Kaposiform hemangioendothelioma complicated by Kasabach-Merritt phenomenon: ultrastructural observation and immunohistochemistry staining reveal the trapping of blood components.

Kaposiform hemangioendothelioma (KHE), a borderline tumor of endothelial origin, is associated with Kasabach-Merritt phenomenon, characterized by profound thrombocytopenia and consumptive coagulopathy resulting from the localized intravascular coagulation (LIC) in the tumor. Previous studies have suggested that the trapping of blood components, including platelets, may underlie the LIC in KHE. However, more evidence is needed to support this hypothesis. In this study, one case of a Chinese infant with a KHE in the left arm was complicated by Kasabach-Merritt phenomenon. The tumor was partially resected and the sample was used for ultrastructural observation and immunohistochemistry staining of Glut-1. Ultrastructural observation found the trapping of erythrocytes, platelets, macrophages, and lymphocytes in the slit-like channels of the tumor nodules, and phagocytic vesicles in the cytoplasm of neoplastic cells. Immunohistochemistry staining further showed numerous Glut-1(+) erythrocytes in the channels. In conclusion, our results provided compelling morphological evidence of the trapping of blood components in KHE, which may interpret the LIC in the tumor and subsequent consumptive coagulopathy.

Primary kaposiform hemangioendothelioma of a long bone: two cases in unusual locations with long-term follow up.

Kaposiform hemangioendothelioma (KHE) is a rare vascular neoplasm of low malignant potential that mainly affects infants and adolescents. The tumor almost exclusively occurs in somatic soft tissue or the retroperitoneum. We report herein two cases of primary KHE occurring in a long bone without cutaneous changes with long-term follow up in young patients. The patients were a 9-year-old girl and 5-year-old boy presenting with lytic lesions of the femur and humerus, respectively, without cutaneous lesions. Histologically, the neoplasms were comprised of nodules of spindle- to oval-shaped cells growing in an infiltrative fashion. The neoplastic cells formed poorly canalized or slit-like blood vessels alternating with solid spindle areas. Immunohistochemical studies showed that the tumor cells expressed CD31, CD34 and Fli1, but not HHV8, LNA-1 or GLUT1. D2-40 stained the neoplastic spindle cells and lymphatic channels adjacent to vascular lobules. The girl remains well with 15 years and 6 months follow up after a second complete excision. The boy has no signs of recurrence or metastasis nearly 5 years after local complete excision. To our best knowledge, this is the first report in the English literature of primary long bone occurrences of KHE without cutaneous changes with long-term follow up.

Kaposiform hemangioendothelioma arising in the deltoid muscle without the Kasabach-Merritt phenomenon.

Kaposiform hemangioendothelioma (KHE) is a rare tumor that occurs nearly exclusively during infancy and childhood. It has features common to both capillary hemangioma and Kaposi sarcoma and for that reason many terms have been used for these tumors including "Kaposi-like infantile hemangioendothelioma" and "hemangioma with Kaposi sarcoma-like features." KHE typically presents as an ill-defined, red to purple, indurated plaque and is often complicated by the Kasabach-Merritt phenomenon (KMP), a condition of severe thrombocytopenia and consumptive coagulopathy. Knowledge of the radiological findings of this uncommon tumor might be helpful for diagnosis. We present the MRI features of a case of KHE with neither typical skin lesions nor the Kasabach-Merritt phenomenon.

Ultrastructure of Kaposi sarcoma.

Kaposi sarcoma (KS) is a complex disease with aspects of virology (human herpesvirus-8, HHV-8, and human immunodeficiency virus, HIV), immunology (immunodeficiency), hyperplasia (multiple widely spaced de novo lesions), and neoplasia (metastases) that has always been the most common AIDS-defining malignancy. The lesional spindle cell has been classified as being derived from either blood vascular or, more recently, lymphatic endothelial cell origin. This study revealed a spectrum of endothelial cell ultrastructure from lymphatic to blood vascular. It demonstrated frequent Weibel-Palade bodies and gap junctions. The spindle cells were shown to behave as facultative phagocytes, internalizing and processing necrotic cells and leaked red blood cells (RBCs). Fragmented RBCs were equivalent to the "hyaline droplets" seen by light microscopy. The final stages of RBC disintegration were hemosiderin and ferritin. Most significantly, this study disclosed that KS is actually composed of a single type of randomly oriented spindle cell forming vessels of varying size and integrity.

