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.     

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.     

Expression of c-myc and bcl-2 oncogene products in Reed-Sternberg cells independent of presence of Epstein-Barr virus.

AIMS: To evaluate the expression of c-myc and bcl-2 oncogene products in Reed-Sternberg cells in Hodgkin's disease, especially in relation to Epstein-Barr virus infection and expression of EBV encoded latent membrane protein (LMP). METHODS: Tissues from 33 cases of Hodgkin's disease were studied for the presence of EBV DNA by polymerase chain reaction (PCR) and DNA in situ hybridisation (DISH), for the presence of EBER-1 and EBER-2 EBV RNA by RNA in situ hybridisation (RISH); and for the presence of LMP, bcl-2, and c-myc proteins by immunohistochemical staining. RESULTS: A substantial number of Reed-Sternberg cells expressed bcl-2 in 20 of 29 (69%) and c-myc in 30 of 32 (94%) Hodgkin's disease samples. In 18 of the 25 (72%) cases Reed-Sternberg cells expressed both oncogene products. Of these 18 cases, 10 (56%) were EBV-PCR positive; eight (44%) were EBV-PCR negative. CONCLUSIONS: Reed-Sternberg cells in Hodgkin's disease frequently express both bcl-2 and c-myc oncogene products, suggesting that these oncogenes may act in concert in the pathogenesis of the disease. Moreover, the expression of c-myc and bcl-2 proteins in Reed-Sternberg cells is independent of EBV and LMP status.     

Rare expression of T-cell markers in classical Hodgkin's lymphoma.

Hodgkin's and Reed-Sternberg cells of classical Hodgkin's lymphoma are primarily of B-cell origin, although there are instances of T-cell antigen expression suggesting T-cell origin. We comprehensively analyzed expression of various T-cell antigens in 259 classical Hodgkin's lymphoma cases using the tissue microarray technique. Expression of the T-cell antigens CD2, CD3, CD4, CD5, CD7 and CD8 was assessed by immunohistochemistry. Hodgkin's and Reed-Sternberg cells of T-cell marker-positive cases were microdissected and analyzed by a multiplex polymerase chain reaction for clonal immunoglobulin heavy chain- and T-cell receptor gamma gene rearrangements. In all, 12 cases (5%) expressed at least one T-cell marker in the following order: CD2 in 11 cases, CD4 in five, CD3 in two, and CD5 and CD8 in one case each; there were no CD7-positive cases, and five cases (2%) expressed more than one T-cell antigen. In positive cases, a mean fraction of 40% of the Hodgkin's and Reed-Sternberg cells (range 20-100%) expressed the analyzed T-cell markers. Two cases (<1%) evidenced clonal T-cell receptor gamma gene rearrangement. Phenotypic expression of T-cell antigens in Hodgkin's and Reed-Sternberg cells of classical Hodgkin's lymphoma is rare (5%), while genotypically, less than 1% of classical Hodgkin's lymphomas are of possible T-cell origin. Therefore, T-cell antigen expression on Hodgkin's and Reed-Sternberg cells is aberrant in the majority of cases and only infrequently classical Hodgkin's lymphomas are of T-cell origin.     

Epstein-Barr viral DNA in tissues of Hodgkin's disease

Tissue specimens from 21 cases of Hodgkin's disease were examined for the presence of Epstein-Barr virus (EBV) and cytomegalovirus DNA by molecular hybridization techniques. EBV DNA was detected in 4 cases, including 2 of 8 cases which had been previously shown to contain clonal immunoglobulin gene rearrangements. Two of the cases containing EBV DNA were of the nodular sclerosing type and had received prior therapy; the other 2 were classified as mixed cellularity Hodgkin's disease and had not received therapy before the biopsy tissue was obtained. Analyses of the terminal portions of EBV genomes indicated a monoclonal or oligoclonal proliferation of EBV-infected cells in the tissues studied. In contrast, none of the 21 cases had detectable cytomegalovirus DNA sequences. The identification of EBV DNA may reflect the proliferation of lymphoblastoid cells due to the reduced immune competence frequently noted in Hodgkin's disease or may indicate the presence of EBV genomes within Reed-Sternberg cells.     

