Anatomic Pathology / HHV-8 STAINING
نویسندگان
چکیده
Human herpesvirus 8 (HHV-8) is recognized as a major causal agent of Kaposi sarcoma (KS), and it has been detected in all epidemiologic variants of KS. Until now, detection of HHV-8 in paraffin-embedded sections was done mostly by using reverse transcriptase– polymerase chain reaction. To assess the sensitivity and specificity of an anti–HHV-8 antibody and its potential usefulness for separating KS from its mimickers, we immunostained 72 KS samples and 108 samples of potential mimickers of KS with the monoclonal antibody latent nuclear antigen-1 (LNA-1; Advanced Biotechnologies, Columbia, MD). Cases of KS included all epidemiologic variants of the disease. Non-KS lesions included 34 angiosarcomas, 4 kaposiform hemangioendotheliomas, and 70 various benign vascular lesions. Immunostaining for CD31, CD34, and/or von Willebrand factor (factor VIII) also were performed in all cases. All but 1 case of KS (sensitivity, 99%) and none of the non-KS lesions (specificity, 100%) stained with the LNA-1 anti–HHV-8 antibody. The LNA-1 anti–HHV-8 antibody is a reliable marker of all variants of KS. Because KS mimickers are consistently negative for this marker, its use for diagnostic purposes is recommended. Kaposi sarcoma (KS) is a vascular, often multicentric lesion characterized by 4 epidemiologic manifestations: classic, iatrogenic, endemic (ie, African), and associated with AIDS.1 The classic form of KS initially was described by Moriz Kaposi as a cutaneous sarcoma of elderly people (most patients are men and older than 50 years).2 Classic KS is observed chiefly in the Mediterranean region, Eastern Europe, and the Middle East. The iatrogenic variant is observed characteristically in a context of organ transplantation, long-term cytotoxic drug administration, and the use of corticoids or in autoimmune diseases. It generally occurs 24 months after the onset of therapy.2 In endemic (African) KS, male predominance is also a characteristic feature, but patients are usually younger (younger than 40 years). An often fatal, lymphadenopathic form occurring in children has been described in this KS form.2 Among the KS cases associated with acquired immunodeficiency states, AIDS-associated KS is the most frequent. Among HIV-infected patients, homosexuals and bisexuals are high-risk groups.3 KS is the main proliferating lesion in patients with AIDS, of whom 15% to 20% develop it.4 The clinical course and histopathologic manifestations of these 4 epidemiologic forms are roughly similar. Tumor sites include skin, mucosa (oropharynx and digestive tract), lymph nodes, and visceral organs. In 1994, Chang et al4 identified 2 unique herpesviruslike DNA sequences in AIDS-associated KS, sequences corresponding to human herpesvirus 8 (HHV-8), a virus of the γ subtype of the herpesvirus family. This virus is akin to herpesvirus saimiri, a virus that is responsible for lymphoproliferative lesions in new-world primates.5 HHV-8 DNA has been detected in virtually all epidemiologic forms of KS but not in the adjacent uninvolved skin of patients with KS or in Anatomic Pathology / ORIGINAL ARTICLE Am J Clin Pathol 2004;121:330-334 331 331 DOI: 10.1309/96U16LRRAN5HWWVE 331 © American Society for Clinical Pathology unaffected subjects.6 Interestingly, whereas about 5% of the population worldwide probably is infected with HHV-8, the incidence of KS is less than 1/100,000.5 The presence of HHV-8 is now considered to be a major initiating event for the development of KS.7,8 It is observed in the spindle cells of KS and in endothelial cells and also has been found in tumor leukocytes by some3,9 but not all8 authors. Until now, HHV-8 has been detected in KS by using polymerase chain reaction (PCR)6,10,11 and only seldom by using immunochemical analysis applied to paraffinembedded tumor tissue samples.8,12,13 By using PCR, HHV-8 has been detected in 95% to 100% of KS cases. This finding is important because it permits a firm diagnosis of KS in cases of exiguous or otherwise inadequate histologic material.5 Conflicting results have been reported in the literature about the presence of HHV-8 in angiosarcoma. By using PCR, some