Metastasis is a defining feature of malignant tumors and is the most common cause of cancer-related death. However, the genetics of metastasis are poorly understood. Uveal melanoma (UM) is the most commonprimary cancer of the eye and the second most common form of melanoma. UMs have a highly characteristicpattern of metastasis to the liver that is resistant to conventional chemotherapy and is usually fatal. UMs haveremarkably little genomic instability, few cytogenetic alterations, and rare genetic mutations. Thus, whenmutations are found in these tumors, they are highly likely to be driver rather than passenger mutations. UMscan be grouped according to risk of metastatic death into class 1 (low risk) and class 2 (high risk) based on avalidated gene expression signature. A major focus of research has been to identify the specific geneticchanges that confer metastatic competency in UM. The class 2 signature is usually accompanied by loss ofone copy of chromosome 3 (monosomy 3), which has led to the widespread expectation that loss of one copyof chromosome 3 in UM cells unmasks recessive inactivating mutations in a gene (or genes) on the remainingcopy of chromosome 3 that confer metastatic capacity. Other changes include gain of chromosome 8q andloss of chromosome 8p. In the class of class 1 tumors, gain of chromosome 6p is common. The)investigatorsof this proposal are pioneers in the analysis of the molecular genetics of UM. We were the first group to applythe recently described technique of exome capture followed by massively parallel sequencing to identify BAP1as the metastasis suppressor mapping to chromosome 3 in UM. In the current study we will capitalize on ourrecent success with these technologies to identify additional tumor suppressor genes and oncogenes mutatedin UM. In Aim 1 we will generate additional exome sequences of both class 1 and class 2 tumors, comparingthem with their matched germline DNA. Potential tumor suppressor genes harboring deleterious mutations,and oncogenes harboring potential activating mutations driving the development of UM will be confirmed withSanger sequencing and evaluated in an additional 10-30 class 1 and class 2 tumors and matched germlineDNA. In Aim 2 we will use targeted capture to re-sequence newly identified additional mutations within thesegenes in additional samples (>100 of each tumor type) to determine their contribution to UM. In Aim 3 we willperform limited functional studies of 5 newly identified genes in cell lines. We will perform binding assays toevaluate the effects of mis-sense and in-frame coding changes on interactions with known and novel partners,and over-express activating oncogenes and knockdown tumor suppressors to determine their effect on cellmorphology and gene expression. Information on molecular alterations in tumors will then be incorporated withclinical information to begin to develop a prognostic classification of UM. This is a collaborative proposal from amultidisciplinary collaborative group from the departments of Ophthalmology and Genetics that has the provenexpertise to complete the aims of this proposal. )