Induction and progression of human lymphoproliferative lesions by Epstein-Barr virus.

Epstein-Barr virus (EBV) is involved in numerous lymphoproliferative diseases. In addition to classical lesions such as endemic Burkitt's lymphoma and infectious mononucleosis, there are other disorders of the lymphoid system that are discussed in relation to EBV: B-cell lymphomas in immunosuppressed individuals. Hodgkin's disease and, to some extent, primary extranodal lymphomas. Studies of the EBV expression in classical and nonclassical lesions could lead to the better understanding of different EBV mechanisms in lymphomagenesis.


Introduction
Epstein-Barr virus (EBV) is intimately linked with some well-described lesions of the lymphoid system (1). However, the basic mechanisms of EBV-associated lymphomagenesis are not yet fully understood. The study of EBV-induced lymphoproliferative disease (LPD) in immunodeficiency (2) has been very helpful in this context. Recently, EBV involvement in tumors of the lymphoid system other than the classical ones is being discussed again. One of the lesions under consideration is Hodgkin's disease (3); another condition is Tcell lymphoma (4). Further, the involvement of EBV in extranodal lymphomas of various origins is suggested (5). In this report we discuss the distribution of EBV in lymphoid lesions. In addition, morphological evidence of different EBV expression in reactive and tumorous lesions is presented.

Materials and Methods
Five hundred thirty-nine cases with an eventual association with EBV were collected from the ifies of the Institute of Pathology Wurzburg or were a gift from various other colleagues to whom we are greatly indebted. Most of the cases were lymphomas and LPD. Five nasopharyngeal carcinomas and six cases of infectious mononucleosis (IM) were included for control purposes. African lymphomas and lymph nodes came from Uganda, Rwanda, and Tanzania. All other lesions were from Central Europe. The results presented were obtained with lymphomas in AIDS, LPD in inborn immunodeficiencies in children, and extranodal lymphomas.
All lesions were studied morphologically and immunohistochemically. They were investigated for the presence of EBV DNA by hybridization (dot or Southern blot) with whole cellular DNA obtained from fresh or paraffin material and appropriate EBV probes. In parallel, tissue sections were used for in situ hybridization (ISH) to localize EBV DNA in the tissue. Molecular biology studies made use of standard techniques (6). DNA extraction from paraffin-embedded material was carried out as described by Goelz et al. (7), ISH with 35S-labeled probes and consecutive autoradiographic development for 1 to 5 weeks. Controls were as follows: Raji cell DNA served as positive controls and IM and nasopharyngeal carcinoma as positive cases. Negative controls included unrelated lymphoid tissues (42 lymphadenitis) and hybridization with a cytomegalovirus probe and omission of the specific probe.

Results
Lymphomas in AIDS, African Burkitt's Lymphomas, and LPD in Inborn Immunodeficiency Histological classification and the main clinical data are shown in Table 1. The LPD in children with inborn immunodeficiencies were diagnosed according to Frizzera's proposition (8). Lymphomas were evaluated using the Kiel classification. The results obtained using ISH in all the cases already positive in blotting assays are listed in Table 2. In ISH studies two different patterns were found. In IM, the EBV-positive cells are distributed throughout the tissue section. Approximately 10 to 20% of the cells are labeled. These EBVpositive cells are often gathered around areas of necrosis. Still, the majority of the paracortical lymphoid tissue in IM cases remains unstained (Fig. 1).
The second pattern is found in classical endemic Burkitt's lymphomas. All tumor cells are labeled in a homogeneous way. Each cell shows about the same number of silver grains. Starry sky macrophages, adjacent structures, and vessels are not labeled and serve as an endogeneous negative control (Fig. 2). Of the EBVpositive AIDS-associated lymphomas, four have the homogeneous distribution pattern of EBV; in the three remaining AIDS lymphomas the EBV distribution resembles IM in that only some tumor cells are labeled intensively and that these cells are scattered. But also in the four lymphomas with a homogeneous EBV positivity there are some areas with a more scattered EBV distribution.
Four children with inborn immunodeficiencies and EBV-positive LPD had either polymorphic diffuse Bcell hyperplasia (PDBH) or polymorphic B-cell lymphoma (PBL). In both the PDBH or the PBL, the EBV pattern was heterogeneous throughout all the tissues investigated in each case. There were no differences between lymphoma and lymphoid hyperplasia. One PBL was monoclonal, the other polyclonal by immunophenotyping. All endemic African Burkitt's lymphomas had the homogeneous pattern. These lymphomas were from malaria-free regions (Rwanda) as well as from regions with endemic malaria.