Reactivation of Kaposi's sarcoma-associated herpesvirus by natural products from Kaposi's sarcoma endemic regions.

Kaposi's sarcoma (KS) and its causative agent, Kaposi's sarcoma associated herpesvirus (KSHV/HHV-8), a gamma2 herpesvirus, have distinctive geographical distributions that are largely unexplained. We propose the "oncoweed" hypothesis to explain these differences, namely that environmental cofactors present in KS endemic regions cause frequent reactivation of KSHV in infected subjects, leading to increased viral shedding and transmission leading to increased prevalence of KSHV infection as well as high viral load levels and antibody titers. Reactivation also plays a role in the pathogenesis of KSHV-associated malignancies. To test this hypothesis, we employed an in vitro KSHV reactivation assay that measured increases in KSHV viral load in KSHV infected primary effusion lymphoma (PEL) cells and screened aqueous natural product extracts from KS endemic regions. Of 4,842 extracts from 38 countries, 184 (5%) caused KSHV reactivation. Extracts that caused reactivation came from a wide variety of plant families, and extracts from Africa, where KSHV is highly prevalent, caused the greatest level of reactivation. Time course experiments were performed using 28 extracts that caused the highest levels of reactivation. The specificity of the effects on viral replication was examined using transcriptional profiling of all viral mRNAs. The array data indicated that the natural extracts caused an ordered cascade of lytic replication similar to that seen after induction with synthetic activators. These in vitro data provide support for the "oncoweed" hypothesis by demonstrating basic biological plausibility.

The three-dimensional microvascular architecture of the human Kaposi sarcoma implanted in nude mice: a SEM corrosion casting study.

The human Kaposi sarcoma represents one of the most common skin lesions associated with AIDS. Its clinical presentation and anatomopathological structure seem to demonstrate a particularly rich vascularity. The latest therapies aim to limit its intrinsic angiogenic activity in an attempt to reduce vascular density and the formation of new vessels. For these reasons, we decided to study the microvascular architecture of Kaposi sarcoma in three dimensions. We used a corrosion casting technique applied to nude mice previously transplanted subcutaneously with human modified neoplastic Kaposi sarcoma cells. The cooption of host vessels made by the tumor was demonstrated by three-dimensional scanning electron microscopy (SEM) images. At high magnification several angiogenic patterns were observed in the form of potato-shaped vessels, sprouts, intussusceptions and mouse tailed end tipped capillaries along with some ultrastructural features such as intercellular extravasations and endothelial cell modifications. Our investigation allowed us to build a detailed map of tumor vasculature in human Kaposi sarcoma. Furthermore, this study want to shed light on the sharp morphological three-dimensional conformation of angiogenic sprouts so to be able to better understand their modifications occurred during time and after antiangiogenic experimental therapies, by now observed only by immunohistochemical or immunofluorescent assays.

Endothelial infection with KSHV genes in vivo reveals that vGPCR initiates Kaposi's sarcomagenesis and can promote the tumorigenic potential of viral latent genes.

The Kaposi's sarcoma herpesvirus (KSHV) has been identified as the etiologic agent of Kaposi's sarcoma (KS), but initial events leading to KS development remain unclear. Characterization of the KSHV genome reveals the presence of numerous potential oncogenes. To address their contribution to the initiation of the endothelial cell-derived KS tumor, we developed a novel transgenic mouse that enabled endothelial cell-specific infection in vivo using virus expressing candidate KSHV oncogenes. Here we show that transduction of one gene, vGPCR, was sufficient to induce angioproliferative tumors that strikingly resembled human KS. Endothelial cells expressing vGPCR were further able to promote tumor formation by cells expressing KSHV latent genes, suggestive of a cooperative role among viral genes in the promotion of Kaposi's sarcomagenesis.

Morphogenesis of HHV8 in primary human dermal microvascular endothelium and primary effusion lymphomas.

