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Is Hodgkin's disease an infectious disease?
Annals of Oncology 5 (Suppl. I): S105-S111, 1994. © 1994 Kluwer Academic Publishers. Printed in the Netherlands.
Original article Is Hodgkin's disease an infectious disease? J. Wolf & V. Diehl Department of Internal Medicine I, University of Cologne, Cologne, Germany
Summary
Introduction
Key words: atypical immune response, Epstein-Barr virus, Hodgkin's disease
cells. The recently described association of HD with a human DNA tumor virus (EBV) might be persuasive enough to wind up the debate on the true nature of this disease.
Since the first description 'on some morbid appearances of the absorbent glands and spleen' by Thomas Hodgkin (1832) [1], the concept of Hodgkin's disease as a true neoplastic disorder of the lymphatic system has repeatedly been questioned. Wilks presented a col- Hodgkin's disease — true neoplastic disorder or atypilection of 15 cases of HD in 1865 [2] and was con- cal immune response? vinced that 'the disease of Hodgkin is clearly separable from lardaceous disease, from cancer and tubercle, al- Clinically, HD presents with pronounced signs of an inthough these affections may bear a relation to one an- flammatory process - fluctuating fever, chills, nightother.' Dorothy Reed, one of the first pathologists de- sweats, monocytoses, lymphopenia, and elevated levels scribing thoroughly the histopathology of the Hodgkin of soluble IL-2 receptor in the patient's serum [5]. lesion, stated in 1902: 'Histologically, it might be Histologically, in remarkable contrast to malignant thought that the changes resemble those of a malignant disorders, there is no homogenous malignant cell growth, but we believe that closer study will show a population which represents the majority of cells in the greater similiarity to inflammatory processes' [3]. Ac- tumor tissue. Rather, a heterogenous mixture of lymcordingly, a search for infectious agents, including bac- phocytes, histiocytes, eosinophils, plasma cells, fibroteria, fungus, and viruses paralleled Hodgkin's re- blasts, and others is found in affected lymphatic tissue. search, but was unsuccesful until 1987, when Weiss and Among them, the H/RS cells which are believed to be coworkers were the first to detect Epstein-Barr virus the malignant substrate of HD, represent, at least in DNAinHDtissue[4]. initial stages, only a minority of 0.1%-l% [6]. The situation becomes even more puzzling considIndependent of scientific controversies on the pathogenesis of Hodgkin's disease, the concept of HD ering the results of early cell kinetic studies. In vitro as a true neoplasia of the lymphatic system has pre- and in vivo labeling with tritiated thymidine in vailed in the clinical setting: Anticancer treatment strat- Hodgkin's tissue revealed the presence of two major egies like radiation therapy alone or in combination proliferating-cell populations in HD: cells identified as with polychemotherapy led to cure rates of about two- transformed lymphocytes with a high labeling index and Hodgkin cells with a lower labeling index [7]. thirds of all patients suffering from HD. This impressive success in treatment is still counter- Reed-Sternberg cells were found to incorporate only faced by a major lack of understanding of the patho- rarely tritiated thymidine in these studies. Therefore, genetic events leading to Hodgkin's disease. No con- the 'normal' lymphocytes, not the 'malignant' Hodgkin vincing model exists either to define the cell of origin of or RS cells, represent the majority of dividing cells in HD or to explain the interaction between the putative HD tumor masses. malignant H/RS cells and the surrounding bystander Despite the high labeling index of the lymphocytes
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Several lines of evidence question the concept of Hodgkin's disease (HD) starting as a true malignant disorder of the lymphatic system: (i) In early stages HD exerts pronounced clinical and biological features of an atypic immune response, (ii) Despite extensive investigations, the Hodgkin/ReedSternberg (H/RS) cells have not been unequivocally determined as the definite malignant cell population in HD. (iii) The epidemiological pattern of HD strongly resembles that of an infectious disease, (iv) About 50% of HD cases can be
linked to infection with Epstein-Barr virus. Hodgkin's disease in early stages might thus be understood as the unsuccessful attempt of the organism to eliminate a cell expressing a putative (cellular or viral) target antigen. A stepwise transformation of this antigen-carrying cell, possibly triggered by an inherent genetic instability, might then lead to outgrowth of a malignant cell clone in late stages of the disease.
106
Hodgkin/Reed-Sternberg cells — the malignant substrate of Hodgkin's disease?
