Latency-associated nuclear antigen expression and human herpesvirus-8 polymerase chain reaction in the evaluation of Kaposi sarcoma and other vascular tumors in HIV-positive patients

Department of Pathology and Laboratory Medicine, Emory University Hospital, Atlanta, GA, USA.
Modern Pathology (Impact Factor: 6.19). 05/2005; 18(4):463-8. DOI: 10.1038/modpathol.3800221
Source: PubMed


Human herpesvirus-8 (HHV-8) latency-associated nuclear antigen (LANA) is expressed in endothelial and spindle cells of nearly all Kaposi sarcomas, and the presence of this antigen in serum is strongly correlated with the risk of developing Kaposi sarcoma in immunocompromised individuals. Studies of vascular tumors occurring in the general population show LANA expression to be specific for Kaposi sarcoma. No study to date, however, has examined whether non-Kaposi sarcoma vascular tumors arising in immunocompromised patients may express LANA, possibly reflecting origin from an HHV-8-infected endothelial progenitor cell. The objective of this study was to evaluate the specificity of LANA expression for Kaposi sarcoma in immunocompromised patients by LANA immunohistochemistry and real-time polymerase chain reaction (PCR) for HHV-8. A total of 13 cases of non-Kaposi sarcoma vascular tumors (12 hemangiomas and one epithelioid hemangioendothelioma) and 24 cases of Kaposi sarcoma, all from known HIV-positive patients, were immunostained for LANA and evaluated for the presence of HHV-8 DNA by real-time PCR. LANA expression was seen in 22 of 24 (92%) of Kaposi sarcoma cases and in 0 of 13 non-Kaposi sarcoma cases. Real-time PCR detected HHV-8 in all of the Kaposi sarcoma cases and in four of the non-Kaposi sarcoma cases (all hemangiomas). LANA expression appears to be a highly sensitive and specific marker of Kaposi sarcoma in both the general population and in HIV-positive patients. This is in contrast to HHV-8 PCR, which is positive in a small subset of non-Kaposi sarcoma vascular tumors, most likely due to detection of HHV-8 within intratumoral blood mononuclear cells by the highly sensitive real-time PCR technique. For this reason, LANA immunohistochemistry is preferable to HHV-8 PCR for the evaluation of problematic vascular proliferations in HIV-positive individuals.

