Frequent expression of Epstein-Barr virus latent membrane protein-1 in tumour cells of Hodgkin's disease in HIV-positive patients.

Epstein-Barr virus (EBV) is believed to be implicated in the aetiology of non-Hodgkin's lymphomas developing in immunodeficient individuals including AIDS patients. EBV has also been associated with Hodgkin's disease (HD), where the genomes have been demonstrated in the Hodgkin and Reed-Sternberg cells in some of the cases. Recent evidence has shown that EBV genomes are transcribed in these cells, because the EBV-encoded latent membrane protein-1 (LMP-1) can be demonstrated in the tumour cells in about half of the HD cases in HIV-negative patients using immunohistochemistry. LMP-1 is of special interest as a possible oncogenic agent because of its strong transforming capacity in vitro. In this study we have examined the expression of LMP-1 in HD of HIV-positive patients compared with HD in HIV-negative patients. We investigated 18 lymph nodes from 16 HIV-positive patients with HD (eight mixed cellularity, nine nodular sclerosis, one unclassified) using the CS.1-4 anti-LMP-1 monoclonal antibodies, which can usually be applied successfully to archival biopsy material. In each case, 50-90 per cent of the tumour cells were labelled. Staining was excellent for both fixatives used (4 per cent buffered formalin, Bouin's fluid). It is concluded that EBV-encoded LMP-1 is firmly associated with HD of HIV-positive patients. This is most conspicuous in the nodular sclerosing subtype HD in HIV-positive patients, in which 100 per cent were LMP-1 positive as compared with 32 per cent of nodular sclerosis HD in HIV-negative cases in a previously published series. This difference is statistically significant (P < 0.001). The possible biological and clinical significance of this difference should therefore be studied in larger series.     

Oesophageal mesenchymal tumours: clinicopathological features and absence of Epstein-Barr virus

BACKGROUND: Recent studies have suggested that the Epstein-Barr virus (EBV) is associated with smooth muscle tumours (leiomyoma and leiomyosarcoma) in patients with human immunodeficiency virus and in organ transplant recipients. Leiomyoma is the most common mensenchymal tumour found in the oesophagus. AIM: To report a single institution experience on oesophageal mesenchymal tumours and to determine whether EBV is associated with these tumours. METHODS: 40 sporadic oesophageal mesenchymal tumours were studied and their diagnosis confirmed on pathological review and immunohistochemical studies. Formalin fixed, paraffin was embedded tissues from these tumours were analysed for EBV using in situ hybridisation for two messenger RNA (mRNA) probes, EBER and BamH1 W. RESULTS: The oesophageal mesenchymal tumours comprised 36 leiomyomas, two undifferentiated stromal tumours, and two gastrointestinal autonomic nerve tumours (GANTs). Median age of the patients with leiomyoma (26 men, 10 women) was 62 years (range 30 to 85) and 81% of them had an asymptomatic lesion. The median longitudinal size was 1.2 cm. Multiple leiomyomas were seen in 11% of the patients and calcification was noted in one tumour. Coexisting squamous cell carcinoma was found in one third of cases. The stromal tumours were small, asymptomatic, and located in the lower third of the oesophagus, while the GANTs were large, symptomatic, and found in the upper third of the oesophagus. EBV mRNAs were not detected in all these tumours. CONCLUSIONS: The clinicopathological features of oesophageal leiomyoma, undifferentiated stromal tumour, and GANT were different. Some oesophageal leiomyomas were associated with oesophageal squamous cell carcinomas. EBV is not associated with sporadic oesophageal mesenchymal tumours.     

Modulation of interleukin-6 expression in Hodgkin and Reed–Sternberg cells by Epstein–Barr virus

Variable proportions of Hodgkin's disease (HD) cases are associated with the Epstein–Barr virus (EBV), but the role of EBV in HD is not entirely clear. Hodgkin and Reed–Sternberg (HRS) cells of EBV-associated HD are characterized by expression of the EBV gene product LMP1. In other cellular environments, LMP1 has been shown to induce interleukin (IL)-6. In this study, 105 HD cases were tested for differences in IL-6 expression among LMP1-positive and -negative cases. Isotopic in situ hybridization and correlation with the presence of EBV gene products revealed significantly higher proportions of cases with IL-6-expressing tumour cells in LMP1-positive (31 of 37, 84 per cent) as compared with LMP1-negative HD cases (35 of 68, 51 per cent). Thus, although not exclusive to EBV-positive HRS cells, IL-6 expression appears to be upregulated in EBV-associated HD. IL-6 receptor (CD126) expression was tested by in situ hybridization and found in a broad spectrum of cell types, regularly including HRS cells. Superinduction of IL-6 expression may be among the mechanisms by which EBV confers a growth advantage on virus-infected HRS cells and by which the virus may contribute to the morphological and clinical peculiarities of HD. © 1997 John Wiley & Sons, Ltd.     

