M M Kennedy

Identification of HHV8 in early Kaposi's sarcoma: implications for Kaposi's sarcoma pathogenesis.

AIMS: Kaposis sarcoma is a vascular tumour of uncertain pathogenesis possibly caused by an infectious agent, identified in high risk groups. Accumulating solution phase polymerase chain reaction (PCR) and seroepidemiological data suggest that a previously undescribed herpes DNA virus (human herpesvirus 8 (HHV8)) is the causative agent. Using a unique cohort of early Kaposis sarcoma, the precise cell type infected with HHV8 in such lesions was identified to elucidate further the role of HHV8 in the pathobiology of Kaposis sarcoma. METHODS: Sixteen cases of early Kaposis sarcoma (derived from skin and lymph node) were assessed for the presence of HHV8 using both standard solution phase PCR and TaqMan PCR to the KS330 Bam region of HHV8. In situ amplification was also performed on a selected group in an attempt to identify the candidate infected cells. RESULTS: Using both conventional solution phase and TaqMan PCR, 87% of cases were positive. In addition, HHV8 amplicons were localised in situ to endothelial and spindle cell proliferations in early Kaposis sarcoma. The HHV8 viral load varied from lesion to lesion. CONCLUSIONS: The presence of HHV8 in early lesions supports a role for HHV8 in the pathogenesis of Kaposis sarcoma. Coupled with recent seroepidemiological studies, these results suggest that HHV8 is the aetiological agent of Kaposis sarcoma. Its precise interaction with other factors known to be involved in the development of Kaposis sarcoma, including cytokines and anti-apoptosis genes, requires elucidation.

Cellular localisation of HHV-8 in Castleman's disease: is there a link with lymph node vascularity?

Aims—Human herpesvirus 8 (HHV-8) has been identified in multicentric Castlemans disease and in angioimmunoblastic lymphadenopathies. However, the presence of the virus does not necessarily indicate an aetiological role in these conditions. This study investigates the cell types infected by HHV-8 in Castlemans disease and examines the correlation between HHV-8 and Castlemans disease lymph node angiogenesis. Methods—Sixteen formalin fixed, paraffin wax embedded samples from patients with Castlemans disease (six multicentric, 10 solitary) were examined for the presence of HHV-8 using the polymerase chain reaction (PCR), non-isotopic in situ hybridisation, PCR in situ hybridisation (PCR-ISH), and real time quantitative TaqMan PCR to HHV-8 open reading frame 26 (ORF-26), and viral (v)-cyclin encoding regions. Vascularity was assessed using CD34, CD31, and factor VIII immunocytochemistry, and lymph nodes were scored as “low” or “high”. Results—Five multicentric Castlemans disease and two solitary Castlemans disease biopsies were positive for HHV-8. HHV-8 was identified in approximately 10% of intranodal B lymphocytes, in endothelial cells, and in subcapsular spindle cell proliferations. The copy number of HHV-8 was low at 10–50 copies/1000 cells. The highest copy number was in subcapsular spindle cells. There was no correlation between vascularity score and HHV-8 status. Conclusion—The preferential localisation of HHV-8 in subcapsular spindle cell proliferations (where early intranodal Kaposis sarcoma initiates) and endothelial cells in Castlemans disease might finally explain the link between intranodal Kaposis sarcoma and Castlemans disease.

HHV8 and Kaposi's sarcoma: a time cohort study.

AIMS: The recent finding that human herpes virus 8 (HHV8) is found in the majority of Kaposis sarcoma (KS) cases supports the epidemiological observation that the tumour may be caused by an infectious agent. This study aimed to address when and how HHV8 evolved. METHODS: A cohort of African endemic KS (49 samples from 45 patients) and European KS (18 samples from 13 patients), spanning 27 years, was assessed for the presence of HHV8 by both standard solution phase polymerase chain reaction (PCR) and the newly described technique of TaqMan PCR. RESULTS: HHV8 was present in approximately 49% (24 of 49 tissue samples) of the African cases and in more than 90% (16 of 18 tissue samples) of the European cohort, in keeping with recent seroepidemiological data. CONCLUSIONS: HHV8 is strongly linked to the development of KS; however, in some patients, other factors may operate. The utility and flexibility of TaqMan PCR in detecting low copy viral target in human tissues was demonstrated.

Human herpes virus 8 (HHV-8) in Kaposi's sarcoma: lack of association with Bcl-2 and p53 protein expression.

