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e22090 Background: Optical proteomics quantifies interactions between proteins and post-translational modifications by measuring Förster resonance energy transfer (FRET) quantified by fluorescence lifetime imaging microscopy (FLIM). This project aims to derive multiple high throughput optical proteomic markers, to predict metastatic risk at first diagnosis, and to perturb 'high risk' protein-protein interactions using targeted therapeutics. This initial step develops robust FRET/FLIM assays, suitable for use in formalin fixed paraffin embedded (FFPE) tissue to be correlated with patient outcome. METHODS: Fluorophore-conjugated antibodies to proteins involved in cell migration and survival, were applied to tissue microarrays (TMA), created from archived FFPE invasive ductal breast carcinoma samples. Where fluorophores are located within nanometer proximity, FRET occurs, thus allowing quantification of protein-protein interaction. Ezrin and PKCα phosphorylation, distribution, and interaction were imaged on four TMAs (patients diagnosed with early breast cancer 1984 -1987: 20 years follow-up data). RESULTS: 71 patient samples were optically imaged. Patients were clustered based on the pairwise distances between 18 optical variables 'input data'. Data are represented on self organising maps and dendrograms and correlated with clinical outcome 'output data', displaying a heatmap distribution. CONCLUSIONS: Ezrin and PKCα phosphorylation, distribution, and interaction imaged optically within FFPE contain prognostic information regarding metastatic outcome in breast cancer, thus stepping ever closer to individualising prognosis. These advanced optics-based parameters informing on metastatic potential will be validated in prospective studies in conjunction with FRET/FLIM assays measuring HER2/HER3 dimerisation, and EGFR and HER2 ubiquitination in order to improve patient selection for targeted therapy. No significant financial relationships to disclose.


Journal article


J Clin Oncol

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