌患者对侧正常鼻咽粘膜之间感染EBV的几率、潜伏感染EBV的数量没有显著性差异(P>0.05);鼻咽癌及其癌旁、对侧正常粘膜组织三者之间,鼻咽癌与癌旁组织之间,癌旁和对侧正常粘膜组织之间EBV拷贝数存在显著性差异(P<0.01)。EBER-1原位分子杂交发现,对侧正常粘膜上皮细胞未检测到EBER-1的表达信号,癌旁靠近癌一侧的部分不典型增生细胞检测到EBER-1弱阳性信号,而在癌组织中的所有癌细胞均有EBER-1强阳性信号。结论无论是鼻咽癌患者还是健康人,EBV在鼻咽组织中的潜伏感染是一种普遍现象,但从鼻咽癌患者对侧正常鼻咽粘膜到癌旁再到癌组织EBV感染的量发生了改变,说明鼻咽上皮细胞中EBV感染量和表达信号的改变很可能是EBV致鼻咽癌的重要环节。     

Detection of Epstein-Barr Virus DNA in Hodgkin- and Reed-Sternberg-Cells by Single Cell PCR

The Epstein-Barr virus (EBV) can be detected in the majority of lymph nodes involved by Hodgkin's lymphoma using the highly sensitive polymerase chain reaction (PCR). However, the rate of EBV-DNA detection by in-situ hybridisation, which allows allocation of EBV to a defined cell population, i.e. the neoplastic H&RS-cells, is lower. In an attempt to combine the advantages of the high sensitivity of the PCR and the possibility of cellular allocation by in-situ hybridisation, we established a single-cell PCR of Hodgkin- and Reed-Stemberg (H&RS)-cells isolated by micromanipulation from biopsy tissues. We amplified EBV sequences from the BamW-region by single-cell PCR. Using this method we were able to detect EBV-DNA in the H&RS-cells from 4 of 6 patients. In EBV positive cases all H&RS-cells of a given patient were positive, proving the high sensitivity and reproducibility of the method. Other cells in the biopsy tissue involved by EBV-positive H&RS-cells were shown to be negative. This indicates that EBV may have a role in the pathogenesis of many but not all cases of Hodgkin's disease.     

Detection of Human Papillomavirus Type 16 in Sexual Partners of Patients Having Cervical Cancer by Polymerase Chain Reaction

The polymerase chain reaction (PCR) was employed to detect human papillomavirus (HPV) 16 and 18 in cytological samples from the uterine cervix and in urine samples from the male consorts. HPV 16 was detected in 2 (25%)of 8 males whose wives were positive for HPV 16, while it was detected in only one (7%) of 14 consorts whose wives were negative for HPV 16 and 18. This is the first report of the detection of HPV 16 in urine. Viral detection in urine samples by the PCR method is a non-invasive, convenient and useful tool for large-scale epidemiologic studies and investigations of the mechanism of virus transmission.