Diagnosing Emergence of Kinase Inhibitor Resistance on a Microchip

描述（由申请人提供）：该项目将开发并发布新诊断平台的研究人员与临床科学家联合起来，临床科学家将指导这种诊断方法的实施，以监测黑色素瘤中靶向激酶抑制剂的耐药性。将优化基于微流控图像细胞术（MIC）平台的信号诊断测试，以监测临床抑制剂治疗期间已知分子定义的耐药机制的临床演变。也就是说，该平台将使用细针抽吸（FNA）活检监测pERK、PDGFR、pAKT和细胞凋亡报告基因裂解的Caspase-3/7。这些测定的临床效用将通过在激酶抑制剂治疗期间连续监测患者的肿瘤来证实-包括在治疗之前和期间以及在进展时进行的多次测量。长期目标是耐药机制的早期临床检测，以及基于信号传导反应的基于患者治疗的肿瘤对特异性激酶抑制剂的反应性预测。 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的、基于细针抽吸（FNA）的方法来表征进行性肿瘤来克服。已建立和发布的MIC平台能够使用200至3，000个细胞对多种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PI 3 K信号通路中的四种关键信号蛋白，对一组19例人脑肿瘤活检进行，以确定临床上不同的患者亚组。与生物信息学分析一起，MIC平台为系统病理学分析和个性化医疗提供了一种强大的、可行的体外分子诊断技术。 激活B-RAFV 600 E激酶突变发生在50%的人类黑色素瘤中。新型突变BRAF选择性抑制剂vemurafenib的早期临床经验表明，B-RAFV 600 E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，获得性耐药性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抑制的获得性抗性的机制包括i）除了MAPK之外还激活RTK（PDGFRb）依赖性存活途径，或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的信号传导试验，以监测黑色素瘤中与BRAF抑制剂耐药性相关的信号传导变化。随后，微流体黑素瘤信号传导测定将应用于用BRAF抑制剂治疗前和治疗后的患者细针抽吸活检。黑色素瘤特别具有侵袭性的性质，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。Graeber，H. R.曾河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。 公共卫生相关性：本研究的目的是基于MIC（Microfluidic Image Cytometry）平台开发一种用于黑色素瘤的微流控信号诊断测试。该平台将监测黑色素瘤患者临床抑制剂治疗期间已知分子定义的耐药机制的临床演变。最终，所提出的微流体信号诊断平台将与临床实践相结合，以实现耐药机制的早期检测，以及基于患者治疗的肿瘤对特定激酶抑制剂选择的反应性预测。

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. PUBLIC HEALTH RELEVANCE: 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.