Misleading Kaposi's sarcoma: usefulness of anti HHV-8 immunostaining

We report two cases of early Kaposi's sarcoma illustrating the risk of misdiagnosis. Both lesions showed histological features and clinical aspects that mimicked lymphangioendothelioma. The diagnosis of lymphangioma-like Kaposi's sarcoma was not made until a few years later, after the lesions had become more extensive and bilateral. Because of this evolution which is uncommon for a lymphangioendothelioma, immunohistochemical staining with anti-HHV8 antibody was done, and was positive in successive biopsies of our two patients. These results reveal that these vascular lesions had been Kaposi's sarcomas of the lymphangioma-like type since the beginning. In conclusion, it seems essential to search for HHV8 within endothelial cells of any vascular lesion mimicking lymphangioendothelioma or any other vascular proliferation difficult to classify. These results lead us to question whether lymphangioendothelioma is a real entity.     

Latent and lytic HHV-8 mRNA expression in PBMCs and Kaposi's sarcoma skin biopsies of AIDS Kaposi's sarcoma patients

Human herpes virus 8 (HHV-8) is associated with all clinical forms of Kaposi's sarcoma. HHV-8 DNA is present in Kaposi's sarcoma biopsies and is observed regularly in saliva and less consistently in blood of Kaposi's sarcoma patients. The expression pattern of latent (ORF 73) and lytic (vGCR, vBcl-2, and vIL-6) HHV-8 mRNA was studied in peripheral blood mononuclear cell (PBMC) samples and Kaposi's sarcoma skin biopsies from 11 AIDS Kaposi's sarcoma patients with four different nucleic acid sequence-based amplification (NASBA) assays. Patients were divided into groups according to the clinical stage of Kaposi's sarcoma (stage I-IV). All biopsies were positive for two or more of the mRNA measured. No clear difference could be seen in the expression pattern in the lesions of the different clinical stages. In the corresponding PBMC samples, very little or no mRNA was measurable in the patients with Kaposi's sarcoma stage I or II, whereas patients with more advanced Kaposi's sarcoma (stage III or IV) had more detectable mRNA in the PBMCs. Thus, the HHV-8 DNA load in the PBMCs increases in more advanced Kaposi's sarcoma, as does the frequency of mRNA detection in PBMCs.     

Iatrogenic Kaposi's sarcoma following steroid therapy for nonspecific interstitial pneumonia with HHV-8 genotyping

We present a case of iatrogenic Kaposi's sarcoma (KS) in a 64-year-old, human immunodeficiency virus (HIV)-negative woman with nonspecific interstitial pneumonia (NSIP) following steroid therapy. She suffered from longstanding exertional dyspnea, and was diagnosed to have NSIP by a thoracoscopic lung biopsy. After 4 months of steroid therapy, multiple purplish nodular or patchy skin lesions developed throughout the entire body. Microscopically, the skin lesions, consisting of closely packed spindle cells forming slit-like vascular structures, were diagnosed as typical KSs. The tumor cells were positive for CD34, factor VIII-related antigen, and human herpesvirus 8 (HHV-8) immunostainings. Using fresh tumor tissue and peripheral blood, polymerase chain reactions revealed the presence of HHV-8. Phylogenetic analysis of HHV-8 ORF K1 genes disclosed that the strain belonged to the subtype II/C and had the M allele at the right-hand side of the genome. To the best of our knowledge, this case report is the first documentation of iatrogenic KS associated with NSIP. A brief review focusing on the HHV-8 genotypes of iatrogenic KS is also presented.     

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 Kaposi's sarcoma. The mode by which HHV-8 causes Kaposi's 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 Kaposi's sarcoma. Because the HHV-8 genome contains cytokine homologues, this study investigated whether CD40 expression in Kaposi's 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 Kaposi's 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 Kaposi's sarcoma cell survival following infection by mechanisms other than those involving CD40.     

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 Kaposi's sarcoma. The mode by which HHV-8 causes Kaposi's 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 Kaposi's sarcoma. Because the HHV-8 genome contains cytokine homologues, this study investigated whether CD40 expression in Kaposi's 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 Kaposi's 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 Kaposi's sarcoma cell survival following infection by mechanisms other than those involving CD40.     

