The expression of the EBV latent membrane protein (LMP-1) is independent of CD23 and bcl-2 in Reed-Sternberg cells in Hodgkin''s disease

A series of 33 cases of Hodgkin's disease was investigated for the presence of the EBV encoded latent gene product LMP-1 and of CD23 using immunohistochemical techniques. The expression of bcl-2 was examined in a subset of cases. LMP-1 was detected in the Reed-Sternberg cells in 15 cases. Although LMP-1 is known to upregulate CD23 and bcl-2, there was no correlation between the expression of LMP-1 and the detection of CD23 and bcl-2 in Reed-Sternberg cells.     

Presence of Epstein-Barr virus harbouring small and intermediate-sized cells in Hodgkin'S disease. Is there a relationship with Reed-Sternberg cells?

Forty-four cases of Hodgkin's disease (HD), mostly of the nodular sclerosing type, were investigated for the presence of Epstein-Barr virus (EBV) by polymerase chain reaction (PCR) and DNA and RNA in situ hybridization (DISH, RISH), as well as by immunohistochemistry for the detection of latent membrane protein-1 (LMP-1) of EBV. in situ hybridization (ISH) was combined with immunohistochemistry to correlate the presence and activity of the virus at the cellular level. In 18/34 (53 per cent) cases, EBV-DNA sequences could be detected with the PCR method. In 12/18 positive cases, DISH and RISH were also positive. In the remaining six EBV-PCR positive cases, two were also positive with RISH and LMP-1, whereas no positive signal with DISH could be obtained. All DISH and/or RISH positive cases were also positive for LMP-1. With RISH, not only the Reed-Sternberg cells and their mononuclear variants (RS cells) stained positive, but also small and intermediate cells frequently reacted with the EBV-specific probes (EBER-1 and -2). Double staining with cellular markers (CD3, CD20, CD45, CD45RO, CD68, and the lectin PNA) revealed that most of the smaller EBER-positive cells frequently did not express T, B, or histiocytic markers, but that they, as well as the RS cells, showed cytoplasmic and membranous staining with PNA. These smaller EBER-positive cells were not found in EBV-PCR negative HD. EBER-positive RS cells were almost always LMP-1 positive, as well as a substantial proportion of the intermediate-sized cells, whereas the majority of the small EBER-positive cells remained LMP-1 negative. In EBV-PCR positive non-malignant lymph nodes, only a few EBER-1 and -2 positive cells could be observed. As in infectious mononucleosis, these cells frequently expressed the B-cell marker CD20. Although we cannot exclude the fact that the majority of the smaller EBV-positive cells in HD belong to reactive EBV-infected lymphocytes, our data favour the hypothesis that at least some of these smaller cells may belong to the reservoir of neoplastic cells in HD.     

鼻咽癌高发区何杰金病EB病毒DNA及潜伏感染膜蛋白检测的意义

为检测鼻咽癌高发区何杰金病中爱泼斯坦-巴尔病毒（EBV）DNA及其表达产物──潜伏感染膜蛋白的存在及探讨其意义，采用热启动聚合酶链反应（PCR）及LSAB免疫组化法结合微波处理技术，检测了选自鼻咽癌高发区的40例何杰金病、20例淋巴结良性病变存档标本中的EBVDNA和潜伏感染膜蛋白（LMP1）。结果显示：65％的何杰金病中EBVDNA阳性，淋巴结良性病变中的阳性率也达50．0％（10例／20例），两者差异无显著性（P＜0．05）。LMP1只在何杰金病肿瘤细胞中表达，其检出率为52．5％（21例／40例）。20岁以下何杰金病中EBVDNA和LMP1的检出率分别为84．6％（11例／13例）和76．9％（10例／13例），皆明显高于20岁以上年龄组（分别为14例／25例，56．0％和10例／25例，40．0％）P＜0．05。本结果表明：鼻咽癌高发区半数以上何杰金病肿瘤细胞中有EBV潜伏感染，并可能在肿瘤的发生发展中起着一定的作用。提示青少年何杰金病和EBV潜伏感染并表达LMP1的关系更为密切。     