This study elucidates the morphology of HHV8 replication in human dermal endothelial cells and primary effusion lymphomas (PEL) and compares it to that seen in Kaposi sarcoma. Primary human dermal microvascular endothelial cells (DMVEC) exposed to the cell-filtered supernatant of the PEL JSC1 and PEL cell lines (KS-1, BCBL-1, BC-1, BC-3) were cultured in the presence or absence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or butyrate. Cells were fixed in neutral-buffered glutaraldehyde, gelled in cooled agar, and processed for TEM. There was a quantitative, but not a qualitative difference in viral expression associated with no treatment or exposure to TPA or butyrate of H HV8 in DMVEC and PEL. Two types of viral-induced intranuclear inclusions (INI) were visible at the light and ultrastructural levels. The more common INI had lighter staining material filling the nucleus, except for a rim of dense chromatin, and could be seen even before viral nucleocapsids (NC) were visible. The second type of INI resembled a target formed by condensation of electron-dense material surrounded by a lighter halo and marginated heterochromatin and containing NC. Collections of coalescing electron-dense granules resembling starbursts were often present in nuclei containing either type of INI. Next to appear in productively infected cells were mature enveloped particles that formed mostly by the budding of NC into cytoplasmic vacuoles. Mature particles were also seen free on the plasma membrane. Tufts of electron-dense intermediate filaments were associated with maturing particles. Mature virions lacked an electron-dense tegument. Viral production was ultimately associated with cell lysis. It appears that HHV8 propagate in DMVEC, with and without stimulation, and have a similar morphogenesis to that seen in PEL cell lines and Kaposi sarcoma lesions. Several unique features characterize cells productively infected by HHV8.

Lytic growth of human herpesvirus 8: morphological aspects.

The human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus, is a gamma herpesvirus associated with AIDS-related body cavity-based lymphomas (BCBL), also called primary effusion lymphomas (PEL). These are a rare form of non-Hodgkin lymphomas in which HHV-8 is present, often associated with Epstein-Barr virus (EBV) infection. HHV-8 is also present in a latent state or in a state of low-level persistence in different primary effusion lymphoma-derived cell lines, such BCBL-1 cells, that lack EBV infection. This cell line was induced to produce mature virions by treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and the characteristic ultrastructural features of HHV-8 lytic replication were identified and compared to those of the other members of Herpesviridae family.

Transgenic expression of the chemokine receptor encoded by human herpesvirus 8 induces an angioproliferative disease resembling Kaposi's sarcoma.

Human herpesvirus 8 (HHV8, also known as Kaposi's sarcoma [KS]-associated herpesvirus) has been implicated as an etiologic agent for KS, an angiogenic tumor composed of endothelial, inflammatory, and spindle cells. Here, we report that transgenic mice expressing the HHV8-encoded chemokine receptor (viral G protein-coupled receptor) within hematopoietic cells develop angioproliferative lesions in multiple organs that morphologically resemble KS lesions. These lesions are characterized by a spectrum of changes ranging from erythematous maculae to vascular tumors, by the presence of spindle and inflammatory cells, and by expression of vGPCR, CD34, and vascular endothelial growth factor. We conclude that vGPCR contributes to the development of the angioproliferative lesions observed in these mice and suggest that this chemokine receptor may play a role in the pathogenesis of KS in humans.

Kaposi's sarcoma: breeding ground of herpesviridae - A tour de force over viral evolution (review).

After reviewing the molecular biological basis of prominent theories for the integration of viruses into the earliest forms of living matter, an account is given on the immunoevasive strategies viruses have had to acquire in order to secure their existence against the most sophisticated anti-viral defensive mechanisms evolving in their hosts. Herpes-viridae and Kaposi's sarcoma illustrate the complexity of host-virus relationship. In following the evolutionary steps of simians and hominoids to Homo, it becomes evident that: a) Epstein-Barr virus evolved in Africa and its ancestral viruses are present in cercopithecines and hominoids; b) human herpes-virus-8-related viruses are present in macaques, in S. American primates and in Homo but such isolates from the great apes are missing. Thus interspecies transfer occurred from lower monkeys to Homo but when and at what geographical location? The human retrolentiviruses also jumped species barriers: this occurred recently in Africa, from great apes (chimpanzee and bonobo) to Homo sapiens (except when HIV-2 was transferred to mankind from sooty mangabeys). The matter is further complicated by the long coevolutionary cooperative interactions between herpes- and retrolentiviruses. Of pathological entities suspected to be etiologically affected by such complex viral cooperation, the origin of Reed-Sternberg cells of Hodgkin's disease is singled out for critical analysis. In this article the senior author summarizes his own 52 years of studentship in virology.

Visualization of human herpesvirus type 8 in Kaposi's sarcoma by light and transmission electron microscopy.