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    • "The primer/probe sequences of our HHV-8 ORF26 qPCR assay have been used by many laboratories [23,31], but problems with false positives have been reported [32]. Hammock et al.[23] perform their PCR assays with up to 50 cycles in order to detect potentially very low viral loads. "
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    ABSTRACT: Background: Human herpesvirus 8 (HHV-8), the aetiological agent of Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL) is rare in Australia, but endemic in Sub-Saharan Africa, parts of South-east Asia and Oceania. While the treatment of external KS lesions can be monitored by clinical observation, the internal lesions of KS, MCD and PEL require extensive and expensive internal imaging, or autopsy. In patients with MCD and PEL, if HHV-8 viraemia is not reduced quickly, ~50% die within 24 months. HHV-8 qPCR is a valuable tool for monitoring HHV-8 viraemia, but is not available in many parts of the world, including those with high prevalence of KS and HHV-8. Methods: A new molecular facility with stringent three-phase workflow was established, adhering to NPAAC and CLSI guidelines. Three fully validated quantitative assays were developed: two for detection and quantification of HHV-8; one for GAPDH, necessary for normalisation of viral loads in tissue and peripheral blood. Results: The HHV-8 ORF73 and ORF26 qPCR assays were 100% specific. All qPCR assays, displayed a broad dynamic range (102 to 1010 copies/μL TE Buffer) with a limit of detection of 4.85x103, 5.61x102, and 2.59x102 copies/μL TE Buffer and a limit of quantification of 4.85x103, 3.01x102, and 1.38x102 copies/μL TE Buffer for HHV-8 ORF73, HHV-8 ORF26, and GAPDH respectively.The assays were tested on a panel of 35 KS biopsies from Queensland. All were HHV-8 qPCR positive with average viral load of 2.96x105 HHV-8 copies/μL DNA extract (range: 4.37x103 to 1.47x106 copies/μL DNA extract): When normalised these equate to an average viral load of 2.44x104 HHV-8 copies/103 cells (range: 2.20x102 to 7.38x105 HHV-8 copies/103 cells). Conclusions: These are the first fully optimised, validated and MIQE compliant HHV-8 qPCR assays established in Australia. They worked well for qualitative detection of HHV-8 in archival tissue, and are well-suited for quantitative detection in whole blood. They are now available for research, for clinical diagnosis of HHV-8 infection, and for monitoring treatment efficacy.
    Full-text · Article · Sep 2012 · BMC Infectious Diseases
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    • "The invariable immunoreactivity for HHV8 [3] [19] detected in the present study was previously reported in KS [12] [13] [20]. Other authors [20] [21] extrapolated that HHV8 LANA-1 detection could be used to distinguish Kaposi's sarcoma from other vascular and nonvascular spindle cell lesions. "
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    ABSTRACT: Kaposi's sarcoma (KS) is an angioproliferative disorder associated with human herpesvirus 8 infection. Classic KS is the most prevalent type of KS in countries of the Mediterranean basin including Egypt. Several in vitro studies have detected c-kit expression in AIDS related-KS however, only a few studies addressed this issue in the classic type with no data on the ethnicity of studied cases. The prospect of installing targeted anti- c-kit treatment to KS patients presents a promising avenue in KS therapeutics. To elucidate the expression of c-kit in classic KS cases and study possible relations with expression of HHV8 latency-associated nuclear antigen-1 (LANA-1) and other clinicopathological parameters. Twenty four cases of classic KS of the plaque and nodular stages in the lower limb were studied. Immunohistochemical detection of HHV8-LANA-1 and c-kit was carried out on archival paraffin embedded tissue, possession of the Pathology and Dermatology Departments, Alexandria School Of Medicine, Egypt. Statistical analysis of possible relations between both antigens and clinicopathological parameters (patient's age and gender and histological stage) was performed. HHV8 expression was detected in 100% of cases while c-kit immunoreactivity was found in 54.2% of cases. There was no correlation between c-kit and HHV8 immunoreactivity or any of the studied clinicopathological parameters. This is the first report of c-kit expression in classic KS in an ethnically homogeneous cohort of Arabs of the Mediterranean region. We detected c-kit expression in about half the cases with no relationship to HHV8 LANA expression or clinicopathological parameters.
    Full-text · Article · Mar 2012 · Journal of the Egyptian National Cancer Institute
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    • "The immunogenic LNA-1 is encoded by Open reading frame (ORF) 73 of Kaposi's sarcoma-associated herpesvirus. It is expressed in endothelial and spindle cells of nearly all Kaposi's sarcomas (Hammock et al., 2005). This antigen maintains the herpesvirus episome and tethers the viral genome to chromatin during mitosis (Radkov et al., 2000). "
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    ABSTRACT: Typically, Kaposi's sarcoma (KS) also presents with immune cell infiltrates. This study does immunophenotype characterization for demographics of some of these cells to test the hypothesis that 'development of end-stage (nodular/tumor) KS is associated with numeric alteration in immune cell infiltrates.' Fifteen cases of classic-KS (nodular/tumor) and 20 normal biopsies were examined using antigen-antibody reactions and immunoperoxidase staining for histiocytes (CD68), B cells (CD20) and T cells (CD3). Variations between normal and lesions were observed with significant increases in immune cell infiltrates (2.3+/-0.6 vs. 6.4+/-0.4); CD68(+) (4.0+/-1.0 vs. 7.0+/-0.5); CD3(+) (3.0+/-1.1 vs. 10.1+/-0.8); and CD20(+) (0.0+/-0.0 vs. 0.3+/-0.0). Increased density of immune cells in the nodular lesions of KS may reflect increased antigenicity of these lesions; the increase in T cells suggests enhanced T cell activity in KS. However, these issues should be tested by further studies.
    Preview · Article · Feb 2008 · Cell Biology International
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