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The clinical and histological presentations of angioimmunoblastic T-cell lymphoma (AITL) often mimic an infectious process. Epstein–Barr virus (EBV) and human herpes virus (HHV6) are known to be associated with AITL, but whether these viral infections play a role in its pathogenesis is unclear. It also remains to be investigated whether there might be other viruses associated with AITL. We first screened 26 well-characterised cases of AITL for herpesvirus by polymerase chain reaction (PCR) with universal primers and found evidence of only EBV and HHV6B infection. Subsequent PCR using virus-specific primers demonstrated EBV and HHV6B infection in 40/49 biopsies (36/42 cases) and 21/49 biopsies (19/42 cases) of AITL respectively with both viral infections found in 17/49 specimens (15/42 cases). Importantly, simultaneous infection with both viruses was found only in specimens showing histological pattern II (n = 2) or III (n = 15). Interestingly, among specimens containing both viruses, there was a tendency towards an inverse correlation between the EBV and HHV6B viral load as shown by quantitative PCR. In specimens positive only for EBV, the viral load was significantly higher in specimens with histological pattern III than those with pattern II. High EBV load was also significantly associated with B-cell monoclonality. Double EBV encoded small RNA (EBER) in situ hybridisation and immunohistochemistry indicated that EBV-infected B cells had a late postgerminal centre immunophenotype. Our results demonstrate an association between EBV and HHV6B infection and the histological progression of AITL, suggesting that these viruses may play a role in the pathogenesis of this lymphoma.
Angioimmunoblastic T-cell lymphoma (AITL) is a neoplasm of mature T cells which occurs mostly in the middle-aged and elderly (Dogan et al, 2003). Patients typically present with a systemic illness, characterised by B-symptoms (such as fever and night sweats), generalised lymphadenopathy, and often hepatosplenomegaly and a skin rash, in many cases mimicking an infectious process (Dogan et al, 2003). Abnormal haematological and immunological laboratory indices are frequent and many patients exhibit autoimmune phenomena and immune dysfunction (Dogan et al, 2003). Histologically, the lymphoma is characterised by partial or complete effacement of the normal lymph node architecture by a polymorphic lymphoid infiltrate including neoplastic T-cells that are often CD10 positive, various chronic inflammatory cells, prominent high endothelial venules and expanded follicular dendritic cell (FDC) networks (Attygalle et al, 2002).
The histological architecture of AITL may be classified into three overlapping patterns (Attygalle et al, 2002). In pattern I, the lymph node architecture is largely preserved. Hyperplastic B-cell follicles are surrounded by an expanded paracortex containing a polymorphic infiltrate of lymphocytes, transformed large lymphoid blasts, plasma cells, macrophages and eosinophils within a prominent vascular network. FDC show little or no evidence of expansion outside of the follicles. Pattern II is characterised by loss of the normal lymph node structure, except for the presence of occasional regressed/depleted follicles. On immunostaining, these cases show FDC proliferation beyond the follicles. The remainder of the node shows a polymorphous infiltrate and vascular proliferation similar to that of pattern I. In pattern III, the normal lymph node structure is completely effaced, with prominent irregular and perivascular proliferation of FDCs, extensive vascular proliferation and a diffuse polymorphic infiltrate similar to that seen in patterns I and II. These morphological patterns are associated with increasing numbers of neoplastic CD10-positive T cells, which spread from an intra- or peri-follicular location in pattern I to a more diffuse distribution in patterns II and III. In several patients in whom sequential biopsies have been examined, we and others have observed a transition from pattern I histology to pattern III histology over time (Ree et al, 1998; Attygalle et al, 2002; Attygalle et al, 2007a). Thus, these histological patterns appear both to reflect quantitative differences in the number of neoplastic cells present and to represent temporal progression of disease.
Infectious agents, both directly oncogenic [e.g. Epstein–Barr Virus (EBV) in classical Hodgkin lymphoma] and co-stimulatory [e.g. Helicobacter pylori in gastric mucosa-associated lymphoid tissue (MALT) lymphoma] have been associated with numerous lymphoproliferative disorders (Wotherspoon, 1997; Kuppers, 2003). Both the clinical presentation and polymorphic histological appearances of AITL raise the possibility of a role for infectious agents in its pathogenesis. Several infectious agents including EBV, human herpesvirus (HHV) 6, HHV8 and hepatitis C virus have been reported to be associated with AITL. Among them, EBV, HHV6 and HHV8 appear to be most relevant as they are not only lymphotropic but also oncogenic. EBV is found in most cases of AITL as shown by EBV encoded small RNA (EBER) in situ hybridisation (ISH) (Weiss et al, 1992; Khan et al, 1993; Attygalle et al, 2007b), while HHV6 was reported in 22–58% cases of AITL by polymerase chain reaction (PCR) (Luppi et al, 1993; Vrsalovic et al, 2004). However, neither EBV nor HHV6 is found in the neoplastic T-cells themselves, suggesting a lack of a direct cell autonomous role in lymphomagenesis. HHV8 was detected in occasional cases of AITL by PCR in a single study (Luppi et al, 1996), but subsequent studies failed to show any evidence of HHV8 involvement in AITL by both PCR and immunohistochemistry (Chadburn et al, 1997). Nonetheless, it remains to be determined whether infectious agents play a role in the pathogenesis of AITL, perhaps by inducing microenvironmental conditions which promote lymphoma progression, even if not playing a direct initiating role. It also remains to be investigated whether there are other viruses, which might be associated with the development of AITL.
