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Uveal melanoma (UM) is a highly metastatic cancer that, in contrast to cutaneous melanoma, is largely unresponsive to checkpoint immunotherapy. Here, we interrogate the tumor microenvironment at single-cell resolution using scRNA-seq of 59,915 tumor and non-neoplastic cells from 8 primary and 3 metastatic samples. Tumor cells reveal novel subclonal genomic complexity and transcriptional states. Tumor-infiltrating immune cells comprise a previously unrecognized diversity of cell types, including CD8 + T cells predominantly expressing the checkpoint marker LAG3, rather than PD1 or CTLA4. V(D)J analysis shows clonally expanded T cells, indicating that they are capable of mounting an immune response. An indolent liver metastasis from a class 1B UM is infiltrated with clonally expanded plasma cells, indicative of antibody-mediated immunity. This complex ecosystem of tumor and immune cells provides new insights into UM biology, and LAG3 is identified as a potential candidate for immune checkpoint blockade in patients with high risk UM.

Cancer is thought to arise through the accumulation of genomic aberrations evolving under Darwinian selection. However, it remains unclear when the aberrations associated with metastasis emerge during tumor evolution. Uveal melanoma (UM) is the most common primary eye cancer and frequently leads to metastatic death, which is strongly linked to BAP1 mutations. Accordingly, UM is ideally suited for studying the clonal evolution of metastatic competence. Here we analyze sequencing data from 151 primary UM samples using a customized bioinformatic pipeline, to improve detection of BAP1 mutations and infer the clonal relationships among genomic aberrations. Strikingly, we find BAP1 mutations and other canonical genomic aberrations usually arise in an early punctuated burst, followed by neutral evolution extending to the time of clinical detection. This implies that the metastatic proclivity of UM is "set in stone" early in tumor evolution and may explain why advances in primary treatment have not improved survival.