Multicentric Castleman’s disease (MCD) is a rare disease without a clearly defined treatment. We compared several such assays with a panel of 88 specimens from human immunodeficiency virus (HIV)-infected patients with Kaposi’s sarcoma (KS) (current-KS patients; n = 30), HIV-infected patients who later developed KS (later-KS patients; n = 13), HIV-infected patients without KS (no-KS patients; n = 25), and healthy blood donors (n = 20). Focus formation, stress fiber dissolution, and activation of the ERK-1/2 MAP kinase signal cascade were reverted by the cytohesin-1 E157K mutant, which is deficient in catalyzing guanine nucleotide exchange. We hypothesized that among the cellular genes, vascular endothelial growth factors (VEGFs) and their cognate receptors may be involved in viral-mediated transformation. HHV8 vFLIP gets recruited to a approximately 700-kDa IkappaB kinase (IKK) complex and physically associates with IKKalpha, IKKbeta, NEMO/IKKgamma, and RIP. The specimens were routinely processed and stained. Evaluation of HHV-8 DNA levels in tumour tissues, thus, indicates a correlation between virus load and KS stage.
These data also suggest that potential use of anti-COX-2 and anti-EP receptor therapy may not only ameliorate the chronic inflammation associated with KS but could also lead to elimination of the KSHV latent infection and the associated KS lesions. In all, 21/21 cases of Kaposi sarcoma showed strong, diffuse, nuclear staining for human herpes virus 8 latent nuclear antigen-1 (100%), whereas all cases of spindle cell hemangioma, cutaneous angiosarcoma, dermatofibrosarcoma protuberans, vascular transformation of lymph node, pilar leiomyoma, stasis dermatitis, pyogenic granuloma, and spindled melanoma were negative for this antigen. The monoclonal antibody to human herpes virus 8 latent nuclear antigen-1, open reading frame-73, is a highly sensitive and specific marker of human herpes virus 8 infection in paraffin-embedded tissue sections of Kaposi sarcoma. As such, it is an extremely useful tool for differentiating between Kaposi sarcoma and other vascular and nonvascular spindle cell lesions, which do not express human herpes virus 8 latent nuclear antigen-1. Distinguishing Kaposi sarcoma from other benign or malignant vascular tumors, as well as other nonvascular spindle cell soft-tissue neoplasms, can be challenging. The differential diagnosis may include cutaneous angiosarcoma, spindle cell hemangioma, dermatofibrosarcoma protuberans, vascular transformation of lymph nodes, pilar leiomyoma, stasis dermatitis, pyogenic granuloma, and spindled melanoma among others. Thus, immunohistochemical detection of HHV-8 in fixed tissues would be diagnostically useful, enabling one to differentiate Kaposi sarcoma from these entities.
Recently, a monoclonal antibody to HHV-8 latent nuclear antigen-1 (LNA-1), open reading frame-73 (ORF-73), has become commercially available, which is suitable for immunohistochemistry in fixed tissues. HHV-8 LNA-1 is a protein encoded for by ORF-73 of the virus’ genome. The protein is expressed predominantly during viral latency and appears to play a role in viral integration into the host genome.13, 14 It also interferes in apoptosis via interactions with p53.15 Antibodies to LNA-1 have been used in formalin-fixed, paraffin-embedded tissues previously.9, 11, 12, 16, 17 At the time of this writing, we know of no study evaluating the utility of HHV-8 LNA-1, ORF-73 antibodies in distinguishing Kaposi sarcoma from other histologically similar vascular and nonvascular spindle cell neoplasms. For this reason, we sought to study the sensitivity and specificity of this antibody in the detection of HHV-8 LNA-1 in Kaposi sarcoma. A CT scan revealed diffuse lymphadenopathy (axillary, mesenteric root, and retroperitoneum) and mild hepatosplenomegaly. (A) Alignment of the nucleotide sequences of the 2 highly variable regions (VR1 and VR2) of the orf-K1 gene. This suggests that the main routes of HHV-8 transmission in non-homosexual populations are from mother to child and between siblings.
MIB-1/Ki-67 (Fig 2D, ×400) demonstrated a high proliferation index with positive stain in 80% of cells. Primary antibody dilution was 1:1000 with an incubation time of 32 min. In vivo, the majority of spindle and endothelial cells in KS lesions maintain a latent HHV8 infection with virus in only a small percentage of cells spontaneously entering the lytic replication cycle (33,36, 51, 52, 57). Strong, diffuse, nuclear staining in >10% of the tumor cells was considered a positive result. A cell block of the primary effusion lymphoma cell line BC-3 and a case of Kaposi sarcoma were used as positive controls. Characteristic histologic features of Kaposi sarcoma included spindle-shaped tumor cells surrounding hyperemic vascular slits, often accompanied by extravasated erythrocytes, hemosiderin, and fibrosis. The sites of the 21 Kaposi sarcoma specimens are summarized in Table 1.
Similar to Kaposi sarcoma, cases of spindle cell hemangioma were composed of bland spindle cell proliferations between vascular lumens with extravasated erythrocytes. However, unlike Kaposi sarcoma, vacuolated cells were sometimes noted lining lumens, as well as epithelioid endothelial cells. A naturally occurring soluble form of the IL-6-R (sIL-6-R) has been found in various body fluids [23,24]. Infiltrating, anastomosing vascular channels lined by numerous plump spindled-to-epithelioid cells with large hyperchromatic nuclei characterized these cases. Infiltrative lesions with bland spindle cells in a tight, storiform pattern, were the rule for cases of dermatofibrosarcoma protuberans. The lymph node with vascular transformation demonstrated conversion of nodal sinuses into numerous capillary-like spaces containing some erythrocytes. Pilar leiomyomas were composed of nodular, dermal aggregates of poorly circumscribed, intersecting fascicles of eosinophilic spindle cells with plump, cigar-shaped nuclei.
