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Purpose: Mutations in TP53 induce autoantibody immune responses in a subset of cancer patients, which have been proposed as biomarkers for early detection. Here, we investigate the association of p53-specific autoantibodies with multiple tumor subtypes and determine the association with p53 mutation status and epitope specificity., Experimental Design: IgG p53 autoantibodies (p53-AAb), were quantified in 412 serum samples using a programmable ELISA assay from patients with serous ovarian, pancreatic adenocarcinoma, and breast cancer. To determine if patients generated mutation-specific autoantibodies we designed a panel of the most relevant 51 p53 point mutant proteins, to be displayed on custom programmable protein microarrays. To determine the epitope specificity we displayed 12 overlapping tiling fragments and 38 N- and C-terminal deletions spanning the length of the wild-type p53 protein., Results: We detected p53-AAb with sensitivities of 58.8% (ovarian), 22% (pancreatic), 32% (triple negative breast cancer), and 10.2% (HER2+ breast cancer) at 94% specificity. Sera with p53-AAb contained broadly reactive autoantibodies to 51 displayed p53 mutant proteins, demonstrating a polyclonal response to common epitopes. All p53-AAb displayed broad polyclonal immune response to both continuous and discontinuous epitopes at the N- and C-terminus as well as the DNA-binding domain., Conclusion and Clinical Relevance: In this comprehensive analysis, mutations in tumor p53 induce strong, polyclonal autoantibodies with broadly reactive epitope specificity., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: We aim to develop a protein microarray platform capable of presenting both natural and denatured forms of proteins for antibody biomarker discovery. We will further optimize plasma screening protocols to improve detection., Experimental Design: We developed a new covalent capture protein microarray chemistry using HaloTag fusion proteins and ligand. To enhance protein yield, we used HeLa cell lysate as an in vitro transcription translation (IVTT) system. Escherichia coli lysates were added to the plasma blocking buffer to reduce nonspecific background. These protein microarrays were probed with plasma samples and autoantibody responses were quantified and compared with or without denaturing buffer treatment., Results: We demonstrated that protein microarrays using the covalent attachment chemistry endured denaturing conditions. Blocking with E. coli lysates greatly reduced the background signals and expression with IVTT based on HeLa cell lysates significantly improved the antibody signals on protein microarrays probed with plasma samples. Plasma samples probed on denatured protein arrays produced autoantibody profiles distinct from those probed on natively displayed proteins., Conclusions and Clinical Relevance: This versatile protein microarray platform allows the display of both natural and denatured proteins, offers a new dimension to search for disease-specific antibodies, broadens the repertoire of potential biomarkers, and will potentially yield clinical diagnostics with greater performance., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)