Pathology Update

Pathology Update: Human Herpesvirus-8 DNA in Visceral Kaposi's Sarcoma of AIDS and Other Immunocompromised Patients in Hungary

K. Nagy, MD¹; A. Horváth, MD; M. Medveczky, Z. Szabó, MD³;A. Tóth, MD⁴;B. Szende, MD⁴; B.G. Yangco, MD²; and P. Medveczky, MD² National Institute of Dermato-Venereology, Hungary;¹ the University of Sourth Florida;² St. László Hospital³ and Semmelweis Medical University,⁴ Hungary

This regular feature presents special issues in oncologic pathology.

Introduction

Kaposi's sarcoma (KS), first described over a century ago as angiosarcoma idiopathicum haemorrhagicum,[1] is a neoplasm of uncertain histogenesis. KS rarely occurs in elderly individuals whose origins are in eastern Europe, the Mediterranean region, and the Middle East. It occurs more frequently in immunosuppressed organ transplant recipients.[2-5] KS is a common neoplasm in patients infected with the human immunodeficiency virus (HIV) and in those with acquired immunodeficiency syndrome (AIDS).[6,7] The risk of KS development in homosexual men with AIDS is 20 times higher than in HIV-infected children and hemo-philiacs.[2] Epidemiologic data suggest that sexually transmitted agents may be involved in the pathogenesis of KS,[6,8] but to date, no causal role has been proven for any known infectious agent.

DNA sequences of a novel herpesvirus were recently discovered in biopsies of KS patients by representational difference analysis.[9,10] DNA sequences were closely homologous to the minor capsid and tegument protein genes of Herpesvirus saimiri (51% homology) and Epstein-Barr virus (39% homology) of the Gammaherpesvirinae subfamily. The new virus was referred to as KS-associated herpesvirus or HHV-8. Subsequently, HHV-8 sequences were found in the majority of AIDS-associated KS lesions[9,11,12] and in various epidemiologic forms of KS other than AIDS (ie, classic European KS,[13] endemic African KS in HIV-negative individuals, HIV-negative homosexual men,[2,11,13] and posttransplant KS cases [unpublished data, K.N. and N.T., 1996]).[14] The viral genome also has been identified in a rare and distinctive subset of B-cell lymphomas called body-cavity–based lymphomas[15] (recently renamed "primary effusion lymphoma") that occurs in HIV-infected individuals (usually in association with Epstein-Barr virus). It also occurs in Castleman's disease, a multicentric angiolymphoproliferative hyperplasia that often presents with KS.[14] HHV-8 sequences were detected in peripheral blood of HIV-infected individuals without KS, and it has been suggested that this virus might predict subsequent development of KS.[16] HHV-8 was also demonstrated in lymphoproliferative disorders[17] and blood mononuclear cells of HIV-negative persons.[18] A low level of viral induction and de novo infection in culture by HHV-8 has been reported.[19] Renne et al[20] demonstrated that HHV-8 virion production can be activated in body-cavity–based lymphoma cells by phorbol esters, and they presented ultrastructural visualization of the new virus.[12,21] High-titer serial propagation of HHV-8 in a cell line also has been observed by G. Nabel and B. Nickoloff (personal communication, 1996).

The eastern and central European regions, where KS was originally described and has sporadically been present for more than a century, are affected by the AIDS epidemic at a lesser degree than that of western Europe. AIDS incidence in Hungary remains low (0.2/100,000 people),[20] and although homosexuals comprise the most af-fected risk group, the incidence of KS is less than 10% in patients with AIDS. From the beginning of the AIDS epidemic in 1986 through 1995, AIDS-associated KS was found in 13 (6.4%) of 202 patients with AIDS.[23]

Conversely, KS is the most common malignant tumor in Hungary in immunosuppressed recipients of organ transplants. From 1991 to 1996, KS occurred in eight (22%) of 36 posttransplant neoplasms.[24] Classic KS cases have a disseminated and visceral appearance, and they occur more frequently in patients under 50 years of age.