Epstein-Barr virus genomes in Hodgkin's disease and non-Hodgkin's lymphomas

Seventeen of 40 cases of Hodgkin's disease (HD) and eight of 46 non-Hodgkin's lymphomas (NHL) were associated with Epstein-Barr virus (EBV) infection, judged by the EBER-1 in situ hybridization (ISH) method. Approximately 40% incidence in HD was comparable to previous reports. Young children and elderly HD patients were more prone to be found EBV positive. Fourteen of 17 HD and two of 8 NHL cases with positive EBER-1 ISH were also positive on LMP-1 immunostaining. EBV might have a role in lymph-omagenesis in these cases. The fact that 7 of 8 EBV-related NHL were peripheral T cell lymphoma indicates the necessity of a larger-scale survey on this subject. As the present study revealed four cases with positive LMP-1 immunostaining but negative EBER-1 ISH (1 HD, 3 NHL), LMP-1 alone should not be regarded as a tool to prove EBV infection.     

Epstein-Barr virus (EBV)-associated undifferentiated carcinoma of the parotid gland

A case of undifferentiated carcinoma of the salivary gland occurring in the parotid gland of a southern Chinese was reported. Tumour cells showed immunofluorescence for Epstein-Barr virus (EBV)-associated nuclear antigen, and DNA hybridization demonstrated the presence of EBV-DNA in tumour tissue. The findings in this case, together with previous reports, suggest a causal relationship between EBV and salivary gland carcinoma. The relationships between EBV and undifferentiated epithelial tumours of the salivary glands, nasopharynx and thymus are also discussed.     

Persistence of Epstein-Barr virus in Reed-Sternberg cells throughout the course of Hodgkin's disease.

Non-isotopic in situ hybridization employing digoxigenin-labelled DNA probes has been used to localize Epstein-Barr virus (EBV) in 55 cases of Hodgkin's disease (HD). The virus was found in Reed-Sternberg (RS) and mononuclear Hodgkin's cells in nine patients (16 per cent). Further samples taken at different times from three patients also showed the presence of EBV in the malignant cell population. Estimations of the number of EBV genomes present per cell suggested wide variations between different patients, but relatively constant amounts in different samples from the same patient. These findings are compatible with a stable infection of the neoplastic cells and support the notion that EBV may play a role in the development of HD in these patients. We also found evidence for the presence of EBV in a small percentage of non-neoplastic cells in 8 of the 55 samples. This suggests that isolation of EBV from HD tissue does not always signify a pathogenetic role for the virus. Furthermore, it is apparent that a high percentage of HD tissues do not contain demonstrable EBV, and the virus is therefore unlikely to be a causative agent for all cases of HD.     

Angioimmunoblastic T-cell lymphoma (angioimmunoblastic lymphadenopathy with dysproteinemia [AILD]-type T-cell lymphoma) followed by Hodgkin's disease associated with Epstein-Barr virus

A patient is described with angioimmunoblastic T-cell lymphoma (AIL] (angidmmunoblastic lymphadenopathy with dysprotelrrrpmia [AILD]-type T-cell lymphoma), which was later followed by Hodgkin's disease. At the time of the initial diagnasis, histological examination of a cervical lymph node showed a typical picture of AIL with abundant clear calls which were CD45RO+, CD43+, and CD20^--, and there was no evidence of a monoclonal B-cell proliferation by Immunohistochemical analysis. In situ hybridization for Epstein-Barr virus (EBV) was negative. Interposed by a bout of recurrence, the patient developed, 16 years later, a left subparotid mass which showed histologic features of Hodgkin's disease, mixed cellularity type. Diagnostic Reed-Sternberg cells and their variants were CD30+, CD15^-- and CD20+. Neither rearrangement of TCR beta and gamma chain genes nor of immunoglobulin heavy chain and kappa light chain genes was detected in DNA extract from fresh material. In situ hybridization showed the presence of EBV within the Reed-Sternberg cells. The data show that EBV was not etiologically related to AIL in this case. Further, the deficit in cellular immunity that accompanied AIL conceivably permit primary EBV infection or reactivation of latent infection, which eventuated in development of Hodgkin's disease, but the exact pathogenesis remains uncertain.     

