Immune responses to autoantigens are prevalent in melanoma. Autoantibodies and tumor specific cytotoxic T-lymphocytes (CTL) have been identified, isolated and characterized. While these spontaneous immuneresponses are often insufficient to induce tumor regression, they can be harnessed for diagnosis, assesing prognosis, active immunotherapy, and selection of patients for immunotherapy. A major obstacle in achieving these goals has been the variability in patients'responses, as the experience so far is that only a fraction of patients share immune responses to the same antigens. Our preliminary results employing highdensity protein microarrays (ProtoArrays from Invitrogen) suggest that a great number of serological responses exist that have not been detected by prior methods. Our data confirm that a proteome-wide screen can reveal melanoma-associated serological profiles (antibodies and antigens) that can be used as the basis for a comprehensive, informative and clinically applicable screening test. We will therefore expand our interrogation of ProtoArrays composed of thousands of recombinant human proteins for serological immune- profiles in a global unbiased fashion. In Aim 1 we will employ the high-density Invitrogen human ProtoArrays to screen for serum autoantibodies and discover new antigens in patients with melanoma compared to patients with lung cancer and control healthy individuals. In Aim 2 we will validate the serological profiles with the ProtoArrays in a large number of melanoma patients with primary and metastatic disease. In Aim 3 we will assess the specificity of the antibodies and the presence of the antigens in serum, tumors and cells by performing Western blotting, reciprocal immunoprecipitation/immunoblotting analyses, sandwich immunoassays and immunohistochemistry. We will assess sensitivity, accuracy and reproducibility in discriminating between sera from patients and healthy individuals, and patients with different disease stages. The results from our studies will be the basis for the development of a serum immunome-profile test for diagnosis of melanoma. The test could also be developed to assess propensity to recur, responses to therapy, and to monitor disease progression. It will enable new therapeutic, diagnostic, and prognostic options, and assist in selection of patients for adjuvant therapy and vaccine therapies.