The presence and prevalence of KS-associated human herpesvirus-8 (HHV-8) DNA were determined in cutaneous and visceral lesions of various forms of KS in AIDS and other immunocompromised patients in Hungary. This study includes all AIDS-associated KS observed in Hungary from 1986 to 1995. HHV-8 DNA was detected in 92% of AIDS-associated KS, in all cases of HIV-negative immunosuppressed organ transplant recipients with KS, and in classic KS patients studied. All samples were tested by polymerase chain reaction (PCR) with specific primers to amplify KS330₂₃₃, an HHV-8 DNA sequence. HHV-8 DNA was present not only in the cutaneous KS lesions, but also in the majority of visceral KS lesions. Since AIDS-associated KS occurs in less than 10% of AIDS patients, and since KS is responsible for 22% of all posttransplant tumors in this region of eastern central Europe, this suggests that HHV-8 is circulating in the general population as a persistent infection and is controlled by the immune system.

Following is our report on the detection and presence of HHV-8 sequences in various forms of KS associated with AIDS, posttransplant KS, and classic KS in Hungary.

Materials and Methods

Kaposi's Sarcoma Samples

We investigated the presence of HHV-8 DNA in a total of 20 patients (Table). Of the 35 KS lesions obtained from these individuals, 24 specimens of cutaneous and visceral KS lesions were acquired from 13 patients with AIDS (representing all documented AIDS-associated KS patients observed in Hungary from 1986 through 1995). All specimens were formalin-fixed, paraffin-embedded KS tissue from 14 different organs. Five specimens of formalin-fixed, paraffin-embedded KS tissue samples from three immunosuppressed renal transplant patients were also analyzed, as well as four cutaneous KS biopsies from classic KS cases and from two samples of uninvolved skin. Tissue specimens were fresh-frozen immediately after removal and stored at –80 degrees C. The diagnosis of KS was confirmed in all cases by histopathologic examination.

DNA Preparation

DNA was purified after deparaffinization of the tissues,[25] proteinase K digestion and phenol and phenolchloroform extraction, and ethanol precipitation and resuspended in deionized, distilled water and stored at 4 degrees C.

Polymerase Chain Reaction Detection of HHV-8

PCR primers KS1 and KS2 were synthesized, which amplifies a 233-bp DNA fragment KS330₂₃₃, located from base pairs 987 to 1219 of the KS330 Bam fragment.[9] This sequence is homologous to portions of the minor capsid genes ORF26 of H. saimiri and BDLF1 of Epstein-Barr virus.[10] PCR reaction was performed in 25 mL of solution for 35 cycles of amplification[10] in an Ericomp Thermal Cycler (San Diego, Calif). The PCR products were analyzed on 2% agarose/ethidium-bromide gel and were transferred to a nylon membrane. They were then subjected to Southern blot hybridization using specific radiolabeled- probe DNA cloned from an HHV-8-positive AIDS-associated KS lesion.

To determine if the DNA of KS specimens were amplifiable, PCR was also performed with a primer pair specific for the human dihydrofolate reductase (DHFR) gene that resulted in amplification of a 240-bp-long fragment of DHFR.[26] DNA from HUT102 cells that contained the DHFR gene was used as a positive control. Representative amplification product specifically hybridized in the expected 233-bp region was sequenced by standard methods. Antibodies to HIV-1 and HIV-2 were detected by enzyme-linked immunosorbent assay in the sera of patients.

Results

A representative Southern blot hybridization analysis of PCR-amplified products (Figure) showed a 233-bp fragment of KS330 characteristic of HHV-8 DNA in all three patient groups with various forms of KS (classic KS: lane 3; AIDS-associated KS: lanes 4 and 5; and HIV-negative posttransplant KS: lanes 6 and 7). However, this was not found in samples of uninvolved skin (classic KS: lanes 1 and 2). In lanes 5, 7, and 8, an incomplete additional band is visible that may be due to the partial hybridization of the radiolabeled-probe DNA (originated from an HHV-8-positive AIDS-associated KS lesion) to the DNA of KS of mesentherial and liver origin.