非霍奇金淋巴瘤EB病毒特异DNA序列的检测

目的　探讨EB病毒 (EBV)与中国南方地区非霍奇金淋巴瘤 (NHL)的相关性 ,以及EBV与不同类型NHL的关系。方法　采用PCR技术 ,检测 2 0 6例石蜡包埋的NHL组织及 2 3例反应性增生的淋巴组织中的EBV特异DNA序列。结果　(1 ) 2 0 6例NHL组织中 ,94例PCR扩增出EBV特异的DNA序列 ,阳性率 4 5 6 % ;对照组反应性增生的淋巴组织 2 3例中 ,5例阳性 ,阳性率 2 1 7% ;两者差异有显著性 (P <0 0 5 )。 (2 ) 2 0 6例NHL中B NHL 1 2 8例 ,EBV阳性者 4 8例 ,阳性率 37 5 % ;T NHL 78例 ,EBV阳性者 4 6例 ,阳性率 5 9 0 %。两者差异有显著性 (P <0 0 5 )。结论　EBV与中国南方地区NHL ,特别是T NHL有一定的相关性     

Budd-Chiari syndrome and Epstein-Barr virus (EBV) associated plasmacytoma in a patient with chronic active EBV infection

A 42-year-old Japanese man with chronic active Epstein-Barr virus (EBV) infection initially responded to treatment with interleukin-2 (IL-2). Six months later he developed thrombosis in the hepatic veins, and Budd-Chiari syndrome associated with severe hepatic damage was diagnosed. He also developed a solitary EBV-positive plasmacytoma in the right femur. Since these rare complications occurred after long-term IL-2 therapy, the possibility that long-term IL-2 therapy might cause Budd-Chiari syndrome and liver damage as well as EBV-associated plasmacytoma is discussed.Abbreviations EBV Epstein-Barr virus - EBER1 EpsteinBarr virus-encoded RNA-1 - IL-2 interleukin-2 - NK natural killer - LMP latent membrane protein - LAK lymphokine activated killer     

Detection of Epstein-Barr virus in lymphoid tissues of patients with infectious mononucleosis by in situ hybridization.

Although the immunological response during infectious mononucleosis (IMN) has been studied in detail, little is known about the spread of Epstein-Barr virus (EBV) in lymphoid organs or the topographical distribution of the infected cells. In this study, EBV was detected in 11 lymph nodes, 4 tonsils, and 1 spleen of 16 patients with IMN. The predominant cell type positive for the EBV genome was identified as small lymphocytes localized chiefly within typical T areas, preferentially in perifollicular and interfollicular regions of the lymph node. A few endothelia of epithelioid venules were also found to be positive. Furthermore, a small number of sinus lining cells of lymph nodes exhibited labelling. Altogether, only a small number of cells, not exceeding 1 per cent of all cells, were infected with EBV. Our results show that only a small number of lymphocytes carry the EBV and that besides B lymphocytes, other cell constituents of lymphatic tissues are infected by EBV during IMN.     

Detection of the Epstein-Barr virus in primary gastric lymphoma by in situ hybridization

The Epstein-Barr virus (EBV) has been shown to be associated with numerous human malignancies including Burktt's lymphoma and nasopharyngeal lymphoepithelioma. In addition, some typical gastric adenocarcinomas were also recently reported to demonstrate EBV relevance. The present study was designed to detect EBV in primary gastric lymphoma, using the in situ hybridization (ISH) method, in which oligo-nucleotide probes for the EBERl RNA and the EBV DNA W region have been used. Of the 49 cases of primary gastric lymphoma studied, which all showed B cell immunopheno-type, EBER1 sequences could only be found in four cases, including two low-grade cases and two high-grade cases of histological subtypes while the number of positive cells was less than 50% of the tumor cells. In one case of low-grade mucosa associated lymphoid tissue (MALT) lymphoma, the EBER1 -positive neoplastic cells were found in the regional lymph node, but the primary site of the stomach showed no positive signals. The EBV presence was further confirmed by the EBV DNA ISH. Using the ISH method, rare or occasional positive lymphoid cells (probably non-tumorous bystander cells) could be detected in 10 other cases including all histological subtypes. The present study shows that only a small proportion of primary gastric lymphoma is associated with EBV, and such positive cases could be found in both high- and low-grade histological subtypes. It is also suggested that the EBV presence in the neoplastic cells of some cases of primary gastric lymphoma is most likely a secondary phenomenon.     

