This project unites investigators who have developed and published a new diagnostic platform with clinical scientists who will guide the implementation of this diagnostic approach in monitoring targeted kinase inhibitor resistance in melanoma. A signaling diagnostic test based on the microfluidic image cytometry (MIC) platform will be optimized for monitoring the clinical evolution of known molecularly defined resistance mechanisms during clinical inhibitor treatment. Namely, the platform will monitor pERK, PDGFR?, pAKT and the apoptosis reporter cleaved Caspase-3/7 using fine needle aspirate (FNA) biopsies. The clinical utility of these assays will be confirmed by serially monitoring patient's tumors during kinase inhibitor therapy - including multiple measurements made before and during treatment and upon progression. The long-term goal is early clinical detection of resistance mechanisms, and 'in patient-treatment'-based prediction of tumor responsiveness to specific kinase inhibitors based on signaling responses. A key issue in analyzing acquired resistance is the limitation of repeat diagnostic measurement of tumors since it is not always feasible to perform surgical biopsies. This can be overcome by developing a minimally-invasive, fine needle aspirate (FNA)-based approach to characterize progressive tumors. The established and published MIC platform is capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 200 to 3,000 cells. In past work, simultaneous measurement of four critical signaling proteins within the PI3K signaling pathway was performed on a panel of 19 human brain tumor biopsies to identify clinically distinct patient subgroups. Together with bioinformatic analysis, the MIC platform provides a robust, enabling, in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine. Activating B-RAFV600E kinase mutations occur in 50% of human melanomas. Early clinical experience with a novel mutant BRAF-selective inhibitor, vemurafenib, have demonstrated an unprecedented 80% anti-tumor response rate among patients with B-RAFV600E-positive melanomas. However, acquired drug resistance frequently develops after initial responses. Recent studies by the UCLA team unveiled that mechanisms of acquired resistance to B-RAF inhibition include i) activating an RTK (PDGFRb)-dependent survival pathway in addition to MAPK, or ii) reactivating the MAPK pathway via N-RAS mutations. This work will develop a MIC-based signaling assay to monitor signaling changes associated with BRAF inhibitor resistance in melanoma. Subsequently, the microfluidic melanoma-signaling assay will be applied to patient fine needle aspirate biopsies pre- and post-treatment with BRAF inhibitors. The particularly aggressive nature of melanoma, and the established clinical and basic science programs of the UCLA melanoma team (T. Graeber, H.-R. Tseng, R. Lo and A. Ribas), make the melanoma microfluidic diagnostic approach uniquely positioned to impact ongoing clinical trials and therapy.
The goal of this proposal is to develop a microfluidic signaling diagnostic test for melanoma based on the MIC (Microfluidic Image Cytometry) platform. The platform will monitor the clinical evolution of known molecularly defined resistance mechanisms during clinical inhibitor treatment of melanoma patients. Ultimately, the proposed microfluidic signaling diagnostic platform will be coupled to clinical practice to enable early detection of resistance mechanisms, and 'in patient-treatment'-based prediction of tumor responsiveness to particular kinase inhibitor options.
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