Assessment of a combined testing strategy for detection of antibodies to human herpesvirus 8 (HHV-8) in persons with Kaposi's sarcoma,persons with asymptomatic HHV-8 infection,and persons at low risk for HHV-8 infection

The performance of a human herpesvirus 8 (HHV-8) enzyme immunoassay (EIA) and selective subsequent use of an HHV-8 immunofluorescence assay (IFA) was tested in persons unlikely to be infected with HHV-8 and those who had HHV-8 detected in their saliva. The IFA was performed on samples within a range of EIA optical densities (0.15 to 0.35) where there was considerable overlap between HHV-8-infected and uninfected individuals. The sensitivity of the testing strategy was 88%, with a specificity of 97%.     

KSHV/HHV-8 associated Kaposi's sarcoma in lymph nodes concurrent with Epstein-Barr virus associated Hodgkin lymphoma

BACKGROUND: The unusual occurrence of a metastatic Kaposi's sarcoma (KS) in a lymph node affected by Hodgkin lymphoma (HL) was originally reported when knowledge of the specific virological features of these tumours was lacking. AIM: To re-evaluate this case by assessing whether the simultaneous presence of the two tumours was linked with common aetiopathogenetic factors. METHODS: The presence of EBV was investigated by in situ hybridisation, whereas KS associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) was detected by immunohistochemistry. Both viruses were analysed in the case reported, in 30 lymph nodes from patients with classic HL, and in 22 skin biopsies from patients with KS. RESULTS: Consistent with the findings in the HL and KS cases analysed, in the case showing features of both HL and KS in the same lymph node, EBV was detectable only in Reed-Sternberg (RS) cells, but not in KS spindle cells, whereas KSHV/HHV-8 was detectable only in KS spindle cells, and not in RS cells. CONCLUSION: It is probable that the development of KS and HL was related to two independent aetiological cofactors-KSHV/HHV-8 and EBV, respectively-and that the occurrence of the two malignancies in the same patient was merely fortuitous.     

HHV8 and female Kaposi's sarcoma

Kaposi's 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. © 1997 John Wiley & Sons, Ltd.     

Human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) DNA in Kaposi's sarcoma lesions,AIDS Kaposi's sarcoma cell lines,endothelial Kaposi's sarcoma simulators,and the skin of immunosuppressed patients.

We used the polymerase chain reaction on 63 tissue specimens of histologically staged classic Kaposi''s sarcoma (KS) from 40 patients, 14 specimens from 14 acquired immune deficiency syndrome (AIDS)-KS cases (all from the same geographic area over a 10-year period), and peripheral blood mononuclear cells from 1 of the non-AIDS KS patients to amplify a specific 210-bp genomic sequence of the newly discovered KS-associated herpesvirus (KSHV). Also tested were 86 benign and malignant endothelial lesions, which potentially simulated each KS histological stage and were further matched by age approximation and by sex with a classical KS specimen. The lesions included hemangioma, lymphangioma, pyogenic granuloma, and angiosarcoma. KSHV was also sought in multiple well characterized vascular endothelial cell lines from AIDS-KS lesions and in 20 mainly cutaneous benign and malignant lesions from 15 immunosuppressed transplant patients. Overall, 92% of KS tissue specimens, representing 88% of classical KS and 100% of AIDS-KS patients, and in addition the sample of peripheral blood mononuclear cell DNA, were positive as visualized on ethidium bromide gels and confirmed by Southern blot hybridization (only 1 case was negative on gell visualization but positive on Southern blot), thus confirming the close association of KSHV with KS of different clinical forms. None of the various other endothelial lesion, skin lesions in immunosuppressed patients, or AIDS-KS endothelial cell lines contained amplifiable KSHV DNA, which indicates that reactivation of KSHV is not present in the skin lesions of immunosuppressed patients and probably is not a ubiquitous agent that secondarily infects proliferative endothelium. The absence of amplifiable virus DNA in the cultured endothelium of KS suggests that the stimulus for angioproliferation originates in another host cell or under conditions not reproduced in culture. The polymerase chain reaction is a specific and sensitive means of verifying KS in the differential diagnosis of angioproliferative lessons.     

