Development of AIDS associated and endemic Kaposi sarcoma : HHV-8/KSHV viral load in cutaneous and oral tumor cells

Abstract: Kaposi's sarcoma (KS) is a highly and abnormally vascularized tumor-like lesion which usually presents as a cutaneous lesion and eventually progresses to systemic disease usually in the gastrointestinal (G1) tract, lungs, oral cavity and lymph nodes. Whether KS represents a predominantly monoclonal neoplastic cell proliferation or a hyperplastic, reactive polyclonal process is still controversial. KS is the most common neoplasm of HIV+ patients and included in the staging of disease progression to AIDS. The KS-associated herpesvirus or human herpesvirus-8 (KSHV/HHV-8), a gamma-herpes virus was discovered in AIDS related KS (AKS) tumor samples and subsequently in all clinico-epidemiological forms of KS as Classic KS (CKS), Iatrogenic KS (IKS), Endemic KS (EKS) but also in some rare lymphoid disorders such as body cavity-based lymphoma (BCBL/PEL) and multicentric Castleman's disease (MCD). It is considered to be the main pathogenic factor of KS but apparently not sufficient to cause KS and other factors like immunosuppression, production of angiogenic cytokines and HIV-tat in AIDS apparently also play an important role. The consistent demonstration of HHV-8 in tumor lesions, blood cells, scrum and saliva of KS patients validates the pathogenic role of HHV-8 in KS oncogenesis. KS lesions develop from early patch/plaque to late, nodular tumors. The histology is characterized by an early infiltration of mononuclear inflammatory cells, formation of atypical small blood vessels and slits (angiogenesis) and the appearance of an increasing number of tumor spindle cells (SC). Various studies favour an endothelial origin (CD34+) of the KS SC but whether of vascular (VEC) or lymphatic (LEC) origin is not clear. The HHV-8 latency associated nuclear antigen type 1 (LANA-1) protein is the most expressed viral antigen in infected cells. In this study we have used formalin fixed paraffin embedded surgical biopsies from Tanzania (Dar Es Salam) at early and late stages of patch/plaque and nodular AKS / EKS development in, oral and cutaneous lesions. The histopathological studies (paper 1) by triple antibody immunofluorescence showed that: (a) the frequency of LANA+/CD34+ cells increased from early patch to late KS nodular lesions; (b) 25-35% of the CD34+ SC were LANA- in both early and late KS, suggesting a continuous recruitment of noninfected endothelial cell into the KS lesion; (c) LANA+/CD34- cells decrease from early 18.7% to late 29% and most of them expressed LEC markers such as LYVE-1 and D2-40, suggesting that the resident LECs represent an early target of primary HHV-8 infection; (d) The decrease of LANA+/CD34- cells from early to late KS (most cells are LANA+/CD34+) indicate a phenotype switch from LEC to VEC. We also established and optimized a semi-quantitative PCR assay for HHV-8 detection in formalin fixed paraffin-embedded KS biopsies (paper II). Our results indicate a better performance for Chelexextracted DNA compare to Qiagene kit method. In late, nodular stage of both AKS and EKS the virus load per unit tissue actin (HHV-8/actin) is higher than in early stages (patch/plaque), which corroborates our findings from double immunostaining for LANA and CD34 of the same cases. With quantitative real time PCR on DNA from oral and cutaneous AKS and EKS biopsies (paper 111), we have not found significant higher HHV-8 load in AKS compared to EKS in accordance with the immunohistochemistry findings and consistent with the notion of basically similar pathogenic mechanisms for these clinically distinct lesions. HHV-8 load in oral was higher than in cutaneous AKS concordant with the finding of more LANA+ cells and LANA+ granules per nucleus of SC in oral compared to cutaneous AKS. This may reflect an increased effect of LANA since we observed more proliferating SCs (Ki67+ frequency) in oral than cutaneous AKS (PaperIV). The high viral load in oral KS lesions is consistent with the known high risk of HHV-8 horizontal transmission. From our data (paper IV) it appears that oral KS corresponds to about 10% of registered KS cases in Tanzania, a frequency apparently higher than in Southern Africa and North America and highly associated with HIV, severe immunodeficiency, sexually active age-group female gender and advanced (nodular) histological stage. Immunohistochemistry showed higher frequency of HHV-8 infected tumor cells in males vs. females, children vs. adults and oral vs. cutaneous AKS. The results generally suggest that oral AKS may have a worse clinical and biological prognosis than cutaneous AKS indicating differential susceptibility of various tissues to HHV-8 infection and subsequent KS development.