R. There was variable acanthosis and hyperkeratosis. All pyogenic granulomas were nodular proliferations of small capillaries with epidermal ulceration, resembling granulation tissue. Finally, the spindled melanomas had spindled cells with highly atypical nuclei and scant cytoplasm embedded in a fibrotic dermal stroma; cellularity varied. Representative high-power views of immunohistochemical staining for HHV-8 LNA-1 in (a), cutaneous patch/plaque Kaposi sarcoma, (b) cutaneous nodular Kaposi sarcoma; and (c), soft-tissue Kaposi sarcoma. Note that all normal vascular endothelial cells are negative for HHV-8 LNA-1. (d) Representative case demonstrating the absence of reactivity for HHV-8 LNA-1 in a case of pyogenic granuloma.
In their original paper, Chang et al2 used representational difference analysis (RDA) to analyze 27 cases of AIDS-associated Kaposi sarcoma and 142 non-Kaposi sarcoma cases. They discovered unique DNA sequences in 90% of AIDS-associated Kaposi sarcoma.2 Using polymerase chain reaction (PCR), Moore et al4 studied 11 cases of AIDS-related, six cases of classic, and four cases of non-HIV-associated Kaposi sarcoma, along with 21 negative controls for the presence of these sequences. PCR product was found in 20 of 21 (95%) tissue samples from the patients with Kaposi sarcoma and only one of 21 control samples (5%). Subsequently, these sequences were found by Moore et al18 to belong to a new human herpes virus, now designated HHV-8. Distinguishing Kaposi sarcoma from other benign or malignant vascular tumors as well as other nonvascular spindle cell soft-tissue neoplasms may, on occasion, be difficult. The histologic features of these lesions overlap, leading to diagnostic dilemmas. With the discovery of HHV-8 in all forms of Kaposi sarcoma,4 it became possible to consider virus detection as a potential diagnostic test.
Molecular methods have traditionally been used to identify HHV-8 in human tissues. These include PCR amplification,2, 16, 19, 20 direct in situ hybridization,6, 21in situ PCR,22 and reverse transcriptase (RT) in situ PCR.23 These methods are labor intensive, time consuming, and require highly skilled laboratory personnel. In addition, highly sensitive PCR methodologies have led to a recent controversy. Initial studies using these methods suggested a biological association between HHV-8 and multiple myeloma. However, most experts now believe these to be ‘false’ positives attributed to overamplification of ‘bystander’ HHV-8 sequences commonly present in healthy individuals with a low rate of infection.24, 25, 26 Recent commercial availability of a monoclonal antibody to HHV-8 LNA-1 has made cost-effective, tissue-localized identification of HHV-8 in fixed human specimens possible. The reliable detection of HHV-8 in fixed tissues by immunohistochemistry could enable one to differentiate Kaposi sarcoma from other histologically similar entities. For this reason, we sought to study the sensitivity and specificity of this antibody in the detection of HHV-8 LNA-1 in Kaposi sarcoma.
All of our Kaposi sarcoma cases demonstrated strong, nuclear staining for HHV-8 (100%), whereas all cases of spindle cell hemangioma (9), cutaneous angiosarcoma (5), dermatofibrosarcoma protuberans (5), vascular transformation in a lymph node (1), pilar leiomyoma (4), stasis dermatitis (4), pyogenic granuloma (4), and spindled melanoma (3) were negative for this antigen (0%). RT-PCR.HHV8 genes ORF 29 and ORF K12 (kaposin) were used as reverse transcriptase PCR (RT-PCR) targets. Antibodies to HHV-8 LNA-1 have been used previously to identify HHV-8 by IHC.9, 11, 12, 16, 17 In a survey of HHV-8+ lesions, Dupin et al9 used an antibody to HHV-8 LNA-1 to study the distribution of cell types latently infected by HHV-8 in patch/plaque and nodular Kaposi sarcoma, multicentric Castleman’s disease, and primary effusion lymphoma. These authors studied 14 cases of Kaposi sarcoma and found nuclear staining in all stages of Kaposi sarcoma. They noted fewer positive cells in patch/plaque lesions compared to nodular lesions. No staining was found in normal endothelium. Our results are similar to Dupin et al, as we noted a similar pattern of staining with fewer immunoreactive spindle cells noted in patch/plaque lesions compared to nodular lesions.
These authors also found LNA-1 expression in cells of other HHV-8 positive lesions such as multicentric Castleman’s disease and primary effusion lymphoma, but no staining was found in samples of multiple myeloma, prostate cancer, or angiosarcoma.9 Unlike Dupin et al, we also studied examples of spindle cell lesions that might enter the differential diagnosis of Kaposi sarcoma (eg dermatofibrosarcoma protuberans), and found them to be uniformly negative for HHV-8 LNA-1. (B) MBP-vIL-6 fusion protein isolated from the periplasmic space of E. Most recently, McMenamin et al11 reported that four of their cases of reactive angioendotheliomatosis stained positive for HHV-8 LNA in lesional cells. Interestingly, two of the four cases arose in immunosuppressed patients. Cool et al17 have also reported HHV-8+ cells both in and around plexiform lesions in a subset of cases of primary pulmonary hypertension. These results may expand the types of vascular lesions in which HHV-8 may be detected. This assay uses an extract, prepared from sucrose gradient-purified HHV-8 whole virions, that was isolated from the KS-1 cell line.
Furthermore, since it does not rely on amplification of nucleic acid and allows tissue localization, false-positive results or detection of ‘bystander’ virus may be less likely. We believe it to be an extremely useful tool to the surgical pathologist confronted with a lesion in which Kaposi sarcoma is a diagnostic consideration.