The concept of Hodgkin's disease being a true neoplastic disorder is mainly based on the presence of a putative malignant cell population, i.e., the mononucleated Hodgkin cells and their bi- or polynucleated derivatives, the Reed-Sternberg cells. These cells are consistently found in Hodgkin's disease. They have also been described, however, in nonmalignant diseases like infectious mononucleosis, measles, rubeola, or anticonvulsant-induced lymphadenopathy [12]. So far, the origin of H/RS cells has not been determined, although any of the hematopoietic cell types has been proposed as cell of origin in Hodgkin's disease [13]. Lymphocyte-predominant HD seems to be an
exception, since in this histological subtype the H/RS cells express a B-cell phenotype as a rule [14]. Immunophenotyping of H/RS cells in the other subtypes of HD, as well as on the few established cell lines, has revealed a more complex pattern. Some antigens are consistently expressed, the activation markers Ki-1 (CD30) and the interleukin-2 receptor (CD25), among others. Both are also expressed on activated lymphocytes or monocytes. In contrast, no homogenous pattern is found for lineage-specific antigen expression: the H/RS cells may express B- or T-cell-specific surface antigens or none of them (0-phenotype) [13]. In analogy, the HD-derived cell lines show either (complete or incomplete) Ig or TCR rearrangements, which might point to an immature lymphoid origin of these cells [15]. In Hodgkin's lymph nodes, clonal Ig or TCR rearrangements have also been described, although the interpretation of these studies is complicated by the scarcity of tumor cells in the biopsy specimens as discussed below. Similarly, no consistent deregulation of a cellular oncogene or inactivation of a suppressor gene could be detected [16]. What are the criteria for considering this heterogenous cell population as the malignant cell population in Hodgkin's disease? Fundamental attributes of neoplastic cells are aneuploidy and clonal origin. Cytogenetic studies in HD are difficult to perform and to interpret owing to the scarcity of the H/RS cells, the low number of obtainable mitoses, and their poor chromosome-banding qualities. Also, many of the mitotic figures in short-term cultures of HD lymph nodes derive from the lymphoid cells of the reactive stromal tissue (bystander cells) [17]. In a recently published review, only 40 well-documented cases of complete karyotype-banding studies in HD were found [18]. In these studies, the proportion of karyotype abnormalities varied from 22% to 83%, including a broad spectrum of numerical and structural abnormalities. No specific chromosomal marker, like, for instance, the translocation (9/22) in chronic myelogenous leukemia or (8/14) in Burkitt's lymphoma was detected. Similarly, in a recent published study [19], 60 lymph nodes from untreated HD patients were analyzed: In 82% of the cases analyzable metaphases were obtained; in 55% chromosomal abnormalities were detected. Most cases showed numerical as well as structural aberrations. Although some aberrations appeared to occur nonrandomly (e.g., loss of chromosome 13 or involvement of band 13pll-13), no consistent pattern was present. It also remains an open question whether these chromosomal aberrations exclusively originate from H/RS cells or whether they are also present in bystander cells. The latter may be evidenced by experiments performed in our laboratory. Stimulated by the successful transplantation of HD-derived cell lines in SCID mice [20], we have attempted to transplant primary lymphatic tissue affected by HD. EBV-positive tumors of B-cell origin grew out preferentially in lymphatic tissue
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indicative for their mitotic activity the overall growth rate of the lymph nodes affected by HD is remarkably low [7]. This discrepancy might be caused by a high rate of cell death and/or a high proportion of lymphocytes leaving the Hodgkin lesion. It is very conceivable that the mechanism underlying this observation is an antigenic triggering of the lymphocytes, resulting in the formation of mitotically active immunoblasts. Usually, an antigen-directed immune response leads to a benign self-limiting lymphocyte proliferation in the lymph node. This might underlie the spontaneous fluctuations of the size of affected lymph nodes which occasionally can be observed in the initial stages of the disease. The overall progredient course of Hodgkin's disease might then be understood as a non-self-limiting immune response. More information has now been obtained about the cellular and humoral factors participating in the inflammatory process taking place in Hodgkin's lymph nodes. A high percentage of activated T-helper lymphocytes surround the H/RS cells. Expression of CD4, CD45RO, and CD45RB characterizes these T cells as Thl cells, which exert delayed type hypersensitivity activity. While CD38 expression indicates activation of these T cells, HLA class II is negative in contrast to normal activated T lymphocytes [8]. A variety of cytokines as well as adhesion molecules are likely to be involved in the interaction between the H/RS cells and the surrounding bystander cells. Interleukins 1,6, and 9 produced by RS cells, for instance, may act as autocrine growth factors as well as stimulators for bystander cells. Vice versa, the interleukins 2 and 6 produced by T cells, may stimulate RS cells [9, 10]. Despite accumulating data describing the network of cytokine interaction in the Hodgkin's lymph node, we are far away from understanding the mechanisms underlying this process which DeVita termed a 'lymphocyte civil war' [11]. The characteristics of HD summarized so far, however, might support the model of an atypical immune response rather than that of a true neoplastic process, at least in early stages of the disease.
107 nant cell, aneuploidy and clonal origin, cannot be demonstrated consistently in H/RS cells. Furthermore, in many cases where karyotype anomalies or Ig/TCR gene rearrangements were detected, their derivation from H/RS cells could not be proved. These findings support the conclusion drawn above, that in early stages of the disease HD shares more characteristics with an atypical immune response than with a true neoplastic disorder.
Virus infection in Hodgkin's disease — the contribution of epidemiology
A bimodal age incidence curve with differences dependent on the demographic situation is typical for Hodgkin's disease. Three epidemiological patterns are found [32]. Type I, in developing countries, shows a first peak in childhood, a low incidence in the third decade, and a second peak in older adults. Type HI is found in industrialized countries, with a very low incidence in children, a first peak in young adults, and a second peak in older age groups. Additionally, in rural areas of industrialized countries, an intermediate type II has been described. Based on these epidemiological data, McMahon suggested in 1966 [33] that HD consists of three different entities corresponding to the onset of clinical disease: HD of children; HD of young adults, which he supposed to be caused by an infectious agent; and HD of the older age groups. The difference of HD in young and older adults may be underlined by the predominance of the nodular sclerosing subtype, as well as by the frequent mediastinal involvement in the younger age group. The epidemiological finding that children with a low socioeconomic standard (type I, first peak), as well as young adults with a high socioeconomic standard (type III, first peak), have a high risk for HD resembles remarkably the epidemiology of poliovirus infection and suggests a pathogenetic role of an infectious agent quite common in childhood [34]. This hypothesis has been confirmed by studies showing an increased risk of HD in young adults living in small families with a high socioeconomic status, whereas people with frequent common contagious illnesses in childhood were found to have a lower risk for HD [35]. First evidence for a possible causal role of EBV in HD came from the observation that persons with a history of infectious mononucleosis have a two- to three fold increased risk of HD [36]. Furthermore, in numerous studies elevated levels of antibodies to EB viral capsid antigen (VCA) and early antigen (EA) have been described in HD patients [37-39]. Finally, elevated levels of IgG and IgA antibodies against EBV capsid antigen and nuclear antigen (EBNA) were shown to be associated with a significant higher relative risk for developing HD [40].
