Diagnosing Emergence of Kinase Inhibitor Resistance on a Microchip

诊断微芯片上激酶抑制剂耐药性的出现

• 批准号: 8536250
• 负责人: THOMAS G GRAEBER
• 金额: $ 18.89万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-24 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8536250
• 关键词: Aftercare; Apoptosis; BRAF gene; Basic Science; Bioinformatics; Biological Assay; Biopsy; Brain Neoplasms; Cell Line; Cells; Cleaved cell; Clinical; Clinical Sciences; Clinical Trials; Collection; Coupled; Cutaneous; Data; Detection; Diagnosis; Diagnostic; Diagnostic tests; Disease; Drug resistance; Early Diagnosis; Event; Evolution; Fine needle aspiration biopsy; Flow Cytometry; Future; Goals; Human; Image Cytometry; In Vitro; Location; MAP Kinase Gene; Measurement; Measures; Medicine; Melanoma Cell; Microfluidic Analytical Techniques; Microfluidics; Molecular; Molecular Profiling; Monitor; Mutation; NRAS gene; Nature; Neoplasm Metastasis; Operative Surgical Procedures; Outpatients; PDGFRB gene; Pathology; Pathway interactions; Patient Monitoring; Patients; Phase; Phosphorylation; Phosphotransferases; Positioning Attribute; Process; Proteomics; Proto-Oncogene Proteins c-akt; Publishing; Punch Biopsy; Receptor Protein-Tyrosine Kinases; Reporter; Research Personnel; Resistance; Resistance development; Sampling; Scientist; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Protein; Subgroup; System; Techniques; Technology; Therapy Clinical Trials; Time; Treatment outcome; Work; base; caspase-3; clinical practice; combinatorial; design; experience; inhibitor/antagonist; kinase inhibitor; melanoma; microchip; minimally invasive; mutant; novel; novel diagnostics; programs; resistance mechanism; response; therapy design; therapy development; tumor; tumor progression

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.

描述(由申请人提供)：该项目将开发和发布新诊断平台的研究人员与临床科学家联合起来，他们将指导这一诊断方法的实施，以监测黑色素瘤的靶向激酶抑制剂耐药性。基于微流控图像细胞仪(MIC)平台的信号诊断测试将被优化，以监测在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)活检来监测PERK、PDGFR、PAKT和凋亡报告裂解的Caspase-3/7。这些分析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而得到证实--包括在治疗前、治疗期间和进展时进行的多项测量。长期目标是及早临床发现耐药机制，并根据信号反应预测肿瘤对特定激酶抑制剂的反应性。分析获得性耐药的一个关键问题是肿瘤的重复诊断测量的局限性，因为手术活检并不总是可行的。这可以通过开发一种基于微创、细针抽吸(FNA)的方法来表征进展性肿瘤来克服。建立和发布的MIC平台能够仅使用200到3000个细胞对多个信号分子进行定量的单细胞蛋白质组分析。在过去的工作中，对19例人脑肿瘤活检组织同时测量了PI3K信号通路中的四个关键信号蛋白，以确定临床上不同的患者亚组。与生物信息学分析相结合，MIC平台为系统病理分析和个性化医学提供了强大的、使能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。使用新型突变型BRAF选择性抑制剂维莫拉非尼的早期临床经验表明，在B-RAFV600E阳性黑色素瘤患者中，Vemurafenib的抗肿瘤反应率达到前所未有的80%。然而，获得性耐药往往在最初的反应后发生。加州大学洛杉矶分校的研究小组最近的研究揭示，获得性抵抗B-RAF抑制的机制包括：i)除MAPK外，还激活依赖RTK(PDGFRb)的生存途径，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的信号分析来监测与黑色素瘤中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患者使用BRAF抑制剂治疗前后的细针抽吸活组织检查。黑色素瘤的特别侵袭性，以及加州大学洛杉矶分校黑色素瘤团队(T.Graeber、H.-R.Tseng、R.Lo和A.Ribas)确立的临床和基础科学计划，使黑色素瘤微流控诊断方法具有独特的定位，可以影响正在进行的临床试验和治疗。