Overexpression of p53 in Hodgkin's disease: Lack of correlation with Epstein–Barr virus infection

Recent work has shown that p53 gene mutations are frequently found in Epstein-Barr virus (EBV)-positive and EBV-negative cases of Burkitt's lymphoma but not in EBV-associated undifferentiated nasopharyngeal carcinomas (NPCs). Similar viral gene expression patterns are observed in undifferentiated NPCs and in EBV-positive cases of Hodgkin's disease (HD), suggesting that the contribution of the virus to the pathogenesis of these malignancies may also be similar. We have analysed 116 cases of HD for EBV association and for immunohistologically detectable overexpression of p53. p53 overexpression was detected in the tumour cell population of 37 (32 per cent) of the cases. Fifteen cases showed p53-specific labelling of more than 40 per cent of tumour cells; in six of these, virtually all tumour cells were stained. In eight cases, between 5 and 40 per cent of tumour cells were labelled, and in another 14 cases, less than 5 per cent of tumour cells expressed detectable amounts of p53. EBV-positive HD cases were found in all groups with different levels of p53 overexpression as well as amongst p53-negative cases. While a more detailed analysis of the p53 gene in HD is required, these data show that overexpression of p53 in HD is heterogeneous and that there is no simple correlation between EBV infection and p53 overexpression.     

Epstein–Barr virus and carcinogenesis: beyond Burkitt's lymphoma

Subsequent to its discovery over 45 years ago, Epstein–Barr virus (EBV) has been associated with numerous human carcinomas. Approximately 95% of the world's population sustain an asymptomatic life-long EBV infection. EBV persists in the memory B cell pool of normal healthy individuals and any disruption of this interaction results in virus-associated B cell tumours. The association of EBV with epithelial cell tumours, specifically nasopharyngeal carcinoma and EBV-positive gastric carcinoma, is less clear and is currently considered to be a consequence of the aberrant establishment of virus latency in epithelial cells displaying pre-malignant genetic changes. Although the precise role of EBV in the carcinogenic process is currently poorly understood, the presence of the virus in all tumour cells provides opportunities for the development of novel therapeutic and diagnostic approaches. The study of EBV and its role in carcinomas continues to provide insights into the carcinogenic process that are relevant to a broader understanding of tumour pathogenesis and to the development of targeted cancer therapies.     

Epstein-Barr virus in gastric adenocarcinomas: association with ethnicity and CDKN2A promoter methylation

AIMS: It has been shown previously (by immunohistochemistry) that gastric adenocarcinomas harbouring Epstein-Barr virus (EBV) frequently lose p16 protein. This study aimed to examine the mechanisms of inactivation of the CDKN2A gene and correlate the results with clinicopathological features. METHODS: Methylation specific polymerase chain reaction was used to detect CDKN2A promoter methylation in gastric adenocarcinomas from American patients. In addition, immunohistochemistry was used to detect the loss of the p16 protein and in situ hybridisation was used to detect the presence of EBV. The tumours were also analysed for the presence of microsatellite instability. RESULTS: Eleven (10%) of 107 tumours harboured EBV in the malignant cells. In gastric cancers without EBV, 32% exhibited CDKN2A promoter methylation and 26% had p16 protein loss. In contrast, 91% of the tumours containing EBV had CDKN2A promoter methylation (p = 0.0003) and 90% showed p16 protein loss (p = 0.0001). The presence of EBV was also associated with male sex (p = 0.03) and was more common in tumours from Texas Hispanics than from non-Hispanic whites or African-Americans (p = 0.01). EBV was not associated with microsatellite instability, histological subtype, stage, or grade of the tumour, or age or survival time of the patient. CONCLUSIONS: The presence of EBV in gastric adenocarcinomas is strongly associated with CDKN2A inactivation by promoter methylation. In addition, these findings suggest that there are ethnic differences in tumour virology and pathogenesis.     

