Diagnosing Emergence of Kinase Inhibitor Resistance on a Microchip

描述（由申请人提供）：该项目联合研究人员开发并发布了一个新的诊断平台，临床科学家将指导该诊断方法在黑色素瘤中监测靶向激酶抑制剂耐药性的实施。基于微流控图像细胞术（MIC）平台的信号诊断测试将被优化，用于监测临床抑制剂治疗过程中已知分子定义的耐药机制的临床演变。也就是说，该平台将通过细针抽吸（FNA）活检监测pERK、PDGFR¿、pAKT和凋亡报告基因cleaved Caspase-3/7。这些检测的临床应用将通过在激酶抑制剂治疗期间对患者肿瘤的连续监测来证实，包括在治疗前、治疗期间和治疗进展时进行的多次测量。长期目标是早期临床检测耐药机制，并根据信号反应预测肿瘤对特定激酶抑制剂的反应。分析获得性耐药的一个关键问题是肿瘤重复诊断测量的局限性，因为进行手术活检并不总是可行的。这可以通过开发一种微创、细针抽吸（FNA）为基础的方法来表征进展性肿瘤来克服。已建立和发布的MIC平台能够仅使用200至3,000个细胞对多个信号分子进行定量，单细胞蛋白质组学分析。在过去的工作中，同时测量PI3K信号通路中的四种关键信号蛋白，在19个人类脑肿瘤活检中进行，以确定临床不同的患者亚组。结合生物信息学分析，MIC平台为系统病理分析和个性化医疗提供了强大的体外分子诊断技术。激活B-RAFV600E激酶突变发生在50%的人类黑色素瘤中。一种新型突变型braf选择性抑制剂vemurafenib的早期临床经验表明，b - rafv600e阳性黑色素瘤患者的抗肿瘤反应率达到了前所未有的80%。然而，获得性耐药往往在最初的反应后发展。UCLA团队最近的研究揭示了对B-RAF抑制获得性耐药的机制包括i)激活RTK （PDGFRb）依赖于MAPK的生存途径，或者ii)通过N-RAS突变重新激活MAPK途径。这项工作将开发一种基于mic的信号分析，以监测黑色素瘤中与BRAF抑制剂耐药性相关的信号变化。随后，微流控黑色素瘤信号分析将应用于BRAF抑制剂治疗前后的患者细针抽吸活检。黑色素瘤的侵袭性，以及加州大学洛杉矶分校黑色素瘤研究小组建立的临床和基础科学项目(T.格雷伯，H.-R。Tseng， R. Lo和A. Ribas)，使黑色素瘤微流体诊断方法独特地定位于影响正在进行的临床试验和治疗。

DESCRIPTION (provided by applicant): 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.