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and were composed of three different histological lesions: lymphoproliferative disease (LPD), anaplastic large-cell lymphoma (ALCL), and Hodgkin-like lesions (HDLL) [21]. After short-term recultivation of these B-cell tumors, both structural and numerical chromosomal aberrations were detected at a high frequency. The SCID mouse tumors obtained after transplantation of HD tissue might therefore derive from EBV-positive bystander cells, which differ significantly from normal EBV-infected B cells, owing to an inherent genetic instability, and which might be amplified in SCID mice. Whether these bystander cells represent a (semi-) malignant cell population present in HD lymph nodes in addition to H/RS cells remains to be clarified. In any case, these data underline that karyotype abnormality might not be sufficient to define H/RS cells as the definite malignant cell population in HD. Conflicting data also exist with regard to clonality of H/RS cells. Two lines of evidence support their clonal origin: First, clonal rearrangements of immunoglobulin (Ig) and T-cell antigen receptor (TCR) genes were described in affected lymphatic tissue [22] or within H/RS cells enriched lymph node preparations [23]. Second, in cases with in situ identification of EBV genomes in H/RS cells, a constant number of EBV terminal repeats (TR) were found by Southern blot analysis [24, 25]. In contrast, absence of clonal Ig or TCR rearrangements was described in numerous cases with a high abundance of H/RS cells (i.e., above the sensitivity threshold of Southern blot analysis) [22, 26, 27] and also in cases where the H/RS cells have been enriched [23]. Similarly, in a recent published study, no correlation could be found between abundance of H/RS cells and presence of Ig or TCR rearrangement [28]. Absence of clonal Ig or TCR gene rearrangements does not exclude monoclonal proliferation of an immature lymphoid precursor cell without rearrangement or of a nonlymphoid cell. Alternatively, these data may point to a polyclonal proliferation of H/RS cells in early stages of the disease. Vice versa, clonal Ig or TCR rearrangements have been reported in nonmalignant lymphoproliferative or lymphoepithelial lesions [29]. The detection of monoclonal EBV in HD lymph nodes might also not be sufficient to demonstrate conclusively the monoclonal origin of H/RS cells. First, using highly sensitive hybridization techniques, all HD lymph nodes with EBV-positive H/RS cells have been shown to harbor additionally EBV-positive bystander lymphocytes [30, 31]. Therefore, the origin of monoclonal EBV genomes cannot be unequivocally demonstrated. Furthermore, clonal episomal EBV genomes have also been detected in nonmalignant hyperplastic lymph nodes [30]. Taken together, H/RS cells are not specific for Hodgkin's disease. In contrast to non-Hodgkin's lymphomas and nearly all other malignancies, no normal cell counterpart has been determined despite extensive investigations. The classical criteria defining a malig-
108 EBV in Hodgkin's disease — transformation or modulation?
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In 1987 Weiss and colleagues were the first to demonstrate the presence of EBV DNA in HD tissue specimens using the cloned BamHl W fragment of EBV, a sequence repeated several times in the viral genome, as hybridization probe [4]. Earlier attempts to detect EBV DNA in HD had failed, probably owing to less sensitive hybridization techniques [41,42]. The presence of EBV DNA in HD tissue and its location in H/RS cells was confirmed by several groups. Differences in the percentage of EBV-positive HD cases mainly depended on the methods used. Southern blot analysis and in situ hybridization using the BamHl W fragment as hybridization probe yielded 20%-40% positive cases [4, 25, 43-45]. With the highly sensitive PCR method, the highest percentage of positive cases has been obtained (up to 60%) [28, 30, 46-48]. This method, however, despite being highly sensitive, lacks specificity and also allows EBV sequences from nonmalignant bystander cells to be amplified. This problem has been overcome now by use of in situ hybridization with EBER-specific singlestranded RNA probes (EBER-ISH). EBER1- and 2RNAs are small EBV-encoded nonpolyadenylated transcripts with a high abundance of about 1 x 106 copies/viral genome [49]. Using EBER-ISH, 18%50% of HD cases have been reported with EBV genomes located in H/RS cells [50-52]. In contrast to initial reports about the exclusive localization of EBV in H/RS cells, the viral genome is also found in small cells in the surrounding of H/RS cells, in cases with EBV-positive as well as with EBV-negative H/RS cells. Most of these EBER-positive bystander cells have been shown to express CD20 and immunoglobulin light chain, thus exerting a B-cell phenotype; others could not be assigned to a specific lineage [52]. Several investigators have demonstrated the clonality of EBV in HD tissue by hybridization with the viral terminal repeats [24, 25]. These findings indicate clonal expansion of single EBV-infected cells and further underline a possible etiological role of EBV in a proportion of HD. Others have critically discussed this interpretation and pointed to the similarities of EBV structure and presence in HD and hyperplastic lymphoid processes: Both contain a high number of EBV genome copies, and both contain episomal clonal, episomal nonclonal, and linear viral genomes [30]. Furthermore, only a poor correlation of the amount of H/RS cells with the amount of EBV genomes has been found [28]. EBV is not a silent passenger in H/RS cells, but shows a specific latent-gene-expression pattern. In 50%-60% of EBV-positive HD cases, the viral latent membrane protein (LMP1) is expressed in H/RS cells, whereas the EBV nuclear protein 2 (EBNA2) is not expressed [53, 54]. EBNA1 expression is difficult to prove, owing to the lack of appropriate monoclonal antibodies. Transcriptional analysis in fresh HD biop-
