Immune responses to autoantigens are prevalent in melanoma. Autoantibodies and tumor specific cytotoxicT-lymphocytes (CTL) have been identified, isolated and characterized. While these spontaneous immuneresponsesare often insufficient to induce tumor regression, they can be harnessed for diagnosis, assesingprognosis, active immunotherapy, and selection of patients for immunotherapy. A major obstacle inachieving these goals has been the variability in patients' responses, as the experience so far is that only afraction of patients share immune responses to the same antigens. Our preliminary results employing highdensityprotein microarrays (ProtoArrays from Invitrogen) suggest that a great number of serologicalresponses exist that have not been detected by prior methods. Our data confirm that a proteome-widescreen can reveal melanoma-associated serological profiles (antibodies and antigens) that can be used asthe basis for a comprehensive, informative and clinically applicable screening test. We will therefore expandour interrogation of ProtoArrays composed of thousands of recombinant human proteins for serologicalimmune- profiles in a global unbiased fashion.
In Aim 1 we will employ the high-density Invitrogen humanProtoArrays to screen for serum autoantibodies and discover new antigens in patients with melanomacompared to patients with lung cancer and control healthy individuals.
In Aim 2 we will validate theserological profiles with the ProtoArrays in a large number of melanoma patients with primary and metastaticdisease.
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 reproducibilityin discriminating between sera from patients and healthy individuals, and patients with different diseasestages. The results from our studies will be the basis for the development of a serum immunome-profile testfor diagnosis of melanoma. The test could also be developed to assess propensity to recur, responses totherapy, and to monitor disease progression. It will enable new therapeutic, diagnostic, and prognosticoptions, and assist in selection of patients for adjuvant therapy and vaccine therapies.
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