authors detected viral DNA in some angiosarcomas,14 whereas others did not.11,12,15 In addition to KS, HHV-8 viral DNA also has been detected in a subset of myofibroblastic inflammatory tumors,16 in primary effusion lymphomas, and in multicentric Castleman disease.16-19 The role of HHV-8 in the histogenesis of multiple myeloma is questionable.20,21 Evidence of HHV-8 DNA also has been detected in a few nonHodgkin lymphomas22 and in 1 Hodgkin lymphoma.23 Materials and Methods We retrieved 72 cases of KS and 108 of non-KS vascular lesions from the files of the Oscar Lambret Cancer Center, Lille, France; the University Institute of Pathology, Lausanne, Switzerland; the François Baclesse Cancer Center, Caen, France; and the Bergonié Institute, Bordeaux, France. We then immunostained the samples for HHV-8. The diagnosis of KS was based on epidemiologic data, morphologic features, and reactivity for endothelial cell markers (CD34, CD31, and/or von Willebrand factor). All cases of KS for which epidemiologic data were not defined clearly were discarded from the series. Early-stage cases of cutaneous KS were diagnosed in the context of an HIV infection or in HIV-negative, elderly Mediterranean patients. These patients had multiple synchronous lesions or a history of excised KS. Distribution of KS according to epidemiologic manifestations is shown in ❚Table 1❚. Non-KS lesions included 34 angiosarcomas with a spindle cell component, 4 kaposiform hemangioendotheliomas, and 70 benign vascular lesions ❚Table 2❚. Fifty KS samples were fixed in 4% buffered formalin, 14 in conventional aqueous Bouin fluid, and 8 in Holland Bouin fluid. Histologic diagnoses were based on the examination of H&E-stained slides. Additional paraffin-embedded sections of all 180 cases were evaluated immunohistochemically according to the streptavidin-biotin-peroxidase method of Hsu et al24 (LSAB kit, DAKO, Glostrup, Denmark). The following antibodies were used: CD34 (monoclonal, clone Qbend 10, dilution 1:200, 30minute incubation, Dakopatts, Glostrup, Denmark), CD31 (monoclonal, clone 1C/70A, dilution 1:320, 30-minute incubation, Dakopatts), von Willebrand factor (factor VIII; polyclonal, dilution 1:800, 30-minute incubation, Immunotech, Marseille, France), and HHV-8 (monoclonal antibody to latent nuclear antigen [LNA-1] open reading frame 73, dilution 1:1,000, 60minute incubation, Advanced Biotechnologies, Columbia, MD). Tissue sections were submitted to microwave oven heating (20 minutes in EDTA buffer [pH 8]). Then, the sections were immunostained using the LSAB kit (DAKO) in an automated immunostainer (TechMate Horizon, DAKO). All steps were performed at room temperature, and diaminobenzidine was used as a chromogen. Appropriate positive and negative control samples were used throughout. ❚Table 1❚ Kaposi Sarcoma (KS) Breakdown According to Manifestations AIDS-Associated Endemic (African) Iatrogenic Characteristics Classic KS (n = 45) KS (n = 16) KS (n = 8) KS (n = 3) Total (N = 72) Male 37 16 8 2 63 Female 8 0 0 1 9 Median age (range), y 72 (52-92) 41 (31-62) 32 (17-47) 64 (49-80) 60 (31-92) Location Lower extremities (skin) 29 (1 patch-stage KS) 3 (2 patch-stage KS) 5 0 37 Upper extremities (skin) 10 (1 patch-stage KS) 3 (1 patch-stage KS) 1 0 14 Head and neck* 3 5 (one patch stage KS) 1 9 Genital† 2 1 1 1 5 Lymph node (cervical/inguinal) 1 3 0 0 4 Mediastinum 0 1 0 0 1 Retroperitoneum 0 0 1 0 1 Skin (multiple lesions) 0 0 0 1 1 * Skin, laryngeal mucosa, tonsils. † Scrotum, glans penis, collum glandis. Robin et al / HHV-8 STAINING IN KAPOSI SARCOMA 332 Am J Clin Pathol 2004;121:330-334 332 DOI: 10.1309/96U16LRRAN5HWWVE © American Society for Clinical Pathology For anti–HHV-8 staining, the positive control sample consisted of a lymph node involved by Castleman disease. Labeling for HHV-8 was considered positive if the staining was observed exclusively in the tumor cell nuclei (including spindle cells and endothelial cells), had a granular appearance, and had no concomitant cytoplasmic staining. It was considered negative when no staining was observed in the tumor cell nuclei, as opposed to the positive control samples.
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