sies, however, revealed EBNA1 transcripts [55]. This latent-gene-expression pattern (EBNA1+, EBNA2", LMP1+) is also found in undifferentiated nasopharyngeal carcinoma (NPC) and differs from the latent-geneexpression pattern in Burkitt's lymphoma (EBNA1+, EBNA2", LMP1") and immunoblastic B-cell lymphomas in immunocompromised individuals (EBNA1+, EBNA2 + ,LMPl + )[56]. The biological function of LMP1 expression in HD is still not understood, since the known effects of this latent protein to a certain extent may be regarded as controversial. On the one hand, LMP1 has a transforming potential, as was shown by tumorigenic transformation of epithelial cells after transfection of the LMP1 gene [57]. Furthermore, via up-regulation of the bcl-2 gene, LMP1 might protect B lymphocytes from apoptosis [58]. On the other hand LMP1 (and all other latent viral proteins except EBNA1) might exert a protecting function by representing a target antigen for cytotoxic T lymphocytes (CTL) [56]. LMP1 expression (in part together with EBNA2 expression) also up-regulates numerous cellular genes, including activation-associated B-cell antigens (CD23, CD30, CD39) and adhesion molecules (ICAM-1, LFA-1, LFA-3) [59, 60]. It is thus very conceivable that LMP1 might also indirectly render a cell more 'immunogenic' by inducing expression of cellular surface antigens. EBV-positive lymphoid malignancies either downregulate latent genes (except EBNA1) in a host with an intact immune system (Burkitt's lymphoma) or express the complete set of EBV latent proteins in a host with severe T-cell deficiency (immunoblastic B-NHL) [56]. It remains to be established whether nonexpression of EBNA2, coupled with expression of LMP1 on the background of an altered T-cell function, contributes to the atypical immune response defined as Hodgkin's disease. Surprisingly, less attention has been given so far to a possible transforming function of EBNA1, a DNAbinding protein effecting maintenance of the episomal state of the virus [61]. Several lines of evidence suggest a role for EBNA1 in the transformation process: First, it is the only viral protein being consistently expressed in all EBV-associated malignancies (including EBER+, LMP" HD). Second, it is the only latent viral protein for which no target function for CTLs has yet been demonstrated. Finally, in endemic BL (96% EBV-positive cases), EBNA1 is the only viral latent protein found in tumor cells, whereas the other latent viral genes are down-regulated, thus possibly permitting the cell to escape the host's immune response [56]. Another open question addresses the physical state of the virus in H/RS cells. EBV-positive cells usually harbor multiple copies of episomal EBV genomes [62]. Integration of the virus into the host cell chromosome was generally assumed to be a rare exception [63]. Recently, using fluorescence in situ hybridization to metaphase chromosomes, more cases of EBV integra-
109
EBV-positive HD — linkage to specific subgroups?
Different results have been reported concerning the association of EBV with a histological subtype of HD. Several studies demonstrated more EBV DNA positive cases in the mixed cellularity subtype (HDMC) compared to nodular sclerosis (HDNC) [4, 43-45]; others could not confirm these results [24, 25]. Using monoclonal antibodies against LMP1, a significantly higher percentage of EBV-positive cases in HDMC compared to HDNC and the lymphocyte-predominant subgroup (HDLP) was described [54]. A significant association has also been described between EBV positivity and age under 15 years (7/13) on the one hand and over 50 years (27/38) on the other. In contrast, only a minority of EBV-positive cases was found in young adults (4/28) in this study [45]. If these results can be reproduced with larger case numbers, they confirm the conclusions of epidemiological data drawn by McMahon, as discussed above. HD of childhood and older age then might represent EB-associated HD entities different from HD of young adults, where a still unknown virus might be involved.
Conclusions That Hodgkin's disease represents a distinct disease entity is questionable in several quite different aspects. Epidemiological data provide evidence for the existence of at least two HD entities, i.e., HD of young adults and HD of childhood and older age groups. Analysis of the EBV status in HD tissue distinguishes EBV-negative and EBV-positive cases, the latter subdividing in LMP expressing and nonexpressing. Molecularbiological and cytogenetic analysis have failed to detect any consistent common genetic aberration in Hodgkin's disease. Histopathologically, there is a strong interrelationship between HD and benign lesions like lymphoid hyperplasias producing immunoblastic proliferations (e.g., infectious mononucleosis, drug hypersensitivity reactions) on the one side and some hematological malignancies like anaplastic large-cell lymphoma, peripheral T-cell lymphomas, or lymphomatoid papulosis on the other. Furthermore, as outlined above, several lines of evidence suggest that HD starts as an atypical immune response rather than as a true malignant disorder: (i) the clinical and biological features of a pronounced immune reaction, (ii) the failure to unequivocally determine the H/RS cells as the definite malignant cell population in HD, and (iii) the epidemiological pattern of an infectious disease. Thus, in early stages Hodgkin's disease might be understood as the unsuccessful attempt of the organism to eliminate a cell expressing a yet undefined target antigen. This antigen might have been introduced into the cell by viral infection, but could also be encoded by a cellular gene. In cases of EBV-positive HD, LMP1 might represent this target antigen itself or up-regulate cellular surface antigens. The cell expressing the putative target antigen might be a precursor cell of any hematopoetic lineage. Growth promotion of this cell could be mediated by viral transformation and/or cytokine stimulation from the reactive environment. In the course of the disease, inability of an altered immune system to eliminate this target-antigen-expressing cell coincides with a stepwise transformation, probably triggered by an inherent genetic instability, thus leading to outgrowth of a fully transformed malignant cell clone in late stages of the disease (Table 1).
Table 1. Gradual transformation in Hodgkin's disease.
INFECTION NEOPLASM Atypic immune response
Gradual transformation
Outgrowth of a fully transformed clone
• HD-specific target antigen (viral, cellular)? • immune defect (failure to eliminate the target antigen carrying cell)?
• inherited genetic instability? • mutagenic events (radiation, chemotherapy)?
• suppressor inactivation? • oncogene activation? • cytokine deregulation?
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tion have been described in BL cell lines and EBV immortalized lymphoblastoid cells [64-67]. Integrated and episomal copies were sometimes found together in the same cell. Thus, integration might be more frequent than commonly assumed and yet not detected because of technical reasons. Viral integration might contribute to cellular transformation by several mechanisms, e.g., alteration of viral transcription, modulation of cellular gene transcription, or induction of chromosomal breaks. The EBV integration site in a BL cell line analyzed in our laboratory, for instance, was found to be located near the breakpoint of a chromosomal translocation (11/19) [66]. Furthermore, this integrated viral genome has a large deletion, including among others the terminal repeats and the sequences encoding for EBER1, 2, and LMP [68]. EBV genomes carrying this deletion cannot be detected by EBER-ISH or by staining with LMP monoclonal antibodies. Whether defective genomes are present in part of the EBV-negative HD cases remains to be established.