CD40 expression in Hodgkin's disease.

CD40 is a transmembrane protein that belongs to a superfamily of proteins related to nerve growth factor receptor. CD40 is expressed on B cells and some B cell malignancies. It appears to be involved in B cell proliferation and the prevention of apoptosis in germinal center cells, which is accompanied by expression of bcl-2. Its expression is up-regulated by the EBV protein latent membrane protein-1 and cytokines interleukin-4 and interferon-gamma. The expression of CD40 in 37 cases of Hodgkin's disease and 23 cases of non-Hodgkin's lymphoma (11 T cell lymphomas and 12 B cell lymphomas) was examined by paraffin section immunohistochemistry using the BB-20 monoclonal antibody. In 26 of 37 cases of Hodgkin's disease the Reed-Sternberg cells showed strong membrane or cytoplasmic expression of CD40. Only 3 of 23 non-Hodgkin's lymphomas showed any expression of CD40 and then only weakly. There was no correlation between expression of bcl-2 or latent membrane protein-1 with CD40 expression. These results show that there is probable hyperexpression of CD40 in Hodgkin's disease and suggest that dysregulation of CD40 expression may play a role in the pathogenesis of Hodgkin's disease.     

B细胞淋巴瘤与EB病毒关系的观察

为了了解我国非免疫缺陷相关B淋巴瘤是否与EBV有关，我们采用EBVencodedsmallRNA（EBER－1）原位杂交对127例非免疫缺陷相关B淋巴瘤进行了研究。结果显示，其中8例瘤细胞核内有EBER－1的表达（中心母细胞型4例；淋巴浆细胞样型、浆细胞型、免疫母细胞型和不能分型的高度恶性B细胞淋巴瘤各1例），检出率为6．3％。这一结果与欧美的情况一致（5％左右）．但明显低于我国何杰金病（82％）及T淋巴细胞（62％）的检出率，因此提示EBV在非免疫缺陷B淋巴瘤发病中的作用是有限的，主要致病因素还有待进一步研究。     

Immunohistochemical demonstration of the Epstein-Barr virus-encoded latent membrane protein in paraffin sections of Hodgkin's disease.

Paraffin sections from 46 cases of Hodgkin's disease were examined for the presence of the Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP) using a sensitive (double layer alkaline phosphatase-anti-alkaline phosphatase) immunohistochemical method. LMP was detected in 22 cases, the majority of positive cases being of nodular sclerosis (12/24), mixed cellularity (6/7), and lymphocyte depletion (3/3) subtypes. Only one of 12 cases of lymphocyte predominant disease was positive. In all cases, reactivity was confined to Hodgkin's and Reed-Sternberg cells. These results provide further evidence for an association between EBV and Hodgkin's disease and indicate that LMP may be readily detected in archival material.     

CD40 ligand is constitutively expressed in a subset of T cell lymphomas and on the microenvironmental reactive T cells of follicular lymphomas and Hodgkin's disease.