Epstein Barr virus-associated tumours: an update for the attention of the working pathologist

Epstein-Barr virus (EBV) is a herpesvirus associated with approximately 1% of tumours worldwide. EBV is the epitome of B lymphotropic viruses, but the spectrum of tumours it is associated with extends to T lymphocyte and NK cell malignancies, various types of carcinomas and smooth muscle tumours. Ubiquitous EBV infection in humans implies that most individuals carry EBV-infected cells. Therefore, mere detection of the virus in individuals with a tumour is not sufficient for establishing a causal relationship between both events, but instead requires unequivocal detection of viral nucleic acids or viral proteins in the tumour cells. Recent controversies about EBV infection in several carcinomas mainly resulted from such technical issues. The gold standard remains in situ EBER detection, but detection of EBNA1 would be an interesting alternative. EBV detection can be helpful for diagnostic, prognostic and therapeutic purposes. The rate of EBV association with entities such as NK/T cell tumours of the nasal type is so high that absence of detection of the virus in such a lesion should cast doubt of the accuracy of the diagnosis. Similarly, diagnosis of EBV-associated follicular pseudo-tumour obviously requires detection of the virus. EBV-positive common gastric adenocarcinomas seem to have a better prognosis than their EBV-negative counterparts and identification of the virus in B cell lymphoproliferations in immunocompromised individuals will guide therapeutic options. In conclusion, EBV-associated tumours are common enough to be relevant for the pathologist in everyday practice, but there is a need to facilitate detection of the virus (eg EBNA1 antibody).     

PHENOTYPIC MODULATION OF HODGKIN AND REED–STERNBERG CELLS BY EPSTEIN–BARR VIRUS

Expression of the Epstein–Barr virus (EBV) gene product LMP1 is found in tumour cells in varying proportions of Hodgkin's disease (HD) cases. It is not clear which cellular genes are influenced by EBV in HD. A total of 387 HD cases were tested for differences among LMP1-positive and -negative cases with respect to age, sex, histotype and immunophenotypic parameters (CD2, CD3, CD4, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD43, CD45RA, CD45R0, CD70, HLA-DR, T-cell receptor β-chain, and p53 expression). Comparison of patient age and sex as well as distribution of histotype and tumour cell immunophenotype with published data suggests that the cases in this study are representative of the spectrum of HD in developed countries. LMP1 expression was found in 131/387 HD cases (36·4 per cent) with non-homogeneous distribution among HD histotypes, the mixed cellularity type (HDmc) being most frequently EBV-associated (71/129 cases, 55 percent). No relationship was found to age and sex. Significant phenotypic differences were restricted to the HDmc histotype, where the tumour cells expressed the activation marker CD30 in a larger proportion, and CD20 in a smaller proportion, when harbouring EBV. These results suggest that EBV may influence the tumour cell phenotype in HD.     

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.     

Low grade gastric B-cell lymphoma of mucosa associated lymphoid tissue in immunocompromised patients

An increased incidence of non-Hodgkin's lymphoma is seen in patients with immunodeficiency from any cause. The majority of these are high grade B-cell lymphoma and most are associated with the Epstein-Barr virus (EBV). In post-transplant lymphoma/lymphoproliferative disorders the tumour may regress following reduction of immunosuppression but in AIDS the lymphomas show a characteristic aggressive course and poor prognosis. We describe low grade B-cell gastric lymphoma of mucosa associated lymphoid tissue (MALT) in three immunocompromised patients (two post-transplant, one HIV positive). In each case, the tumour showed classical morphological features of gastric MALT lymphoma and was not associated with EBV. Helicobacter pylori was identified in each case. Clinical follow-up suggests that the behaviour in these tumours is similar to that seen in MALT lymphomas in immunocompetent patients and not typical of the lymphomas usually associated with immunosuppression. Although the finding of MALT lymphoma in immunosuppressed patients might be coincidental, the association of some MALT lymphomas with autoimmune disease suggests that dysregulation of the immune system might play a role in the pathogenesis of these tumours.     

Absence of Epstein—Barr viral RNA in lymphomatoid papulosis

Epstein-Barr virus (EBV) recently has been implicated in the pathogenesis of Hodgkin's disease (HD). Lymphomatoid papulosis (LyP) is a premalignant cutaneous lymphoproliferative disorder which shares several characteristics with HD. The hypothesis has been made that EBV may be associated with the pathogenesis of LyP. We therefore examined 17 skin biopsy specimens and two lymph nodes from nine patients with LyP for EBV RNA using the highly sensitive and specific EBER method. In all specimens, the large atypical cells were negative for EBV while poly T studies confirmed the presence of adequate RNA for detection of EBER. The negative EBER results were confirmed in seven LyP patients whose biopsies were also stained for latent membrane protein (LMP-1). Interestingly, one patient with clonally related LyP and HD had no EBV RNA detected in any specimen. We conclude that EBV is unlikely to be an important aetiological agent in LyP. If confirmed in other patients, HD associated with LyP may have a different aetiology from HD arising de novo.     