110 References
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Correspondence to: Dr. Jiirgen Wolf Department of Internal Medicine I University of Cologne Josef-Stelzmannstr. 9 50924 Cologne Germany
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Original article Is Hodgkin's disease an infectious disease? J. Wolf & V. Diehl Department of Internal Medicine I, University of Cologne, Cologne, Germany
Summary
Introduction
Key words: atypical immune response, Epstein-Barr virus, Hodgkin's disease
cells. The recently described association of HD with a human DNA tumor virus (EBV) might be persuasive enough to wind up the debate on the true nature of this disease.
Since the first description 'on some morbid appearances of the absorbent glands and spleen' by Thomas Hodgkin (1832) [1], the concept of Hodgkin's disease as a true neoplastic disorder of the lymphatic system has repeatedly been questioned. Wilks presented a col- Hodgkin's disease — true neoplastic disorder or atypilection of 15 cases of HD in 1865 [2] and was con- cal immune response? vinced that 'the disease of Hodgkin is clearly separable from lardaceous disease, from cancer and tubercle, al- Clinically, HD presents with pronounced signs of an inthough these affections may bear a relation to one an- flammatory process - fluctuating fever, chills, nightother.' Dorothy Reed, one of the first pathologists de- sweats, monocytoses, lymphopenia, and elevated levels scribing thoroughly the histopathology of the Hodgkin of soluble IL-2 receptor in the patient's serum [5]. lesion, stated in 1902: 'Histologically, it might be Histologically, in remarkable contrast to malignant thought that the changes resemble those of a malignant disorders, there is no homogenous malignant cell growth, but we believe that closer study will show a population which represents the majority of cells in the greater similiarity to inflammatory processes' [3]. Ac- tumor tissue. Rather, a heterogenous mixture of lymcordingly, a search for infectious agents, including bac- phocytes, histiocytes, eosinophils, plasma cells, fibroteria, fungus, and viruses paralleled Hodgkin's re- blasts, and others is found in affected lymphatic tissue. search, but was unsuccesful until 1987, when Weiss and Among them, the H/RS cells which are believed to be coworkers were the first to detect Epstein-Barr virus the malignant substrate of HD, represent, at least in DNAinHDtissue[4]. initial stages, only a minority of 0.1%-l% [6]. The situation becomes even more puzzling considIndependent of scientific controversies on the pathogenesis of Hodgkin's disease, the concept of HD ering the results of early cell kinetic studies. In vitro as a true neoplasia of the lymphatic system has pre- and in vivo labeling with tritiated thymidine in vailed in the clinical setting: Anticancer treatment strat- Hodgkin's tissue revealed the presence of two major egies like radiation therapy alone or in combination proliferating-cell populations in HD: cells identified as with polychemotherapy led to cure rates of about two- transformed lymphocytes with a high labeling index and Hodgkin cells with a lower labeling index [7]. thirds of all patients suffering from HD. This impressive success in treatment is still counter- Reed-Sternberg cells were found to incorporate only faced by a major lack of understanding of the patho- rarely tritiated thymidine in these studies. Therefore, genetic events leading to Hodgkin's disease. No con- the 'normal' lymphocytes, not the 'malignant' Hodgkin vincing model exists either to define the cell of origin of or RS cells, represent the majority of dividing cells in HD or to explain the interaction between the putative HD tumor masses. malignant H/RS cells and the surrounding bystander Despite the high labeling index of the lymphocytes
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Several lines of evidence question the concept of Hodgkin's disease (HD) starting as a true malignant disorder of the lymphatic system: (i) In early stages HD exerts pronounced clinical and biological features of an atypic immune response, (ii) Despite extensive investigations, the Hodgkin/ReedSternberg (H/RS) cells have not been unequivocally determined as the definite malignant cell population in HD. (iii) The epidemiological pattern of HD strongly resembles that of an infectious disease, (iv) About 50% of HD cases can be
linked to infection with Epstein-Barr virus. Hodgkin's disease in early stages might thus be understood as the unsuccessful attempt of the organism to eliminate a cell expressing a putative (cellular or viral) target antigen. A stepwise transformation of this antigen-carrying cell, possibly triggered by an inherent genetic instability, might then lead to outgrowth of a malignant cell clone in late stages of the disease.
106
Hodgkin/Reed-Sternberg cells — the malignant substrate of Hodgkin's disease?
