KSHV LATENT AND LYTIC INFECTION IN KAPOSI'S SARCOMA LESIONS AND LYMPHOID TISSUE (1997)

Background: KSHV has been associated with virtually all cases of Kaposi's sarcoma (HIV and non-HIV), with a unique type of high-grade lymphoma designated body cavity-based lymphoma (BCBL) and with many cases of multicentric Castleman's disease. With a EBV-KSHV+ BCBL cell line, Renne and colleagues (Nature Medicine (1996) 2. 342) were able to demonstrate a restricted expression of viral genes before TPA induction and a more general expression of viral genes after induction. The TPA-induced expression was correlated with ultrastructural identification of vastly increased herpesvirion production. Two riboprobes suitable for in situ hybridization (ISH) experiments were developed from these experiments - one (T0.7) seemed able to detect latently KSHV infected cells, the other (T1.1 or nut 1) was relatively specific for lytic infection. Methods: Specimens included Kaposi's sarcoma lesions from the skin, spleen, and lymph nodes. KSHV-involved lymphoid tissues included lymph node and spleen with and without Kaposi's sarcoma. Digoxigenin-labeled riboprobes for T0.7 and T1.1 (nut 1) KSHV regions were labeled by standard PCR methodology. Detection of ISH was with alkaline phosphatase anti-digoixgenin followed by NCT/BCIP substrate. Immunohistochemical methods were standard avidin-biotin peroxidase methods. Indicators and methods of ISH and immunohistochemistry were altered to fit specific double-label experiments. The ISH experiments were correlated with systematic ultrastructural analysis and histological examination. Results: The novel observations and important corroboration of existing data include - 1) lytic replication of KSHV could be demonstrated by ISH and EM in all the KS lesions. 2) double label experiments showed that lytic replication was occurring in a fraction of endothelial or spindle cells of KS lesions. 3) "activated atypical" lymphocytes in germinal centers, presumably B-immunoblasts and/or plasmacytes (BIP's), are major sites of viral replication This germinal center KSHV lytic lesion could be the precursor to the hyalinized germinal centers described in KS lymphoid tissue and in multicentric Castleman's disease. 4) BIP's commonly infiltrate KS lesions. 5) visceral KS lesions contain foci of viral replication which on the ultrastructural level feature viral production and phagocytosis of cellular debris by endothelial cells. These lytic foci may be an admixture of BIP's and endothelial cells/spindle cells. 6) these studies have allowed us to reliably identify the intranuclear inclusions of KSHV infected cells by standard light microscopy. Conclusion: Our studies have led us to conclude that the pathogenesis of Kaposi's sarcoma is not a simple cancer model such as acquisition of specific genetic abnormalities in a Vogelstein tumor progression. The early stages of Kaposi's sarcoma may be a complex vasoproliferative phenomena in response to KSHV endothelial infection and lysis. Adjacent and intralesional B-cell infection (so-called BIP's) with consequent activation and cytokine production, viral production, and cellular lysis may be complicating variables contributing to KS pathogenesis.