Spatial and Bulk Transcriptomics Reveal Distinct Molecular Signatures in Kaposi Sarcoma with and Without other KSHV-Associated Diseases.

BACKGROUND: Kaposi sarcoma (KS), caused by Kaposi sarcoma herpesvirus (KSHV), is an angioproliferative tumor that presents as skin lesions in people with HIV. Other KSHV-associated diseases (KAD) including multicentric Castleman disease (MCD), primary effusion lymphoma (PEL), and KSHV-associated inflammatory cytokine syndrome (KICS) may occur concurrently with KS and influence clinical outcomes. New sequencing technologies enabling analysis of archival KS tissues provide mechanisms to define cellular and viral characteristics that contribute to disease heterogeneity. METHODS: We profiled gene expression in 42 confirmed KS formalin-fixed paraffin-embedded (FFPE) skin biopsies using the Nanostring nCounter PanCancer ImmunoOncology panel supplemented with KSHV-specific probes. Spatial RNA profiling was performed on four tissues from participants with KS and concurrent KAD (KS+KAD) using GeoMx digital spatial profiling (DSP) platform. Regions of interest were selected using LANA-1, CD45 and CD31 staining to characterize tumor (LANA-1(+), CD31(+)), vessel (LANA-1(-), CD31(+)) and immune cells (CD45(+)) areas of illumination (AOIs). RESULTS: KS samples were obtained from 42 men with HIV (median age 40 years). Median HIV viral load of 27 copies/mL and median CD4(+) T-cell count was 211 cells/μL. Forty-eight percent had KS alone and 52% had KS+KAD. Patients with KS+KAD had worse survival compared to those with KS alone. Transcriptomic analyses identified increased expression of STC1 (log2FC=2.02, p-adjusted (padj)=0.001), a secreted glycoprotein, and MKI67 (log2FC=1.11, padj=0.02), a common proliferation marker, in KS+KAD lesions, along with lower expression of cytokine-associated pathways. Spatial RNA profiling from 4 KS samples from patients with KS+KAD identified increased abundance of lymphatic endothelial cells, elevated LYVE1 expression in LANA-1+ tumor areas as compared to LANA- areas. CONCLUSIONS: Bulk and spatial transcriptomic profiling of archival HIV-associated KS lesions revealed disease-specific molecular programs associated with concurrent KAD that altered tumor and microenvironment features. These findings demonstrate the heterogeneity of KS lesions that may guide future studies on KS pathogenesis and potential therapeutic targets.