The concept of Hodgkin's disease being a true neoplastic disorder is mainly based on the presence of a putative malignant cell population, i.e., the mononucleated Hodgkin cells and their bi- or polynucleated derivatives, the Reed-Sternberg cells. These cells are consistently found in Hodgkin's disease. They have also been described, however, in nonmalignant diseases like infectious mononucleosis, measles, rubeola, or anticonvulsant-induced lymphadenopathy [12]. So far, the origin of H/RS cells has not been determined, although any of the hematopoietic cell types has been proposed as cell of origin in Hodgkin's disease [13]. Lymphocyte-predominant HD seems to be an
exception, since in this histological subtype the H/RS cells express a B-cell phenotype as a rule [14]. Immunophenotyping of H/RS cells in the other subtypes of HD, as well as on the few established cell lines, has revealed a more complex pattern. Some antigens are consistently expressed, the activation markers Ki-1 (CD30) and the interleukin-2 receptor (CD25), among others. Both are also expressed on activated lymphocytes or monocytes. In contrast, no homogenous pattern is found for lineage-specific antigen expression: the H/RS cells may express B- or T-cell-specific surface antigens or none of them (0-phenotype) [13]. In analogy, the HD-derived cell lines show either (complete or incomplete) Ig or TCR rearrangements, which might point to an immature lymphoid origin of these cells [15]. In Hodgkin's lymph nodes, clonal Ig or TCR rearrangements have also been described, although the interpretation of these studies is complicated by the scarcity of tumor cells in the biopsy specimens as discussed below. Similarly, no consistent deregulation of a cellular oncogene or inactivation of a suppressor gene could be detected [16]. What are the criteria for considering this heterogenous cell population as the malignant cell population in Hodgkin's disease? Fundamental attributes of neoplastic cells are aneuploidy and clonal origin. Cytogenetic studies in HD are difficult to perform and to interpret owing to the scarcity of the H/RS cells, the low number of obtainable mitoses, and their poor chromosome-banding qualities. Also, many of the mitotic figures in short-term cultures of HD lymph nodes derive from the lymphoid cells of the reactive stromal tissue (bystander cells) [17]. In a recently published review, only 40 well-documented cases of complete karyotype-banding studies in HD were found [18]. In these studies, the proportion of karyotype abnormalities varied from 22% to 83%, including a broad spectrum of numerical and structural abnormalities. No specific chromosomal marker, like, for instance, the translocation (9/22) in chronic myelogenous leukemia or (8/14) in Burkitt's lymphoma was detected. Similarly, in a recent published study [19], 60 lymph nodes from untreated HD patients were analyzed: In 82% of the cases analyzable metaphases were obtained; in 55% chromosomal abnormalities were detected. Most cases showed numerical as well as structural aberrations. Although some aberrations appeared to occur nonrandomly (e.g., loss of chromosome 13 or involvement of band 13pll-13), no consistent pattern was present. It also remains an open question whether these chromosomal aberrations exclusively originate from H/RS cells or whether they are also present in bystander cells. The latter may be evidenced by experiments performed in our laboratory. Stimulated by the successful transplantation of HD-derived cell lines in SCID mice [20], we have attempted to transplant primary lymphatic tissue affected by HD. EBV-positive tumors of B-cell origin grew out preferentially in lymphatic tissue
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indicative for their mitotic activity the overall growth rate of the lymph nodes affected by HD is remarkably low [7]. This discrepancy might be caused by a high rate of cell death and/or a high proportion of lymphocytes leaving the Hodgkin lesion. It is very conceivable that the mechanism underlying this observation is an antigenic triggering of the lymphocytes, resulting in the formation of mitotically active immunoblasts. Usually, an antigen-directed immune response leads to a benign self-limiting lymphocyte proliferation in the lymph node. This might underlie the spontaneous fluctuations of the size of affected lymph nodes which occasionally can be observed in the initial stages of the disease. The overall progredient course of Hodgkin's disease might then be understood as a non-self-limiting immune response. More information has now been obtained about the cellular and humoral factors participating in the inflammatory process taking place in Hodgkin's lymph nodes. A high percentage of activated T-helper lymphocytes surround the H/RS cells. Expression of CD4, CD45RO, and CD45RB characterizes these T cells as Thl cells, which exert delayed type hypersensitivity activity. While CD38 expression indicates activation of these T cells, HLA class II is negative in contrast to normal activated T lymphocytes [8]. A variety of cytokines as well as adhesion molecules are likely to be involved in the interaction between the H/RS cells and the surrounding bystander cells. Interleukins 1,6, and 9 produced by RS cells, for instance, may act as autocrine growth factors as well as stimulators for bystander cells. Vice versa, the interleukins 2 and 6 produced by T cells, may stimulate RS cells [9, 10]. Despite accumulating data describing the network of cytokine interaction in the Hodgkin's lymph node, we are far away from understanding the mechanisms underlying this process which DeVita termed a 'lymphocyte civil war' [11]. The characteristics of HD summarized so far, however, might support the model of an atypical immune response rather than that of a true neoplastic process, at least in early stages of the disease.
107 nant cell, aneuploidy and clonal origin, cannot be demonstrated consistently in H/RS cells. Furthermore, in many cases where karyotype anomalies or Ig/TCR gene rearrangements were detected, their derivation from H/RS cells could not be proved. These findings support the conclusion drawn above, that in early stages of the disease HD shares more characteristics with an atypical immune response than with a true neoplastic disorder.
Virus infection in Hodgkin's disease — the contribution of epidemiology
A bimodal age incidence curve with differences dependent on the demographic situation is typical for Hodgkin's disease. Three epidemiological patterns are found [32]. Type I, in developing countries, shows a first peak in childhood, a low incidence in the third decade, and a second peak in older adults. Type HI is found in industrialized countries, with a very low incidence in children, a first peak in young adults, and a second peak in older age groups. Additionally, in rural areas of industrialized countries, an intermediate type II has been described. Based on these epidemiological data, McMahon suggested in 1966 [33] that HD consists of three different entities corresponding to the onset of clinical disease: HD of children; HD of young adults, which he supposed to be caused by an infectious agent; and HD of the older age groups. The difference of HD in young and older adults may be underlined by the predominance of the nodular sclerosing subtype, as well as by the frequent mediastinal involvement in the younger age group. The epidemiological finding that children with a low socioeconomic standard (type I, first peak), as well as young adults with a high socioeconomic standard (type III, first peak), have a high risk for HD resembles remarkably the epidemiology of poliovirus infection and suggests a pathogenetic role of an infectious agent quite common in childhood [34]. This hypothesis has been confirmed by studies showing an increased risk of HD in young adults living in small families with a high socioeconomic status, whereas people with frequent common contagious illnesses in childhood were found to have a lower risk for HD [35]. First evidence for a possible causal role of EBV in HD came from the observation that persons with a history of infectious mononucleosis have a two- to three fold increased risk of HD [36]. Furthermore, in numerous studies elevated levels of antibodies to EB viral capsid antigen (VCA) and early antigen (EA) have been described in HD patients [37-39]. Finally, elevated levels of IgG and IgA antibodies against EBV capsid antigen and nuclear antigen (EBNA) were shown to be associated with a significant higher relative risk for developing HD [40].