Detection of the Epstein-Barr virus in primary adenocarcinoma of the lung with Signet-ring cells

The Epstein-Barr virus (EBV) is directly implicated in the pathogenesis of a variety of undifferentiated carcinomas and has also been identified in conventional adenocarcinomas of the stomach. To date, the association of EBV with non-small cell lung carcinoma is restricted to Asian patients. To evaluate the presence of EBV in lung cancers from Europeans, we investigated primary lung adenocarcinomas with a similar morphological tumour pattern to those of the stomach, specifically rare tumours with components of signet-ring cells. Three tumours of signet-ring cell type were examined by means of polymerase chain reaction (PCR). To localise the virus to the neoplastic cells, in situ hybridisation (ISH) was performed using an antisense Epstein-Barr virus encoded RNA probe. Immunohistochemistry was performed to evaluate the expression of latent membrane protein-1 (LMP-1) and EBV nuclear antigen 2 (EBNA-2). PCR investigation confirmed the presence of EBV in one case. Positive signals confined to tumour cells were present on ISH. None of the tumours showed expression of LMP-1 and EBNA-2. To our knowledge, this is the first report on the presence of EBV in primary adenocarcinoma of the lung in a Caucasian patient. The present study indicates that EBV may infect some lung cancers with a specific tumour pattern.     

Detection of Epstein-Barr virus genome within thymic epithelial tumours in Taiwanese patients by nested PCR,PCR in situ hybridization,and RNA in situ hybridization

Epstein-Barr virus (EBV) is known to be associated with a variety of tumours, including Burkitt's lymphoma, nasopharyngeal carcinoma, and some carcinomas of other organs with similar lymphoepithelioma-like features. The association between EBV and thymic epithelial tumours is inconclusive, as reports in this regard are not entirely consistent and the methods employed are of different sensitivity and specificity. This study examined 78 thymomas and 21 thymic carcinomas in Taiwanese patients, to detect the viral genome at both DNA and RNA levels. The tissue blocks were first screened by nested polymerase chain reaction (PCR) targeting on the first tandem internal repeats. The positive cases were further submitted for viral localization by in situ PCR insitu hybridization (ISH) and Epstein-Barr-encoded RNA-1 (EBER-1) ISH. None of the thymomas showed a detectable EBV genome. Eight thymic carcinomas were positive for EBV by nested PCR, of which six displayed nuclear signals within the tumour cells by in situ PCR ISH and/or RNA ISH, one displayed signals within the lymphocytes, and one showed no discernible in situ signals. Most of them exhibited a lymphoepithelioma-like morphology. These results show that nested PCR is a sensitive method for screening the EBV genome in thymic epithelial tumours. In situ PCR ISH is reliable for localization of the virus, in addition to EBER-1 RNA ISH. Thymomas are not related to EBV, even in this endemic area. Thymic carcinomas, especially the lymphoepithelioma-like thymic carcinomas, are more often associated with the virus.     

Association of Epstein-Barr Virus with Pediatric Hodgkin's Disease

A bimodal age incidence curve has been shown for Hodgkin's disease (HD). In developing countries, the first age incidence peak occurs in childhood; however, this peak is delayed until young adulthood in developed countries. This difference may reflect differences in the age of exposure to infectious agents involved in the development of HD or may suggest different etiological agents. Epstein-Barr virus (EBV) has been implicated in the pathogenesis of a proportion of HD cases. In this study, EBV association was investigated in a series of 55 pediatric HD cases from three geographical locations (United Kingdom, Brazil, and Saudi Arabia) and the relationship between country, age, sex, histological subtype, and EBV positivity was evaluated. EBV was detected in 38 cases using RNA in situ hybridization, Southern blot, or immunohistochemical analysis. No significant difference in EBV positivity by country, age, or sex was observed; however, children under 10 years of age were particularly likely to be EBV-associated. The difference in EBV association in the pediatric group compared with that observed previously for young adult HD was highly statistically significant (P < 0.0001). These results are consistent with the hypothesis that pediatric and young adult HD have different etiologies and suggest that EBV is likely to be involved in the pathogenesis of pediatric HD.     

Epstein-Barr virus infections and Hodgkin's disease: a study of fixed tissues using the polymerase chain reaction.