Although CD40 has been extensively studied in B- and T-cell non-Hodgkin's lymphomas (NHLs)/leukemias, and more recently in Hodgkin's disease (HD), little is known about the expression of its ligand (CD40L) in lymphoproliferative disorders other than T-cell NHLs/leukemias. A series of 121 lymphoma/leukemia samples, including 35 cases of HD, 34 T-cell and 39 B-cells NHLs, 2 cases of adult T-cell leukemia/lymphoma, and 11 cases of T-cell acute lymphoblastic leukemia, were evaluated for CD40L expression by immunostaining of frozen tissue sections and flow cytometry with the anti-CD40L monoclonal antibody M90. CD40L was constitutively expressed by neoplastic cells in 15 of 36 (42%) T-cell NHLs/adult T-cell leukemia/lymphomas, almost invariably those displaying the CD4+/CD8- phenotype, whereas no CD40L-expressing tumor cells could be found in B-cell NHL and HD. Among T-cell acute lymphoblastic leukemias, CD40L was detected only on 2 cases displaying a stem-cell-like phenotype. In follicular B-cell lymphomas a large number of CD40L-expressing CD3+/CD4+ T lymphocytes were found admixed with tumor cells within the neoplastic follicles and in their surrounding areas. In the nonfollicular B-cell lymphomas, CD40L-positive CD3+/CD4+ T lymphocytes were few or absent. In all HD subtypes other than the nodular lymphocytic predominance, CD40L-expressing CD3+/CD4+ T lymphocytes were numerous in the HD-involved areas and were mainly located in close proximity to the Reed-Sternberg cells. Our data indicate that in human lymphomas CD40L is preferentially expressed by a restricted subset of T-cell lymphomas, mostly with CD4 immunophenotype. Finally, we have provided morphological evidence that CD40L may play an important role in the cell contact-dependent interaction of tumor B-cells (CD40+) within the neoplastic follicles or Reed-Sternberg cells (CD40+) in HD-involved areas and the microenvironmental CD3+/CD4+/CD40L+ T lymphocytes.     

Co-expression of Epstein-Barr virus latent membrane protein and vimentin in “aggressive” histological subtypes of Hodgkin's disease

Summary The presence of Epstein-Barr virus (EBV) genome in Hodgkin's and Reed-Sternberg (HRS) cells, as detected using in situ hybridization (ISH) with biotinylatedBamHI V probes, along with the expression of EBV-encoded latent membrane protein (LMP) and vimentin was examined in paraffin-embedded sections of 39 immunomorphologically characterized cases of Hodgkin's disease (HD). ISH demonstrated EBV in HRS cells in 15 of 39 cases, whereas LMP expression was detected in 11 of 39 cases, only in the presence of EBV genome detection. With the exception of 1 case, in which HRS cells expressed B-cell-associated antigens, the LMP-positive cases included specimens in which HRS cells were of non-B, non-T phenotype. LMP expression showed a stronger association with lymphocyte depletion (LD) (3/3) and mixed cellularity (MC) (6/11) than with lymphocyte predominance (0/5) or nodular sclerosis (2/20) subtypes. Vimentin expression on HRS cells was found in all the LMP-expressing cases and only in a fraction (13/28) of LMP-negative cases. This study supports the view that HD represents a heterogeneous group of diseases also in terms of EBV association, LMP expression being strongly related to the aggressive LD and MC histological subtypes. In light of the supposed interactions between vimentin and LMP, their coexpression on HRS cells, as detected in this study, provides further evidence for a significant role of EBV in the development of a proportion of HD cases.     

Relation of follicular dendritic reticulum cells to Reed-Sternberg cells of Hodgkin's disease with emphasis on the expression of CD21 antigen.

Based on observations of 66 cases, in which tissues were specially processed to optimize the simultaneous preservation of cell membrane antigens and morphology, we provide evidence in favor of a relationship between follicular dendritic reticulum cells (FDRC) and Reed-Sternberg (RS) cells of Hodgkin's disease (HD) other than the lymphocyte predominance subtype. RS cells were intimately related to the FDRC network (75% of cases), and the expression of CD21 antigen was frequent (41% of cases). Exclusive expression of CD21 antigen was found in 11 cases of HD, while the expression of other B-cell-associated markers (CD19, CD20, CD22) was both variable and inconsistent. The expression of T-cell antigens (CD3, CD4, CD8) was rare. Null phenotype of RS cells was observed in 27 of 66 cases (41%). Epstein-Barr virus (EBV) nucleic acids were found in 34 of 66 (51.5%) cases. Double labeling techniques showed the presence of EBV-positive RS cells within the FDRC network. A non-B-cell origin of RS cells was supported by the differential expression of EBV latent antigens in HD (latent membrane protein+, EB nuclear antigen 2-), which is unusual in EBV-driven lymphoblastoid cell lines and EBV-positive B-cell lymphomas. FDRC and RS cells are known to share morphological traits (binucleated cells), and both cell types possess Fc receptor for IgG. The hypothesis is further backed by the findings of CD15 antigen expression by occasional RS-like dysplastic FDRC in Castleman's disease (five cases), which is characterized by hyperplasia of FDRC. Whether FDRC might be the only cells involved in the conversion to RS cells by the loss or gain of antigens remains to be determined.     