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and were composed of three different histological lesions: lymphoproliferative disease (LPD), anaplastic large-cell lymphoma (ALCL), and Hodgkin-like lesions (HDLL) [21]. After short-term recultivation of these B-cell tumors, both structural and numerical chromosomal aberrations were detected at a high frequency. The SCID mouse tumors obtained after transplantation of HD tissue might therefore derive from EBV-positive bystander cells, which differ significantly from normal EBV-infected B cells, owing to an inherent genetic instability, and which might be amplified in SCID mice. Whether these bystander cells represent a (semi-) malignant cell population present in HD lymph nodes in addition to H/RS cells remains to be clarified. In any case, these data underline that karyotype abnormality might not be sufficient to define H/RS cells as the definite malignant cell population in HD. Conflicting data also exist with regard to clonality of H/RS cells. Two lines of evidence support their clonal origin: First, clonal rearrangements of immunoglobulin (Ig) and T-cell antigen receptor (TCR) genes were described in affected lymphatic tissue [22] or within H/RS cells enriched lymph node preparations [23]. Second, in cases with in situ identification of EBV genomes in H/RS cells, a constant number of EBV terminal repeats (TR) were found by Southern blot analysis [24, 25]. In contrast, absence of clonal Ig or TCR rearrangements was described in numerous cases with a high abundance of H/RS cells (i.e., above the sensitivity threshold of Southern blot analysis) [22, 26, 27] and also in cases where the H/RS cells have been enriched [23]. Similarly, in a recent published study, no correlation could be found between abundance of H/RS cells and presence of Ig or TCR rearrangement [28]. Absence of clonal Ig or TCR gene rearrangements does not exclude monoclonal proliferation of an immature lymphoid precursor cell without rearrangement or of a nonlymphoid cell. Alternatively, these data may point to a polyclonal proliferation of H/RS cells in early stages of the disease. Vice versa, clonal Ig or TCR rearrangements have been reported in nonmalignant lymphoproliferative or lymphoepithelial lesions [29]. The detection of monoclonal EBV in HD lymph nodes might also not be sufficient to demonstrate conclusively the monoclonal origin of H/RS cells. First, using highly sensitive hybridization techniques, all HD lymph nodes with EBV-positive H/RS cells have been shown to harbor additionally EBV-positive bystander lymphocytes [30, 31]. Therefore, the origin of monoclonal EBV genomes cannot be unequivocally demonstrated. Furthermore, clonal episomal EBV genomes have also been detected in nonmalignant hyperplastic lymph nodes [30]. Taken together, H/RS cells are not specific for Hodgkin's disease. In contrast to non-Hodgkin's lymphomas and nearly all other malignancies, no normal cell counterpart has been determined despite extensive investigations. The classical criteria defining a malig-
108 EBV in Hodgkin's disease — transformation or modulation?
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In 1987 Weiss and colleagues were the first to demonstrate the presence of EBV DNA in HD tissue specimens using the cloned BamHl W fragment of EBV, a sequence repeated several times in the viral genome, as hybridization probe [4]. Earlier attempts to detect EBV DNA in HD had failed, probably owing to less sensitive hybridization techniques [41,42]. The presence of EBV DNA in HD tissue and its location in H/RS cells was confirmed by several groups. Differences in the percentage of EBV-positive HD cases mainly depended on the methods used. Southern blot analysis and in situ hybridization using the BamHl W fragment as hybridization probe yielded 20%-40% positive cases [4, 25, 43-45]. With the highly sensitive PCR method, the highest percentage of positive cases has been obtained (up to 60%) [28, 30, 46-48]. This method, however, despite being highly sensitive, lacks specificity and also allows EBV sequences from nonmalignant bystander cells to be amplified. This problem has been overcome now by use of in situ hybridization with EBER-specific singlestranded RNA probes (EBER-ISH). EBER1- and 2RNAs are small EBV-encoded nonpolyadenylated transcripts with a high abundance of about 1 x 106 copies/viral genome [49]. Using EBER-ISH, 18%50% of HD cases have been reported with EBV genomes located in H/RS cells [50-52]. In contrast to initial reports about the exclusive localization of EBV in H/RS cells, the viral genome is also found in small cells in the surrounding of H/RS cells, in cases with EBV-positive as well as with EBV-negative H/RS cells. Most of these EBER-positive bystander cells have been shown to express CD20 and immunoglobulin light chain, thus exerting a B-cell phenotype; others could not be assigned to a specific lineage [52]. Several investigators have demonstrated the clonality of EBV in HD tissue by hybridization with the viral terminal repeats [24, 25]. These findings indicate clonal expansion of single EBV-infected cells and further underline a possible etiological role of EBV in a proportion of HD. Others have critically discussed this interpretation and pointed to the similarities of EBV structure and presence in HD and hyperplastic lymphoid processes: Both contain a high number of EBV genome copies, and both contain episomal clonal, episomal nonclonal, and linear viral genomes [30]. Furthermore, only a poor correlation of the amount of H/RS cells with the amount of EBV genomes has been found [28]. EBV is not a silent passenger in H/RS cells, but shows a specific latent-gene-expression pattern. In 50%-60% of EBV-positive HD cases, the viral latent membrane protein (LMP1) is expressed in H/RS cells, whereas the EBV nuclear protein 2 (EBNA2) is not expressed [53, 54]. EBNA1 expression is difficult to prove, owing to the lack of appropriate monoclonal antibodies. Transcriptional analysis in fresh HD biop-
sies, however, revealed EBNA1 transcripts [55]. This latent-gene-expression pattern (EBNA1+, EBNA2", LMP1+) is also found in undifferentiated nasopharyngeal carcinoma (NPC) and differs from the latent-geneexpression pattern in Burkitt's lymphoma (EBNA1+, EBNA2", LMP1") and immunoblastic B-cell lymphomas in immunocompromised individuals (EBNA1+, EBNA2 + ,LMPl + )[56]. The biological function of LMP1 expression in HD is still not understood, since the known effects of this latent protein to a certain extent may be regarded as controversial. On the one hand, LMP1 has a transforming potential, as was shown by tumorigenic transformation of epithelial cells after transfection of the LMP1 gene [57]. Furthermore, via up-regulation of the bcl-2 gene, LMP1 might protect B lymphocytes from apoptosis [58]. On the other hand LMP1 (and all other latent viral proteins except EBNA1) might exert a protecting function by representing a target antigen for cytotoxic T lymphocytes (CTL) [56]. LMP1 expression (in part together with EBNA2 expression) also up-regulates numerous cellular genes, including activation-associated B-cell antigens (CD23, CD30, CD39) and adhesion molecules (ICAM-1, LFA-1, LFA-3) [59, 60]. It is thus very conceivable that LMP1 might also indirectly render a cell more 'immunogenic' by inducing expression of cellular surface antigens. EBV-positive lymphoid malignancies either downregulate latent genes (except EBNA1) in a host with an intact immune system (Burkitt's lymphoma) or express the complete set of EBV latent proteins in a host with severe T-cell deficiency (immunoblastic B-NHL) [56]. It remains to be established whether nonexpression of EBNA2, coupled with expression of LMP1 on the background of an altered T-cell function, contributes to the atypical immune response defined as Hodgkin's disease. Surprisingly, less attention has been given so far to a possible transforming function of EBNA1, a DNAbinding protein effecting maintenance of the episomal state of the virus [61]. Several lines of evidence suggest a role for EBNA1 in the transformation process: First, it is the only viral protein being consistently expressed in all EBV-associated malignancies (including EBER+, LMP" HD). Second, it is the only latent viral protein for which no target function for CTLs has yet been demonstrated. Finally, in endemic BL (96% EBV-positive cases), EBNA1 is the only viral latent protein found in tumor cells, whereas the other latent viral genes are down-regulated, thus possibly permitting the cell to escape the host's immune response [56]. Another open question addresses the physical state of the virus in H/RS cells. EBV-positive cells usually harbor multiple copies of episomal EBV genomes [62]. Integration of the virus into the host cell chromosome was generally assumed to be a rare exception [63]. Recently, using fluorescence in situ hybridization to metaphase chromosomes, more cases of EBV integra-
109
EBV-positive HD — linkage to specific subgroups?