AIMS: Human herpes virus 8 (HHV-8) is the infectious agent implicated in the pathogenesis of Kaposis sarcoma, although its mode of action is unclear. Recent work indicates that the HHV-8 genome encodes a viral Bcl-2 homologue (v-Bcl-2). The aim of this study was to explore Bcl-2 expression in Kaposis sarcoma using a unique set of HHV-8 positive and negative cases, and to determine whether there is a relation with p53 expression. METHODS: Up to 18 specimens from 17 patients were selected. HHV-8 status was determined using nested polymerase chain reaction (PCR) to the open reading frame (ORF) 26, with further confirmation by TaqMan PCR. In addition, Bcl-2 and p53 immunohistochemistry were performed using standard protocols. RESULTS: The results suggest that Bcl-2 and p53 expression is independent of HHV-8 status. In addition, there does not appear to be a direct correlation with disease stage. CONCLUSIONS: HHV8 histopathogenesis is likely to be a multifactorial complex process, which may be mediated in part by viral genes and apoptosis regulating homologues.

Localisation of HHV-8 in AIDS related lymphadenopathy.

Background—Many lymph node abnormalities have been described in AIDS. These include opportunistic infections that sometimes result in spindle cell pseudotumours, Kaposis sarcoma (KS), malignant lymphoma (Hodgkins and nonHodgkins), and florid reactive hyperplasia. Among these, reactive hyperplasia is the most common manifestation of AIDS related lymphadenopathy. Aim—To examine whether human herpesvirus 8 (HHV-8), the aetiological agent of KS, can be localised in AIDS related lymphadenopathy and whether its appearance in such nodes is predictive of Kaposis sarcoma development. Methods—A series of human immunodeficiency virus (HIV) positive men (n = 21) with AIDS related lymphadenopathy who at the time of presentation had KS or subsequently developed KS (n = 5) were examined. The prevalence of HHV-8 was assessed in these patients using solution phase polymerase chain reaction (PCR), real time TaqMan quantitative PCR, and in cell amplification techniques (PCR in situ hybridisation (PCR-ISH) and labelled primer driven in cell amplification). Results—Using standard solution phase PCR in a nested format, only two of the 21 patients with AIDS related lymphadenopathy were positive for HHV-8. The lymph node of one of these patients contained KS lesions. Three HHV-8 positive patients were identified using TaqMan PCR (the original two positive patients and one additional patient). All of the positive patients either subsequently developed KS (n = 2) or had KS at the time of diagnosis (n = 1). Two additional patients subsequently developed KS, but were negative for HHV-8 by solution phase PCR and TaqMan PCR. Using PCR-ISH, HHV-8 amplicons were identified in some lymphoid cells (in one patient) and in spindle cells of the KS lesion in another. The positive lymphoid cells were predominantly concentrated in B cell areas of the affected lymph nodes, confirming the B cell tropism exhibited by HHV-8. Conclusions—The presence of HHV-8 in AIDS related lymphadenopathy is predictive of KS development and probably represents seeding of HHV-8 infected B cells from the peripheral blood. These findings support a role for HHV-8 in the pathobiology of KS.

HHV8 and female Kaposi's sarcoma

Kaposis sarcoma (KS) is an enigmatic tumour of uncertain histogenesis. Epidemiological data have long suggested that KS may be caused by an infectious agent, possibly sexually transmitted. Following the documentation of human herpesvirus 8 (HHV8) and its strong association with all forms of KS, it now appears that the putative agent has at last been identified. As KS is rare in females, a unique group was screened for the presence of HHV8 using both conventional solution‐phase polymerase chain reaction (PCR) and the newly described technique of TaqMan® PCR. The presence of HHV8 was demonstrated in 10/12 of these female patients. This further supports the direct role of HHV8, in conjunction with cytokines and other factors, in the pathogenesis of KS.

CD40 upregulation is independent of HHV-8 in the pathogenesis of Kaposi's sarcoma

AIMS: Human herpesvirus 8 (HHV-8) is now acknowledged as the infective cofactor in the pathogenesis of Kaposis sarcoma. The mode by which HHV-8 causes Kaposis sarcoma is unresolved and it is probable that it acts in conjunction with other factors including cytokines, anti-apoptosis proteins, and cell surface receptors. CD40, a cell membrane receptor belonging to the tumour necrosis factor receptor super family, promotes B cell survival and is expressed constitutively on endothelial cells. It is upregulated on cytokine treatment and has been documented recently in Kaposis sarcoma. Because the HHV-8 genome contains cytokine homologues, this study investigated whether CD40 expression in Kaposis sarcoma correlated with HHV-8 status, using a unique set of HHV-8 positive and negative specimens. METHODS: Twenty one paraffin wax embedded samples of Kaposis sarcoma were selected, of which 18 were screened for the presence of HHV-8 using both conventional solution phase and TaqMan polymerase chain reaction (PCR). CD40 immunohistochemistry was assessed using a biotinylated amplification system. Staining was scored semiquantitatively. RESULTS: The results indicated that the expression of CD40 is independent of viral status, being present in both HHV-8 positive and negative specimens. CONCLUSIONS: This suggests that HHV-8 promotes Kaposis sarcoma cell survival following infection by mechanisms other than those involving CD40.