Primary Extranodal Lymphomas
From our sample of 539 cases, 37 primary lymphomas of the stomach and 7 primary lymphomas of the salivary glands were selected. Fourteen primary gastric lymphomas were of the mucosa-associated lymphoid tissue (MALT) type (9), 18 were high-grade non-Hodgkin lymphomas (NHL). A summary of investigations done and the results obtained is given in Table 3.
Gastric lymphomas were considered EBV positive when two assays (ISH and blotting) were positive. Briefly, there are hints that EBV may be present in some distinct extranodal NHL. This was found in a case with a high-grade NHL and proliferation with Hodgkinlike cells in the adjacent tissue (case 30003/86). In the surrounding of the lymphoma, ISH revealed few EBV-positive lymphoid cells. The nature of these cells remains to be determined. The seven lymphomas of salivary glands (two high-grade and five low-grade NHL) were negative for EBV.

T-Cell Lymphomas
In our material three cases of T-cell lymphomas were EBV positive. One case was a T-immunoblastic lymphoma occurring in AIDS (Table 1). A second case was from a young African woman who developed a rapidly growing anaplastic T-zone lymphoma. Finally, another T-immunoblastic lymphoma in a immunocompetent patient showed the presence of EBV-genome.

Discussion
In addition to classical EBV-associated lesions [Burkitt's lymphoma (BL), IM, nasopharyngeal carcinoma] other diseases of the lymphoid system seem to be related to EBV. Recently, the association of EBV with T-cell lymphomas in patients with chronic EBV infections (4) has been reported. We found EBV in two NHL of the T-phenotype where a chronic EBV infection has not been documented but could be supposed, one patient having an acquired immunodeficiency and the other patient coming from central Africa, a region with numerous environmental factors influencing the immune system. These findings suggest that T-cell tumors possess EBV DNA, which implies that T-cells support EBVreplication (10).
On the other hand, we presented some evidence for an involvement of EBV in the development of extranodal lymphomas. Primary extranodal lymphomas of the gastrointestinal tract have a histologically distinct     (9) that has been called malignant lymphoma of mucosa-associated lymphoid tissue (MALT) because the accepted classifications of NHL were difficult to apply to these tumors. The main component of these lymphomas is the centrocytelike cell (CCL), ofwhich the benign equivalent is characterized by a surface phenotype IgM+, IgA+, CD 22+, CD 23-, CD 5-, and they are positive for both CD 35 (CR1) and CD 21 (CR2), the Blymphocyte EBV receptor (11). We are actually investigating different forms of chronic gastritis with a preponderance of lymphoid cells for the presence of EBV or viral proteins. Up to now, we have found one case of EBV-positive gastric lymphoma, and so there may be a link between MALT lymphocytes, EBV, and the development of certain lymphomas. The fact that we have only demonstrated EBV in one case of gastric lymphoma could also be a question of sensitivity. Recently, Saito et al. (12) clearly demonstrated EBV DNA in tissue biopsies from patients with Sjogren's syndrome, a condition associated with the development of lymphoid tissue in salivary glands and increased frequency of NHL in these patients using the polymerase chain reaction. Using a more sensitive method, the frequency of EBV in primary extranodal lymphoma and MALT tissue could be higher than is found with normal blotting techniques. In lymphomas and LPD of children with immunodeficiencies, as well as in lymphomas of patients with AIDS, we demonstrated two different patterns: one pattern could be called the IM-like pattern and the other one the BL-like pattern. The interpretation of these findings recalls the findings of studies with BL and lymphoblastoid cell lines (LCL) cell lines. LCL are derived from the peripheral blood ofnormal seropositive donors. LCLs differ from BL lines in a number of phenotypic characteristics. The LCLs represent the most likely counterpart of the EBV-transformed blasts that proliferate in IM (13). BL cells may represent the neoplastic counterpart of latently infected normal B-cells with a corresponding phenotype. The differences consist mainly in viral gene expression and B-cell marker expression. BL cells only express EBNA 1 and CALLA and BLA, whereas LCLs express the full set of EBNA 1-6 and LMP as well as-B activation markers (14). Our finding of two different EBV genome patterns may reflect these two EBV-canrying cell types: the more homogeneous expression of EBV would then be related to the BL phenotype; the scattered distribution occurs when LCL-like cells are the predominant cell in the lesion. This hypothesis could be tested by extensive studies on viral gene expression and marker studies.
The acute phase of IM is comparable to a chaotic attack of specific and nonspecific killer cells against a multitude of rapidly multiplying EBV-carrying B-blasts (14). Our observation of an IM-like pattern in PBL and PDBH may be a further indication that the disease pro-cess in these cases is an overwhelming reaction to EBV rather than an overwhelming infection (15).
The fact that EBV-positive AIDS lymphomas have either IM-like or BL-like pattern indicates that there may be two different groups of EBV lymphomas in AIDS. It would be of interest to compare the clinical outcome of the two lymphoma groups with diverging EBV patterns.