Different results have been reported concerning the association of EBV with a histological subtype of HD. Several studies demonstrated more EBV DNA positive cases in the mixed cellularity subtype (HDMC) compared to nodular sclerosis (HDNC) [4, 43-45]; others could not confirm these results [24, 25]. Using monoclonal antibodies against LMP1, a significantly higher percentage of EBV-positive cases in HDMC compared to HDNC and the lymphocyte-predominant subgroup (HDLP) was described [54]. A significant association has also been described between EBV positivity and age under 15 years (7/13) on the one hand and over 50 years (27/38) on the other. In contrast, only a minority of EBV-positive cases was found in young adults (4/28) in this study [45]. If these results can be reproduced with larger case numbers, they confirm the conclusions of epidemiological data drawn by McMahon, as discussed above. HD of childhood and older age then might represent EB-associated HD entities different from HD of young adults, where a still unknown virus might be involved.
Conclusions That Hodgkin's disease represents a distinct disease entity is questionable in several quite different aspects. Epidemiological data provide evidence for the existence of at least two HD entities, i.e., HD of young adults and HD of childhood and older age groups. Analysis of the EBV status in HD tissue distinguishes EBV-negative and EBV-positive cases, the latter subdividing in LMP expressing and nonexpressing. Molecularbiological and cytogenetic analysis have failed to detect any consistent common genetic aberration in Hodgkin's disease. Histopathologically, there is a strong interrelationship between HD and benign lesions like lymphoid hyperplasias producing immunoblastic proliferations (e.g., infectious mononucleosis, drug hypersensitivity reactions) on the one side and some hematological malignancies like anaplastic large-cell lymphoma, peripheral T-cell lymphomas, or lymphomatoid papulosis on the other. Furthermore, as outlined above, several lines of evidence suggest that HD starts as an atypical immune response rather than as a true malignant disorder: (i) the clinical and biological features of a pronounced immune reaction, (ii) the failure to unequivocally determine the H/RS cells as the definite malignant cell population in HD, and (iii) the epidemiological pattern of an infectious disease. Thus, in early stages Hodgkin's disease might be understood as the unsuccessful attempt of the organism to eliminate a cell expressing a yet undefined target antigen. This antigen might have been introduced into the cell by viral infection, but could also be encoded by a cellular gene. In cases of EBV-positive HD, LMP1 might represent this target antigen itself or up-regulate cellular surface antigens. The cell expressing the putative target antigen might be a precursor cell of any hematopoetic lineage. Growth promotion of this cell could be mediated by viral transformation and/or cytokine stimulation from the reactive environment. In the course of the disease, inability of an altered immune system to eliminate this target-antigen-expressing cell coincides with a stepwise transformation, probably triggered by an inherent genetic instability, thus leading to outgrowth of a fully transformed malignant cell clone in late stages of the disease (Table 1).
Table 1. Gradual transformation in Hodgkin's disease.
INFECTION NEOPLASM Atypic immune response
Gradual transformation
Outgrowth of a fully transformed clone
• HD-specific target antigen (viral, cellular)? • immune defect (failure to eliminate the target antigen carrying cell)?
• inherited genetic instability? • mutagenic events (radiation, chemotherapy)?
• suppressor inactivation? • oncogene activation? • cytokine deregulation?
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tion have been described in BL cell lines and EBV immortalized lymphoblastoid cells [64-67]. Integrated and episomal copies were sometimes found together in the same cell. Thus, integration might be more frequent than commonly assumed and yet not detected because of technical reasons. Viral integration might contribute to cellular transformation by several mechanisms, e.g., alteration of viral transcription, modulation of cellular gene transcription, or induction of chromosomal breaks. The EBV integration site in a BL cell line analyzed in our laboratory, for instance, was found to be located near the breakpoint of a chromosomal translocation (11/19) [66]. Furthermore, this integrated viral genome has a large deletion, including among others the terminal repeats and the sequences encoding for EBER1, 2, and LMP [68]. EBV genomes carrying this deletion cannot be detected by EBER-ISH or by staining with LMP monoclonal antibodies. Whether defective genomes are present in part of the EBV-negative HD cases remains to be established.
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Correspondence to: Dr. Jiirgen Wolf Department of Internal Medicine I University of Cologne Josef-Stelzmannstr. 9 50924 Cologne Germany
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