HHV-8 DNA was detected in 12 (92%) of the 13 cases of AIDS-associated KS. The oldest samples were from 1987, and the most recent samples originated from autopsies performed in 1995. The first diagnosis of AIDS in Hungary occurred in 1987 (Patient #1). KS was confirmed histopathologically in skin biopsies (material was not available) and in various visceral organs. HHV-8–related DNA sequences were detected in the KS lesion from the tonsil and the pharynx. Several visceral KS lesions from various organs from Patient #2 were analyzed. HHV-8 DNA was detected in the pleura and lung but not in other KS lesions. The DHFR gene was negative in the liver and the spleen in this patient, suggesting that the absence of the HHV-8 signal is attributable to an absence of proper DNA or inhibition of amplification. In lymph nodes, however, we were unable to detect a positive signal for HHV-8 in spite of DHFR positivity; this may have been due to the low copy number or the absence of the HHV-8 sequence. Tissue sample from Patient #5 was negative for KS330₂₃₃ but was also negative for DHFR.

All three posttransplant cases were positive for the presence of HHV-8 DNA. KS330₂₃₃ sequences could be detected from all visceral KS lesions, while in one case (Patient #3), cutaneous KS was negative.

Cutaneous biopsies from four classic KS cases also were analyzed. HHV-8–related DNA sequences were detected in all cutaneous KS lesions but not in unaffected skin samples from the same patients. All posttransplant and classic KS patients were negative for the HIV antibody.

The HHV-8 probe used in the Southern blots of this study was cloned and sequenced. Sequence analysis revealed differences at two nucleic acid positions compared with the prototype sequence derived from a genomic library made from a KS lesion.[9,19] At position 1033, C was substituted for T and at position 1160, T was substituted for C, showing a 97.5% homology to the published sequence of HHV-8 (KSU 18551).

Discussion

Epidemiologic evidence suggests that various forms of KS are associated with an infectious agent. Cytomegalovirus, Epstein-Barr virus, C-type retroviruses, papillomaviruses, hepatitis B virus, HHV-6 and -7, and other microorganisms were linked with KS.[27] Compared with HIV-negative individuals, those who are infected with HIV have an estimated probability of 40% that cancer (especially KS) will develop.[15,24] This aggressive form of KS is most prevalent in HIV-infected homosexual men in the United States and Europe, but it is rarely seen in patients infected with HIV caused by heterosexual contact, intravenous drug use, or blood transfusion. This suggests that AIDS-associated KS may be a sexually transmitted disease.

HHV-8 belongs to the Gammaherpesvirus subfamily and is genetically related to Epstein-Barr virus and H. saimiri, both of which latently persist in lymphocytes and are associated with malignant lymphomas.[28] HHV-8 also has been detected in body-cavity–based lymphomas of AIDS patients. It has been suggested that HHV-8 may act in conjunction with Epstein-Barr virus to produce transformation,[29] and it also has been speculated that HIV may act additively with HHV-8 in the pathogenesis of KS contributing to the immunosuppression. According to this hypothesis, tat secreted by HIV-infected cells acts synergistically with basic fibroblast growth factor and may cause initial proliferation of endothelial cells.[30] HHV-8 DNA also has been detected in squamous cell skin carcinomas of transplant recipients.[31] These data support the concept that the new HHV-8 plays an important role in the etiopathogenesis of KS.

Finding HHV-8 DNA sequences in peripheral blood mononuclear cells, in B lymphocytes of HIV-positive and -negative patients with KS, in HIV-positive patients without KS,[16] and in peripheral blood mononuclear cells of healthy donors[18] suggests that the virus is widespread. However, as with Epstein-Barr virus, it is probably undetectable in many individuals.[32] This concept is also supported by our findings that HHV-8–related DNA sequences could be detected in 92% to 100% of KS lesions with various origin (ie, AIDS-associated, posttransplant, and classic form) in a relatively homogenous white population of eastern and central Europe.

KS is more common in immunosuppressed transplant recipients in Hungary,[15,24] thus indicating that immunosuppression predisposes to KS and that, if a transmissible agent is involved, it must be present in the general population.[27] The putative KS agent had been expected to establish a persistent infection, controlled by the immune system.[32] However cofactors other than immunosuppression also may be necessary to promote the development of KS.[16] Our finding that KS is not restricted predominantly to AIDS-associated cases also favors an etiologic role for such a latent virus circulating in the general population that preferentially colonizes KS lesions in immunosuppressed patients. Appreciation is expressed to Béla Kemény for initial help on sample handling. This work was supported by a grant from the National Institutes of Health (5R01CA43264-11).

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