High prevalence of Epstein-Barr virus in the Reed-Sternberg cells of HIV-associated Hodgkin's disease.

The Epstein-Barr virus (EBV) has been implicated in the pathogenesis of Hodgkin's disease, with an frequency of 15 to 50% in the immunocompetent host. We studied 12 formalin-fixed, paraffin-embedded cases of Hodgkin's disease occurring in human immunodeficiency virus-infected individuals to determine the frequency of EBV in Hodgkin's disease from this population. EBV DNA-RNA in situ hybridization was performed using a 30-base biotinylated anti-sense oligonucleotide complementary to the EBER1 gene of EBV. EBV RNA was found in the Reed-Sternberg cells and variants in 11 of 12 cases. Double-labeling studies confirmed the presence of EBV RNA in CD15-expressing Hodgkin's cells in all 11 cases, although rare B lymphocytes coexpressing EBV RNA and CD20 were also noted in these cases. The Hodgkin's cells in all 11 EBER-positive cases expressed latent membrane protein. The one case negative for EBV RNA showed the histology of nodular, lymphocyte predominance, a subtype thought to be distinct from other types of Hodgkin's disease.     

Epstein-Barr virus latent and replicative gene expression in oral hairy leukoplakia

Oral hairy leukoplakia is an epithelial lesion of the tongue associated with productive infection by Epstein-Barr virus (EBV). However, no data concerning the pattern of EBV latent gene expression have been reported, and it remains unresolved whether true latent infection occurs in basal cell layers of oral hairy leukoplakia. We have studied six cases of oral hairy leukoplakia using monoclonal antibody immunohistology for EBV latent--EB nuclear antigen (EBNA) 1, EBNA 2 and latent membrane protein 1 (LMP 1); immediate-early (BZLF1); and replicative (EA, VCA, MA) proteins, and for the EBV-receptor (CD21 antigen). EBV DNA was demonstrated by nucleic acid in situ hybridization. Mid- to upper-zone keratinocytes contained EBV DNA and co-expressed EBNA 1, EBNA 2 (5 of 6 cases), LMP 1, BZLF1 protein, EA, VCA and MA. No EBV genome or gene expression could be demonstrated in basal or parabasal cells. Spinous keratinocytes were labelled by anti-CD21 antibodies HB5 and B2, but did not express the EBV-receptor as defined by reactivity with OKB7. The co-expression of latent and replicative infection-associated antigens is striking, indicating possible functional roles for latent proteins during the productive cycle. Our results suggest that oral hairy leukoplakia is caused by repeated direct infection of upper epithelial cells with virus from saliva or adjacent replicatively infected cells, rather than by a latent EBV infection of basal epithelial cells with a differentiation-dependent switch to productive infection as previously proposed.     