Epstein-Barr virus related gastric cancer in Japan: A molecular patho-epidemiological study

Epstein-Barr virus (EBV) involvement in gastric carcinoma has been demonstrated by the presence of EBV genomes and EBV-encoded small RNA (EBER) in the carcinoma cells, monoclonal proliferation of EBV-infected carcinoma cells and elevated antibody titers. The present study was conducted to investigate the prevalence of EBV involvement among gastric carcinomas observed in nine Japanese cities with varying gastric cancer rates. In situ hybridization of EBER-1 was applied to paraffin sections from 1848 carcinomas observed in 1795 cases and EBV involvement was detected based on uniform hybridization in carcinoma cells. Epstein-Barr virus was detected in 6.6% of lesions and 6.7% of cases. The rate of EBV involvement did not vary significantly for each city and there was no correlation with underlying gastric cancer mortality rates. Thus, geographic variation of gastric cancer rates within Japan cannot be explained in terms of EBV involvement. Epstein-Barr virus-related gastric carcinoma is one of the most common EBV-related tumors in Japan. The involvement of EBV was significantly more frequent among males than among females, mainly for cancers occurring in the upper and middle part of the stomach, and exhibited more variation by cell type among males. These observations suggest that other factors yet to be discovered may modulate the causal role of EBV in gastric carcinogenesis.     

EB病毒与乳腺癌的相关性

目的：探讨EB病毒( Epstein-Barr virus, EBV)与乳腺癌发生、发展的相关性。方法随机选取不同发展阶段的乳腺病变组织246例,采用聚合酶链反应( polymerase chain reaction, PCR)、原位杂交( in situ hybridization, ISH)、激光捕获显微切割( laser capture microdissection, LCM)、免疫组化染色EnVision法检测EBV DNA、RNA、蛋白质水平,分析EBV与乳腺癌发生、发展的关系。结果免疫组化标记246例乳腺病变均未检测到EBV潜伏膜蛋白LMP1的表达。 PCR法在乳腺癌(12/23)、原位癌(0/10)以及乳腺良性病变(0/15)中检测到EBV DNA,但用地高辛标记的EBV DNA探针对48例乳腺良、恶性病变组织(包括PCR扩增阳性的12例乳腺癌标本)进行ISH检测,在癌细胞、乳腺上皮细胞和间质淋巴细胞内均未检测到阳性杂交信号。采用LCM PCR检测12例PCR扩增阳性和12例阴性乳腺标本的乳腺上皮细胞和间质淋巴细胞,在12例PCR阳性标本的间质细胞中扩增出EBV DNA,癌组织中未检出EBV DNA。用EBV RNA探针和ISH方法在75例乳腺良恶性病变组织中均未检测到EBER表达。结论在该文选择的标本中,乳腺癌发生、发展与EBV感染无关。     

Immunopathology associated with Epstein-Barr virus (EBV) infection: Evidence for interactions with T-lymphocyte EBV receptor CD21

Epstein-Barr virus (EBV), also termed Human Herpes Virus 4, is the causative agent of infectious mononucleosis and may be a cofactor in some human cancers. The virus has also been suggested to play a role in human autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. X-linked lymphoproliferative syndrome caused by the deficiency of the Sarc homology 2 domain protein 1A, also termed signaling lymphocyte activation marker-associated protein, can result in immune dysfunction and death after EBV infection. The EBV-related immunopathology in X-linked lymphoproliferative syndrome and prototypical autoimmune syndromes is summarized in this review. A novel model of viral interaction with complement receptor CD21, which is also the receptor for EBV, is proposed to account for both the immunological abnormalities of X-linked lymphoproliferative syndrome and autoimmune diseases associated with EBV infection. The pathogenesis of both X-linked lymphoproliferative syndrome and EBV-associated immune diseases is proposed to result from increased direct infection of T cells by EBV through the T-cell complement receptor CD21 expressed on T cells. A prediction of this model is that therapy designed to decrease CD21-mediated EBV infection of T lymphocytes could also be beneficial in the treatment of some autoimmune diseases.     

Absence of Epstein-Barr virus DNA in the tumor cells of European hepatocellular carcinoma

The Epstein-Barr virus (EBV) has recently been associated with hepatocellular carcinoma (HCC) arising in Japanese patients. We analyzed 82 cases of HCC from Germany and the U.K. for the presence of EBV DNA and viral gene products within tumor cells. Initial screening of whole sections using quantitative (Q)-PCR detected EBV DNA in 9/58 U.K. cases and in 9/24 German cases; in positive cases viral load was very low, ranging between 1.4 and 49.1 copies of the EBV genome/1000 cell equivalents, compared to much higher values for EBV-positive Hodgkin's disease and nasopharyngeal carcinoma controls (range, 714-3259/1000 cells). EBV DNA was not detected in the tumor cells of any of the Q-PCR-positive cases either by Q-PCR of pure tumor cell populations isolated by laser capture microdissection or by isotopic in situ hybridization. Furthermore, none of the German or U.K. HCC tumors tested positive for EBER or EBNAI expression in tumor cells. Our results provide strong evidence that HCCs from the U.K. or Germany are not associated with EBV.     