Human herpesvirus 8 in Kaposi's sarcoma and Kaposi's sarcoma-mimicking vascular tumors.

Kaposi''s sarcoma (KS) had been a rare and unusual vascular tumor until a recent epidemic of a disseminated and fulminant form of KS in AIDS patients. Infectious agents have been suspected of causing KS, and recently partial genomic DNA sequences of human herpesvirus 8 (HHV8) have been identified in AIDS-associated KS lesions. Since then, genomic DNA sequences of HHV8 have been isolated in other forms of KS. Although the partial genomic DNA sequence of HHV8 was reported to be, if rare, identified in vascular tumors other than Kaposi''s sarcoma (KS), the presence of HHV8 in a very large fraction of KS indicates that detection of HHV8 by PCR is a useful auxiliary tool in differentiating KS from other KS-mimicking vascular tumors. We examined whether the 233-bp segment of the viral DNA was detected in Korean patients with KS and other KS-mimicking vascular tumors. HHV8 sequences were identified in all of nine classic type of KS but not in three epithelioid hemangioendotheliomas and seven angiosarcomas. Our results confirm the relatively restricted distribution of HHV8 and also argue against the likelihood of secondary colonization of KS cells by HHV8.     

Epidemiology of human herpes virus 8 (HHV-8) or the herpes virus associated with Kaposi's sarcoma (KSHV)

Human herpesvirus 8 (HHV-8), also called Kaposi's sarcoma-associated herpesvirus (KSHV), is not a ubiquitous virus. In countries with a low viral seroprevalence (< 5% in adult general population) as the USA, Northern Europe and Asia, the infection concerns essentially homosexual men. In this latter population, the viral transmission seems to occur during sex. In endemic countries (HHV-8 seroprevalence between 10-70% in the adult general population) as in the Mediterranean basin (Italy, Greece), and Africa (East and Central Africa), men, women and children are infected. In these countries, HHV-8 seroprevalence increases with age and often reaches adult rates before the end of puberty. Viral transmission, in general endemic populations, seems to occur from mother to child and between sibs whereas heterosexual transmission appears to concern essentially groups at risk for sexual transmitted diseases. Saliva is a major reservoir of HHV-8.     

Detection of human herpes virus 8 DNA and sequence polymorphism in classical,epidemic, and iatrogenic Kaposi's sarcoma in South Africa

The aetiology and detection of human herpes virus type 8 (HHV-8) DNA sequences in Kaposi's sarcoma (KS) is a matter of intense investigation. We report on the detection of HHV-8 DNA and sequence polymorphism in different clinicopathological subtypes of cutaneous KS samples from South Africa. The diagnosis was confirmed by histological examination in all cases. Six patients had classic KS (CKS), 3 epidemic KS (EKS), and 3 iatrogenic KS (IKS). A nested polymerase chain reaction (PCR) assay was used to detect HHV-8 DNA in cell lysates, prepared from formalin fixed, paraffin embedded sections. We investigated polymorphism in the HHV-8 DNA using single-stranded conformational polymorphism (SSCP) analysis on the PCR products, followed by direct sequencing. HHV-8 DNA was detected in all the patients with KS, irrespective of the clinicopathological subtype. Direct sequencing was performed on 5 selected cases and showed single base pair substitutions in all. The spectrum of mutations was similar to those described previously. No correlation was found between the different types of KS and sequence variation. The results support the hypothesis that HHV-8 is strongly associated with different clinicopathological subtypes of KS and confirm the occurrence of HHV-8 in patients with CKS, EKS, and IKS in South Africa. J. Med. Virol. 52:168–172, 1997. © 1997 Wiley-Liss, Inc.     

Evaluation of BC-3 and BCP-1 cell lines in immunofluorescence assay to detect HHV-8 antibodies in Kaposi's sarcoma, multiple myeloma and other groups.

