Single-Cell RNA Profiling of Ocular Adnexal Sebaceous Carcinoma Reveals a Complex Tumor Microenvironment and Identifies New Biomarkers.

PURPOSE: Ocular adnexal sebaceous carcinoma (OaSC) is an aggressive malignancy that often necessitates orbital exenteration. Its tumor composition and transcriptional profile remain largely unknown, which poses a significant barrier to medical advances. Here, we report the first in-depth transcriptomic analysis of OaSC at the single-cell resolution and discern mechanisms underlying cancer progression for the discovery of potential globe-sparing immunotherapies, targeted therapies, and biomarkers to guide clinical management. DESIGN: Laboratory investigation with a retrospective observational case series. METHODS: Single-cell RNA sequencing was performed on six patient specimens: three primary tumors, two tumors with pagetoid spread, and a normal tarsus sample. Cellular components were identified via gene signatures. Molecular pathways underlying tumorigenesis and pagetoid spread were discerned via gene ontology analysis of the differentially expressed genes between specimens. CALML5 immunohistochemistry was performed on an archival cohort of OaSC, squamous cell carcinoma, ocular surface squamous neoplasia (OSSN), and basal cell carcinoma cases. RESULTS: Analysis of 29,219 cells from OaSC specimens revealed tumor, immune, and stromal cells. Tumor-infiltrating immune cells include a diversity of cell types, including exhausted T-cell populations. In primary OaSC tumors, mitotic nuclear division and oxidative phosphorylation pathways are upregulated, while lipid biosynthesis and metabolism pathways are downregulated. Epithelial tissue migration pathways are upregulated in tumor cells undergoing pagetoid spread. Single-cell RNA sequencing analyses also revealed that CALML5 is upregulated in OaSC tumor cells. Diffuse nuclear and cytoplasmic CALML5 staining was present in 28 of 28 (100%) OaSC cases. Diffuse nuclear and membranous CALML5 staining was present in 5 of 25 (20%) squamous cell carcinoma and OSSN cases, while diffuse nuclear staining was present in 1 of 12 (8%) basal cell carcinoma cases. CONCLUSIONS: This study reveals a complex OaSC tumor microenvironment and confirms that the CALML5 immunohistochemical stain is a sensitive diagnostic marker.