CD95 (Fas) ligand expression of Epstein-Barr virus (EBV)-infected lymphocytes: a possible mechanism of immune evasion in chronic active EBV infection

The Epstein-Barr virus (EBV) induces infectious mononucleosis (IM) and can be associated with chronic active EBV infection (CAEBV). Cytotoxic T lymphocytes (CTL) play an important role in excluding EBV-infected cells. Two cytotoxic mechanisms of CTL have been demonstrated: one perforin/granzyme-based and the other Fas (CD95)/Fas ligand (FasL)-based. To clarify these two pathways in CAEBV, we analyzed six patients with CAEBV and four patients with IM using immunohistochemical staining of the lymph nodes. In both CAEBV and IM, CD8⁺ T-cells increased in number, but CD56⁺ natural killer cells were rare. In four of six cases with CAEBV, approximately half the lymphocytes were positive for T cell-restricted intracellular antigens (TIA-1), which were recognized by the cytolytic granules of CTL. In IM, the number of TIA-1 positive cells was smaller than that in CAEBV. Fas-positive lymphocytes were frequently encountered in both CAEBV and IM. However, FasL-positive lymphocytes increased in three of six patients with CAEBV, but not in patients with IM. Except for one case with CAEBV, the number of perforin- and/or granzyme-positive cells was small in number in both CAEBV and IM cases. In double-staining FasL and EBV in situ hybridization, FasL-positive EBV-infected lymphocytes were detected in CAEBV but not in IM. In CAEBV, the Fas/FasL pathway and not perforin pathways appears to play an important role in the pathogenesis. The data suggest that EBV-infected lymphocytes may evade immune attack through the expression of FasL.     

Epstein-Barr virus in angioimmunoblastic T-cell lymphomas

Epstein-Barr virus (EBV) has been proposed as a possible infective agent involved in the pathogenesis of angioimmunoblastic lymphadenopathy (AIL), a progressive and often fatal lymphoproliferative disorder. We have studied 19 cases of AIL-like lymphomas for the presence of EBV using a sensitive in situ hybridization technique based on the detection of Epstein-Barr encoded RNAs with digoxigenin-labelled oligonucleotide probes. EBV was found in 11 cases; in seven of these EBV was detected in occasional cells. Immunocytochemical studies to investigate viral gene expression, revealed the presence of EBV-encoded latent membrane protein only in those cases which had appreciable numbers of positive cells by in situ hybridization. The intensity of staining varied from case to case and the overall proportion of cells staining for latent membrane protein in a given case was considerably less than that by in situ hybridization. In situ hybridization for cytomegalovirus and human herpes virus type-6 was negative in all cases. We discuss these findings in the light of the proposed role of EBV in the pathogenesis of AIL and conclude that the presence of EBV is a consequence of the disease rather than the cause.     

Presence of Epstein-Barr virus in Hodgkin''s disease is not exclusive to Reed-Sternberg cells.

Thirty-three cases of Hodgkin's disease (HD) have been studied for the presence of Epstein-Barr virus (EBV) using a novel nonisotopic in situ hybridization procedure, based on the detection of Epstein-Barr encoded RNAs with oligonucleotide probes. An intense and morphologically distinct nuclear staining, sparing the nucleolus was seen in a total of 12 cases (36%). In six of these cases, the signal was located to the Hodgkin and Reed-Sternberg cells (HR-S); in the other six positive cases, the signal was observed only in the non-neoplastic small lymphocytes. These lymphocytes were few in number and immunocytochemistry results were consistent with a B-cell phenotype. The presence of EBV in those cases characterized by nuclear staining of small lymphocytes was confirmed by the polymerase chain reaction (PCR) analysis. The authors report the detection of EBV in small lymphocytes in HD by in situ hybridization and discuss the implications of these findings in relation to the proposed etiologic association between EBV and HD.