In view of the pivotal role of several types of herpesvirus in a number of lymphoproliferative disorders, we screened a series of well-characterised AITL cases for herpesvirus by PCR with universal primers and found evidence of only EBV and HHV6B infection. To understand the role of EBV and HHV6B infection in the pathogenesis of AITL further, we determined the cell type carrying EBV, measured the EBV and HHV6B viral load in lymphoma tissues by quantitative real-time PCR and correlated the viral load with histological progression.
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- Materials and methods
By PCR screening with universal primers targeting known herpesviruses, we found evidence of EBV and HHV6B, but not other herpesviruses, in AITL. Association of HHV6B and EBV with AITL has been previously reported (Weiss et al, 1992; Khan et al, 1993; Luppi et al, 1993; Vrsalovic et al, 2004; Attygalle et al, 2007b). We have now extended the previous observations, finding HHV6B in nearly half of a large series of AITL and EBV in almost all of our cases, and further showed that EBV was present in B cells that exhibit immunohistochemical features of postgerminal centre differentiation. More importantly, we have also revealed a clear association between histological progression and the load (copy number) of both EBV and HHV6B in the lesional tissue. These findings suggest a dynamic interplay between HHV6B and EBV and the progression of AITL and raise the following possibilities: (1) EBV and HHV6B infection may play an active role in the pathogenesis of AITL and may be, at least in part, responsible for the histological progression and clinical features of the disease; (2) the increased EBV and HHV6B viral load during histological progression of AITL may be a consequence of increasing dysfunction of the immune system during lymphoma progression, the viruses being passengers during the disease process; and (3) a combination of both the above possibilities exists.
The importance of EBV as a pathogen in immunocompromised people is well recognised. More recently, it has also been shown that reactivation of HHV6 from latency occurs in immunocompromised states such as post-transplant immunosuppression, and is associated with recognisable clinical syndromes (Wang et al, 2006). Many patients with AITL have features of immunodeficiency, which appears to be a consequence of the tumour itself rather than a pre-existing condition, and is characterised by a reduction in circulating T cells, an inverted CD4/CD8 ratio and defective T-cell function in vivo and in vitro (Pizzolo et al, 1983; Ganesan et al, 1987). This underlying immune dysfunction may well provide the background for infection with, or reactivation of, EBV and/or HHV6 in patients with AITL.
There are several pieces of evidence to suggest that this viral infection may then play an active role in the progression of AITL. In particular, it is significant that the neoplastic T cells in AITL only account for a small proportion, typically 5–30%, of the total cell population in a lymph node (Attygalle et al, 2002; Willenbrock et al, 2005), while the majority of cells are a polymorphous paracortical mix of reactive, often immunoblastic, B and T cells, plasma cells, macrophages and eosinophils within a prominent vascular network. A somewhat similar paracortical expansion by polymorphic lymphoid cells may be seen in non-neoplastic viral lymphadenitis caused by members of the herpesvirus family, including EBV and HHV6 (Schnitzer, 1995; Maric et al, 2004). It is possible that both EBV and HHV6 may play an important role in the development of the tumour microenvironment of AITL. This situation is analogous to that of classical Hodgkin lymphoma, in which scattered neoplastic Hodgkin/Reed-Sternberg (HRS) cells interact with a prominent inflammatory infiltrate to orchestrate tumour formation. Aside from directly promoting the proliferation and survival of HRS cells (Kuppers, 2003), EBV is also thought to modify the function of these infiltrating inflammatory cells (Dukers et al, 2000; Skinnider & Mak, 2002). Interestingly, HHV6 has also been identified in almost one-third of Hodgkin lymphomas (Di Luca et al, 1994), and may play a similar modulatory role. The significant association between EBV and HHV6 viral load and histological progression of AITL may therefore signify a reciprocal causal relationship: on the one hand, EBV and HHV6 could promote recruitment of the inflammatory infiltrate, which contributes to disruption of the normal lymphoid structure and the histological progression of AITL, while on the other hand, destruction of normal lymphoid tissues could promote further deterioration of immune function, thus exacerbating the viral infection. In this regard, it is noteworthy that the highest loads of EBV and HHV6 detected by quantitative RT-PCR were mutually exclusive. If these viruses are simply inactive passengers during the disease process one would expect to see high levels of both viral loads in at least some of the pattern III cases.