Epstein-Barr viral (EBV) infections are associated with Hodgkin's disease (HD). To better characterize this relationship, fixed tissues of infectious mononucleosis, normal and reactive lymph nodes, lymph nodes with progressively transformed germinal centers, and biopsy specimens with the different subtypes of HD were analyzed by polymerase chain reaction (PCR). The presence or absence of EBV, the relative amounts of EBV, and the presence of multiple EBV genotypes as defined by amplification of a polymorphic EBV locus were determined for each specimen. Epstein-Barr virus could be detected from all specimens with infectious mononucleosis (eight of eight cases), generally in relatively large amounts, with multiple EBV genotypes evident in two cases. Epstein-Barr virus could not be detected from normal or reactive lymph nodes (none of 39 cases). Small amounts of EBV could be detected from a minority of cases with progressively transformed germinal centers (two of 16 cases), with multiple EBV genotypes evident in one case. Variable amounts of EBV could be detected from approximately half of the specimens with HD (26 of 50 cases). Epstein-Barr virus was most often detected in the subtypes of mixed cellularity (12 of 15 cases), nodular sclerosis (seven of 14 cases), and lymphocyte depletion (five of seven cases) compared with nodular lymphocyte predominance HD (two of 14 cases). In contrast to specimens with infectious mononucleosis and progressively transformed germinal centers, only one EBV genotype was evident in the specimens with HD. These findings are consistent with the hypothesis that some cases of HD may be directly associated with EBV.     

Analysis of single EBER-positive and negative tumour cells in EBV-harbouring B-cell non-Hodgkin lymphomas.

In situ hybridization for Epstein-Barr virus (EBV) encoded small nuclear RNAs (EBERs) is the method of choice for the detection of EBV infection at the single cell level. With the application of this technique it was shown that non-Hodgkin's lymphomas of B-cell type may be associated with an EBV infection of tumour cells. Interestingly, in many EBV-positive cases, only a proportion of the tumour cell population has been found to be EBER-positive. To clarify whether EBV is absent or whether EBER gene expression is downregulated in EBER-negative tumour cells, single EBER-positive and negative tumour cells were isolated from paraffin sections from four B-cell non-Hodgkin's lymphoma cases with partial EBER expression in the neoplastic cells. These single cells were then screened for the presence of EBV DNA by a nested single cell PCR. EBV DNA was undetectable in all but one of the 86 EBER-negative cells, whereas in the EBER-positive cells EBV-specific DNA amplificates could be generated following single cell PCR. This finding prompts the conclusion that the inability to detect EBERs in a proportion of tumour cells is not due to a down-regulation of gene expression but to a real absence of EBV from these cells. This partial absence of EBV is thought to be caused by a loss of the EBV episomes during cell division, rather than by infection of only a proportion of the tumour cells.     

The spectrum of micronodular thymic epithelial tumours with lymphoid B-cell hyperplasia

AIMS: A rare type of thymoma, micronodular thymoma with lymphoid B-cell hyperplasia, was recently reported by Suster and Moran. Thymic epithelial tumours with a similar pattern but with varied cytological features of the tumour cells are analysed. METHODS AND RESULTS: A total of 11 cases of thymic epithelial tumours characterized by micronodular proliferation of tumour cells separated by abundant lymphoid stroma with prominent germinal centres were reviewed clinicopathologically and examined immunohistochemically. The presence of Epstein-Barr virus (EBV) genome was also examined by in-situ hybridization. Based on the morphology of tumour epithelial cells, cases were subdivided into four groups: group 1 (two cases) having spindle epithelial cells; group 2 (two cases) showing an admixture of spindle and polygonal epithelial cells; group 3 (five cases) having polygonal epithelial cells, with mild to moderate cytological atypia in four cases, and group 4 (two cases) representing lymphoepithelioma-like carcinoma. The degree of cytological atypia and the number of tumour cells positive for MIB-1 and p53 gradually increased towards group 4. The abundant lymphoid stroma in all cases contained many CD20-positive B-cells and CD3 and CD45RO-positive T-cells. CD99-positive immature T-cells were present in all cases of groups 1 and 2 and in most cases of group 3, but not in both cases of group 4 tumours. IgG, IgM and IgD-positive plasma cells and lymphocytes were also present in all cases, more prominent in those of groups 3 and 4. The EBV genome was detected in only a few lymphocytes in five cases. CONCLUSIONS: The tumours in this series belong to a distinct category of thymic epithelial tumours and each of the above groups may constitute a spectrum in the continuum of cytological atypia. The aetiological relationship of EBV with these tumours could not be proved. The lymphoid B-cell hyperplasia may result from a host immune response and may suggest a favourable clinical course of this type of tumour.