Primary natural killer/T-cell lymphomas of the oral cavity are aggressive neoplasms

Thirty-four cases of primary non-Hodgkin’s lymphoma of the oral cavity were investigated for their clinical findings, histopathological features, immunophenotypes and association with Epstein-Barr virus (EBV). Four cases (12%) were natural killer/T-cell lymphomas, 3 (9%) were T-cell lymphomas and 27 (79%) were B-cell lymphomas. Compared with T- and B-cell lymphomas, NK/T-cell lymphomas had a male predominance (M:F 4:0), and most presented as ulceration of the palate and/or maxillary gingiva. Histologically, the lesions showed diffuse infiltration of medium-sized or large lymphoid tumour cells. Angiocentricity and/or angioinvasion were found in all 4 cases. The immunophenotypes of the NK/T-cell lymphomas were CD3+, CD43+, CD45RO+, CD56+ and TIA-1+. EBV was detected in 2 NK/T-cell lymphomas by in situ hybridization (ISH) and polymerase chain reaction (PCR) methods, and was not detected in T- and B-cell lymphomas. The survival rate of patients with NK/T-cell lymphoma was zero, but the survival rates for patients with T-cell and B-cell lymphomas were 67% and 38%, respectively. It appears that NK/T-cell lymphomas of the oral cavity have a predilection for originating in the palate and maxillary gingiva and are aggressive neoplasms. EBV positivity might be associated with more aggressive behaviour. Received: 21 January 1999 / Accepted: 14 April 1999     

Expression of B7 (CD80) and CD40 antigens and the CD40 ligand in Hodgkin's disease is independent of latent Epstein-Barr virus infection

Aim-To examine the expression of CD40 and B7 (CD80) antigens and the CD40 ligand in Hodgkin's disease.Methods-Antigen and ligand expression was studied in 17 cases of Hodgkin's disease using immunohistochemistry. The study included 11 cases of Hodgkin's disease in which latent Epstein-Barr virus (EBV) infection could be demonstrated within tumour cells by in situ hybridisation for the EBV encoded early RNAs (EBERs).Results-In all cases, irrespective of EBV status, Reed-Sternberg cells and their variants (HRS cells) showed strong expression of both B7 and CD40 antigens. CD40 ligand expression was not shown in HRS cells but was confined to a subset of small lymphocytes some of which were seen to be in intimate contact with HRS cells.Paraffin wax sections from a further 60 cases of Hodgkin's disease were examined for CD40 and EBER expression alone. The CD40 antigen was identified in HRS cells in all of these cases irrespective of EBER expression.Conclusions-As CD40 and B7 expression are features of professional antigen presenting cells, these results provide further evidence that HRS cells may have antigen presenting properties and that this may contribute to the characteristic recruitment and activation of non-malignant lymphocytes which is a feature of Hodgkin's disease. The ability of HRS cells to activate T(h) cells may in turn contribute to their own survival through the induction of the gp39/CD40 pathway.     

Epstein-Barr virus and Hodgkin''s disease. A correlative in situ hybridization and polymerase chain reaction study.

The authors studied typical Hodgkin's disease along with the nodular, lymphocyte predominance subtype by both the polymerase chain reaction (PCR) and in situ hybridization for evidence of the Epstein-Barr virus (EBV). By PCR, EBV DNA was detected in 12/23 cases of typical Hodgkin's disease and 2/13 cases of the nodular, lymphocyte predominance subtype. EBV RNA was detected by in situ hybridization studies within Reed-Sternberg cells and variants in 11/23 cases of typical Hodgkin's disease and 0/13 cases of nodular, lymphocyte predominance Hodgkin's disease. Other cells positive for EBV, identified as both B and T cells in double-labeling immunohistochemical/in situ hybridization studies, were found in 20/23 cases of typical Hodgkin's disease, 9/13 cases of nodular, lymphocyte predominance Hodgkin's disease, 4/6 cases of progressive transformation of germinal centers, and 7/10 normal lymphoid tissues. It is concluded that EBV is significantly associated with Reed-Sternberg cells in approximately one-half cases of typical Hodgkin's disease but not in the nodular, lymphocyte predominance subtype. EBV-infected B and T cells are also present in a majority of cases of Hodgkin's disease as well as in reactive conditions.