BACKGROUND: Human herpesvirus type 8 (HHV-8) has been associated with Kaposi's sarcoma, body cavity-based lymphoma (BCBL), and multicentric Castleman's disease through DNA, in situ hybridization, and serologic studies. HHV-8 has been visualized only in HHV-8-positive/Epstein-Barr virus (EBV)-negative/ cytomegalovirus (CMV)-negative BCBL cell lines, but not in HHV-8-positive/EBV-negative/ CMV-negative Kaposi's sarcoma lesions. DESIGN: Kaposi's sarcoma of the skin, lymph node, and spleen from three patients with AIDS were analysed for HHV-8, EBV and CMV DNA by polymerase chain reaction (PCR), for HHV-8 RNA (Tl.1 riboprobe) by in situ hybridization (ISH), for viral inclusions by light microscopy, and for herpesviruses by transmission electron microscopy (TEM). Sections were also labeled with Tl.1 counterstained with CD34, an endothelial cell marker. RESULTS: The skin lesion was DNA PCR-positive for HHV-8 and CMV (nested, but not single PCR), the lymph node was positive for HHV-8 and EBV, and the spleen was positive for only HHV-8. TEM revealed infection by a virus displaying the typical morphology and cytopathicity of herpesviruses. Hexagonal nucleocapsids and mature enveloped virions were present in vasoformative spindle cells and mononuclear cells, often resembling lymphocytes. Extrapolating from TEM to standard light microscopy on hematoxylin and eosin-stained paraffin sections, eosinophilic, targetoid intranuclear inclusions were identified within spindle cells which often lined vascular lumina. The Tl.1-riboprobe labeled CD34+ spindle cells containing intranuclear inclusions, as well as mononuclear cells within Kaposi's sarcoma and residual lymphoid tissue. CONCLUSION: The herpesvirus visualized in Kaposi's sarcoma lesions has morphologic and cytopathic features typical of human herpesviruses, productively infects vasoformative spindle cells and mononuclear cells, and is consistent with HHV-8. It can also form intranuclear inclusions that are identifiable by light microscopy in hematoxylin and eosin sections and by ISH.

Phenotype and proliferation characteristics of cultured spindle-shaped cells obtained from normal human skin and lesions of dermatofibroma, Kaposi's sarcoma, and dermatofibrosarcoma protuberans: a comparison with fibroblast and endothelial cells of the dermis.

Normal human dermis contains mesenchymal cells that are generally referred to as fibroblasts. However the relationships between fibroblasts and endothelial cells with respect to the types of spindle-shaped cells that are present in cultures obtained from tumor bearing-skin is unclear. To explore the potential heterogeneity amongst dermal-derived cells that grow in culture with a spindle-shaped morphology, we compared the immunophenotype and growth characteristics of several types of cells. Besides dermal fibroblasts and microvascular endothelial cells derived from normal adult skin, we also studied large vessel-derived endothelial cells, and spindle-shaped cells derived from three different tumor-bearing dermal-based neoplasms. Kaposi's sarcoma (KS), dermatofibroma (DF), and dermatofibrosarcoma protuberans (DFSP). A broad panel of eight different antibodies were used to immunophenotype the multi-passaged cultured cells. Spindle-shaped cells from all three neoplasms could be distinguished from the normal skin derived fibroblasts by their constitutive expression of factor XIIIa, and the gamma-interferon induced expression of VCAM-1. All seven types of cultured cells stained positive for s-actin and proline-4-hydroxylase, and none of the cells expressed CD34. Both large and small-vessel derived endothelial cells expressed factor VIII, ELAM-1, and VCAM-1. Using two different types of growth media, significant differences were also observed amongst these cultured cell types. Spindle-shaped cells from DFSP did not grow in DMEM containing 10% fetal bovine serum (DMEM-FBS); but they proliferated in KS cell growth medium (KSGM). Spindle-shaped cells from DF grew best in KSGM, but not in DMEM-FBS. KS tumor cells grew well in KSGM, but not in DMEM-FBS. Fibroblasts proliferated in DMEM-FBS, but failed to grow in KSGM; and even when pre-treated with conditioned medium from a transformed KS cell line (i.e. SLK cells), no fibroblast proliferation could be induced in KSGM. These results indicate that KS cell line (i.e. SLK cells), no fibroblast proliferation could be induced in KSGM. These results indicate that even though dermal-derived cells can have an identical spindle-shape by light microscopy, significant heterogeneity can be defined amongst such cells from normal and tumor-bearing human skin. Having established culture conditions to propagate these different cell types and phenotypic criteria to distinguish them from one another, will provide new research opportunities to explore the function and ontogeny of the diverse mesenchymal cells that take on a spindle-shaped morphology in culture.

