Digital morphometry and cluster analysis identifies four types of melanocyte during uveal melanoma progression

BACKGROUND: Several types of benign and malignant uveal melanocytes have been described based on their histological appearance. However, their characteristics have not been quantified, and their distribution during progression from normal choroidal melanocytes to primary tumors and metastases has not been reported. METHODS: A total of 1,245,411 digitally scanned melanocytes from normal choroid, choroidal nevi, primary uveal melanomas, and liver metastases were entered into two-step cluster analyses to delineate cell types based on measured morphometric characteristics and expression of protein markers. RESULTS: Here we show that a combination of the area and circularity of cell nuclei, and BAP-1 expression in nuclei and cytoplasms yields the highest silhouette of cohesion and separation. Normal choroidal melanocytes and three types of uveal melanoma cells are outlined: Epithelioid (large, rounded nuclei; BAP-1 low; IGF-1R, IDO, and TIGIT high), spindle A (small, elongated nuclei; BAP-1 high; IGF-1R low; IDO, and TIGIT intermediate), and spindle B (large, elongated nuclei; BAP-1, IGF-1R, IDO, and TIGIT low). In normal choroidal tissue and nevi, only normal melanocytes and spindle A cells are represented. Epithelioid and spindle B cells are overrepresented in the base and apex, and spindle A cells in the center of primary tumors. Liver metastases contain no normal melanocytes or spindle A cells. CONCLUSIONS: Four basic cell types can be outlined in uveal melanoma progression: normal, spindle A and B, and epithelioid. Differential expression of tumor suppressors, growth factors, and immune checkpoints could contribute to their relative over- and underrepresentation in benign, primary tumor, and metastatic samples.