Previous studies of EBV, HHV6 and lymphoma suggest several ways in which these viruses might promote the progression of AITL. Most notably, each virus both contains genes encoding viral homologues of human cytokines, chemokines and their receptors, and modulates the production of cytokines, chemokines and receptors by lymphocytes and inflammatory cells. For example, HHV6 contains a chemokine gene U83, and chemokine receptor genes such as U12 (Isegawa et al, 1998; Zou et al, 1999). Similarly, the EBV genome contains BCRF1, a homologue of IL-10 (Moore et al, 1990). Several studies have shown that both HHV6 and EBV can modulate the production of, and response to, cytokines and chemokines such as IL-1, IL-2, IL-6, IL-10, IL-12, IL-15, IL-18, IFNγ, TNFα and CCL5 (RANTES) by many cell types (Gosselin et al, 1992; Flamand et al, 1995; Klein et al, 1996; Arena et al, 1999; Arena et al, 2001; Uchihara et al, 2005). Thus, it can be proposed that by modulating the cytokine milieu, EBV and HHV6 might promote the acquisition of the polymorphous infiltrate seen in AITL, contributing to disease progression. Moreover, the ability of many of the cytokines discussed above to promote the survival and proliferation of T cells suggests that EBV and HHV6 might also indirectly promote the expansion of the neoplastic T cells in AITL. Again, this situation is analogous to that seen in classical Hodgkin lymphoma, in which cytokines and chemokines such as IL-13, TARC, eotaxin, IL-10 and TGFβ are thought to be responsible for HRS cell survival, recruitment of T cells and eosinophils, suppression of cytotoxic T-lymphocyte function and fibrosis, respectively (Skinnider & Mak, 2002).
In addition to the above, at least two other mechanisms by which EBV and HHV6 might promote progression of AITL are suggested by our knowledge of the biology of the viruses and of AITL itself. Firstly, in vitro and in vivo data show that HHV6 is able to induce depletion and functional suppression of T lymphocytes (Flamand et al, 1995; Yasukawa et al, 1998; Gobbi et al, 1999). Together with the liberation of anti-inflammatory cytokines such as IL-10, it is possible that such effects could contribute to the immunosuppression seen in AITL. Secondly, the perifollicular localisation and expression of CD10, Bcl-6 and CXCL13 by the neoplastic T cells in AITL suggests that the tumour may originate from follicle centre T cells (Ree et al, 1999; Attygalle et al, 2002; Grogg et al, 2005). The survival and proliferation of these T cells is regulated by interactions with FDC, and indeed FDC expansion is a key feature of AITL. It is therefore intriguing that Luppi et al (1998) identified HHV6 by immunohistochemistry in FDC in cases of Rosai–Dorfman disease. Although we have not been able to verify the specificity of the available anti-HHV6 antibodies, and have therefore not been able to identify which cells harbour HHV6 in our cases, the results of Luppi et al at least raise the possibility that HHV6 might modulate the neoplastic T cells in AITL by affecting the tumour-associated FDC. The principle that an infectious agent may play a central role in lymphomagenesis without being present in the lymphoma cells themselves is now well established. For example, in gastric MALT lymphoma, the growth of tumour B-cells depends critically upon a T-cell-mediated immune response to H. pylori rather than direct stimulation by the bacterium (Hussell et al, 1996). As discussed above, similar indirect mechanisms may apply to the role of EBV and HHV6 in AITL.
There is also clinical evidence to suggest a role for EBV and/or HHV6 in the clinicopathological presentation and outcome of AITL. Battegay et al (2004) reported a case of AITL that showed complete remission following treatment with the antiviral agent valacyclovir alone. They demonstrated that the occurrence and size of enlarged lymph nodes, as well as systemic symptoms such as fatigue and night sweats, were strongly correlated with the EBV load in peripheral blood. Recently, Strupp et al (2002) and Dogan et al (2005) reported impressive treatment responses of AITL to thalidomide, an anti-angiogenic and anti-inflammatory drug. Significantly, Dogan et al (2005) showed thalidomide treatment was associated with the disappearance of EBV positive cells. This was accompanied by a reduction in the extent of the polymorphic infiltrate and by alteration of the vascular architecture. There was a marked reduction in the number of CD10 positive neoplastic T cells in the perifollicular areas, and those that remained occupied the centres of regressed follicles with reduced FDC meshworks. These clinical reports thus suggest that reduction of EBV load in AITL may be of clear clinical benefit. It is also intriguing that many of the clinical features of AITL are similar to those induced by HHV6 reactivation in immunocompromised hosts, including skin rash, arthritis and bone marrow suppression. However, whether HHV6 plays a role in the systemic clinical features of AITL remains a matter of speculation.
In this study, we have demonstrated an association among EBV, HHV6B and the histological progression of AITL, raising the possibility that these viruses may play a role in the pathogenesis of this lymphoma. The present work forms a platform for further studies investigating both the biological mechanisms of such a role and the clinical importance of HHV6B and EBV load in the course and management of AITL patients, and provides a further avenue for understanding this enigmatic disease.