Ultrastructural study of Kaposi's sarcoma.

An electron microscopic (EM) study of 25 Kaposi's sarcomas (KS) (24 cutaneous lesions, 1 lymphnodal) from 23 patients, mainly sporadic in type, has enabled us to study both the spindle and endothelial cells seen by light microscopy (LM) with the conclusion that they have both a fibroblastic-like EM aspect. Both cell types manifested a spindle shape with oval and occasionally notched nuclei and a reticular pattern of nucleoli. The cytoplasm was characterized by numerous rough endoplasmic reticulum cisternae with few other organelles. The plasma membranes consistently lacked an external or basal membrane and failed to show true cell junctions. In cells delimiting erythrocytes containing spaces it was never possible to document Weibel-Palade (W.-P.) bodies or plasmalemmal vesicles. Unusual findings such as ferritin loaded phagosomes, microtubular reticular structures (MTRS), intracisternal crystalline inclusions (ICCI), multivesicular bodies (MVB), acanthosomic vesicles (AV) and test-tube inclusions or ring-shaped forms (TTI/RSF) were occasionally seen in both sporadic and epidemic KS. The authors discuss the non specific nature of these latter findings.

Three-dimensional laser-scanning confocal microscopy of in situ hybridization in the skin.

In situ hybridization is an important tool in molecular and developmental biology to detect specific nucleic acid sequences (either mRNA or DNA) within cells. This technique is especially applicable to tissue sections since it provides information about the spatial distribution of DNA or mRNA sequences. However, previous studies utilizing in situ hybridization in the skin were hampered by a high degree of nonspecific background, which has made interpretation of the results difficult. In this paper, we demonstrate how refinements in in situ hybridization techniques, combined with laser-scanning confocal microscopy, significantly reduce nonspecific background and produce improved resolution of in situ hybridization in skin specimens. The sensitive detection method of laser-scanning confocal microscopy allows three-dimensional localization of S35 radioactive-labeled riboprobes within the emulsion of specimens, which is not possible with conventional bright or dark field light microscopy.

Immunohistochemical and electron microscopic profiles of cutaneous Kaposi's sarcoma and bacillary angiomatosis.

Thirty cases of cutaneous Kaposi's sarcoma (KS) were evaluated and compared with eight cases of acquired immunodeficiency syndrome (AIDS)-related bacillary angiomatosis (BA). The morphologic features of both lesions were studied by light and electron microscopy and by immunohistochemistry with monoclonal endothelial antibodies against CD34, BNH9, and factor VIII-related antigen as well as the lectins Ulex europaeus 1 and Psophocarpus tetragonolobus. Macrophage/monocyte markers used were alpha 1-antitrypsin, lysosome, Kp1 (CD68), and polyclonal factor XIIIa. Electron microscopic studies demonstrated that most of the spindle cells in KS showed a paucity of cell organelles and an absence of Weibel-Palade bodies (WPB), whereas the cells in BA showed activated endothelial cells with WPB. By immunohistochemistry the spindle cells in KS were consistently positive for CD34 only, whereas proliferating cells in BA expressed all endothelial markers used. Numerous cells expressing macrophage/monocyte markers were present surrounding both KS and BA, and a small number of similar cells were entrapped within both lesions. The results demonstrated a restricted immunohistochemical profile for endothelial cell markers in spindle cells of KS (CD34+) distinct from that of endothelial cells in BA. These findings suggest that the spindle cells in KS are poorly differentiated endothelial cells or that they belong to an endothelial cell subset with partial expression of endothelial phenotype.

Kaposi's sarcoma: a review of gene expression and ultrastructure of KS spindle cells in vivo.

The ultrastructural features and the gene expression pattern of Kaposi's sarcoma (KS) spindle cells in vivo suggest that KS is a tumor of the mixed cell type. The expression pattern of cytokines and cytokine receptors in the tumor lesion, together with the results obtained from in vitro characterization of KS-derived cells, provide evidence that paracrine mechanisms of growth factor action are important for the maintenance of KS. The reports on virus infection of KS cells suggest an indirect role of virus infection in the induction of KS, most likely mediated by immunostimulation and subsequent production of cytokines.