BACKGROUND: We describe a comparative study of an immunofluorescence assay using inducible BC-3 and BCP-1 cell lines as sources of HHV-8 antigens. STUDY DESIGN: Detection of both antibodies to proteins expressed in lytic cycle and during latency in sera from HIV-infected patients with KS, HIV-positive patients without KS, normal blood donors, HIV-negative pregnant women and HIV-negative patients with multiple myeloma. Where possible, detection of antibody was associated with nested PCR detection of HHV-8 in peripheral mononuclear cell (PBMC) samples collected from AIDS-KS patients. RESULTS: Immunofluorescence was more intense with the BC-3 cell line than with BCP-1, thus facilitating examination under the microscope. HHV-8 antibodies were detected among 82.75% of AIDS-KS patients, in 27.3% of HIV-infected homosexual men, 2% of blood donors and in 2% of pregnant women. No HHV-8 antibodies were detected in serum samples from HIV-negative patients presenting multiple myeloma. HHV-8 DNA sequences were detected and confirmed by southern blot hybridization in five out of 17 (29.4%) PBMC samples from AIDS-KS patients. Titre of antibodies to proteins expressed in lytic cycle was much higher than the titre of antibodies to proteins expressed during latency. CONCLUSIONS: Both immunofluorescence assays were found useful and HHV-8 seroprevalence rates reported in previous studies were confirmed. In addition, results obtained using these assays tend to provide evidence for a lack of epidemiological association between HHV-8 infection and development of multiple myeloma.     

Human herpesvirus 8 (HHV-8) detected in two patients with Kaposi's sarcoma-like pyogenic granuloma

AIMS: To report the finding of human herpesvirus 8 (HHV-8) in two patients with Kaposi's sarcoma (KS)-like pyogenic granuloma. This form of pyogenic granuloma closely resembles KS histologically and it has been reported that immunohistochemistry in such lesions may be positive for smooth muscle actin and factor VIII related antigen, which are typically negative in KS. In both patients the lesions were positive for CD31, CD34, smooth muscle actin, and factor VIII related antigen, a profile typical of KS-like pyogenic granuloma. The lesions were tested for the presence of HHV-8 DNA, which to date has been consistently found in all types of KS. METHODS: The lesions were tested for the presence of HHV-8 DNA using the polymerase chain reaction (PCR). A known HHV-8 positive KS specimen was used as the positive control. Six samples of non-KS vascular skin lesions were used as negative controls for the PCR reaction. RESULTS: Both lesions were positive on PCR for HHV-8 and the specificity of product was confirmed by direct sequencing. None of the six control vascular skin lesions was positive for HHV-8. These results strongly indicate KS as the true diagnosis and are supported by the reported clinical course in both cases. CONCLUSIONS: Techniques targeting HHV-8 DNA for detection to confirm a diagnosis of KS are both sensitive and specific. In cases where the differential diagnosis includes KS-like pyogenic granuloma, caution should be taken not to diagnose solely on the basis of immunohistochemistry phenotype. In such cases, PCR targeting HHV-8 DNA sequences is a better diagnostic tool.     

Human herpesvirus type 8 infection in an area of Northern Italy with high incidence of classical Kaposi's sarcoma

Previous studies have reported a large variation in the incidence of classical Kaposi's sarcoma across different Districts of the province of Mantua (Northern Italy). To assess whether such differences might be explained by different anti-HHV8 antibody prevalence, a serological study was conducted in 343 healthy elderly individuals resident in two adjacent Districts, at the highest and the lowest classical Kaposi's sarcoma incidence rate, respectively. Qualitative and quantitative determinations of IgG antibodies against both latent and lytic HHV-8 antigens were performed by indirect immunofluorescence assay. The assay's sensitivity was studied in 26 patients with classical Kaposi's sarcoma. Overall, anti-HHV8 antibodies were detected in 25 out of 26 patients (96%), confirming the high sensitivity of this assay. The prevalence of anti-HHV-8 antibodies was higher among individuals living in the District had a high incidence of classical Kaposi's sarcoma compared to those living in the District with low incidence (19.4% vs 9.8%, and 15.9% vs 8%; P<0.05, for latent and lytic antibodies, respectively). Anti-lytic antibody GMT was higher in people living in the District at high incidence rate compared to those of the other area (328.9 vs. 180.4; P<0.01). A higher prevalence of HHV-8 infection was found among persons living in municipalities surrounded by watercourses (OR 2.2, 95% CI: 1.10-4.32). In conclusion, variation in HHV-8 prevalence appears to explain differences in the incidence rates of classical Kaposi's sarcoma observed in different areas of the province.