MICROFLUIDIC DIAGNOSTICS FOR MONITORING OF BRAF INHIBITOR RESISTANCE IN MELANOMA

此PPG中的项目3的目标是开发由我们的 从细针抽吸中量化多个信号事件和基因组损伤的研究小组 (FNA)活检或循环黑色素瘤细胞(CMC)。我们将研究最低限度应用的可行性。 有创采样技术(即FNA活组织检查和外周血CMC浓缩) 在BRAF抑制剂(BRAFi)治疗过程中反复采样黑色素瘤细胞。肿瘤细胞 从FNA活检和巨噬细胞中分离出来的细胞将接受单细胞信号分析技术 包括微流控图像细胞术(MIC)用于多种信号的定量蛋白质组学分析 分子和用于逆转录酶聚合酶链式反应的Fluidigm BioMark系统 (RTPCR)和靶向DNA测序。通过生物信息学分析，我们的微流控诊断技术使 系统病理学方法，能够解剖肿瘤异质性和监测颞叶疾病 进化论。我们的长期目标是对耐药机制进行早期临床检测，并用于患者治疗。 根据信号反应预测肿瘤对关节激酶抑制剂的反应性。 激活BRAFV600E激酶突变发生在50%的人类黑色素瘤中。应用于临床的经验 新型突变型BRAF选择性抑制剂维莫拉非尼发现前所未有的60%-80%的抗肿瘤反应率 在BRAFV600E阳性的黑色素瘤患者中。然而，获得性耐药经常发生。 在几乎所有接受治疗的患者都有初步反应后。我们联合团队最近的研究发现， 对BRAF抑制的获得性抵抗包括MAPK通路的重新激活(例如，通过NRAS 突变)或通过RTK/AKT途径(例如，通过PDGFRp)激活替代信号 过度表达)。为了克服BRAFi抗性，我们需要更好地理解、监测和研究进化 在BRAFi治疗过程中的耐药机制。项目3旨在演示微流控诊断技术 动态监测BRAFi耐药的临床演变。 随着联合研究工作的展开，我们的微流控衍生的单细胞蛋白质组和基因组分析 将被用于检测项目1和项目1中与抗药性相关的基因组和磷酸图谱的发现 2在临床患者样本中帮助指导治疗选择。我们还设想，拟议的微流体 诊断学可以用来评估BRAF抑制剂对免疫疗法的影响(项目4)。 相关性(请参阅说明)： 分析黑色素瘤获得性耐药的一个关键问题是重复诊断的局限性 肿瘤的测量。这可以通过应用微创采样技术来克服 描述治疗过程中进展性肿瘤的特征。本PPG中项目3的目标是 开发用于量化多个信号事件和基因组损伤的微流控诊断工具箱 来自细针抽吸(FNA)活检或循环黑色素瘤细胞(CMC)。

The objective of Project 3 in this PPG is to exploit a microfluidic diagnostics toolbox established by our research team for quantification of multiple signaling events and genomic lesions from fine needle aspirated (FNA) biopsies or circulating melanoma cells (CMCs). We will examine the feasibility of applying minimally invasive sampling techniques (i.e., FNA biopsy and peripheral blood draws for CMC enrichment) to repeatedly sample melanoma cells over the course of BRAF inhibitor (BRAFi) treatment. Tumor cells isolated from FNA biopsies and CMCs then will be subjected to single-cell signaling profiling technologies including microfluidic image cytometry (MIC) for quantitative proteomic analysis of multiple signaling molecules, and the Fluidigm BioMark^'^ system for reverse-transcriptase polymerase chain reaction (RTPCR) and targeted DNA sequencing. With bioinformatic analysis, our microfluidic diagnostics enable a systems pathology approach, capable of dissecting tumor heterogeneity and monitoring temporal disease evolution. Our long-term goal is eariy clinical detection of resistance mechanisms, and 'in patient-treatment' based prediction of tumor responsiveness to articular kinase inhibitors based on signaling responses. Activating BRAFV600E kinase mutations occur in 50% of human melanomas. Clinical experience with the novel mutant BRAF-selectlve inhibitor vemurafenib found an unprecedented 60-80% antitumor response rate among patients with BRAFV600E-positive melanomas. However, acquired drug resistance frequently develops after initial responses in almost all treated patients. Recent studies by our joint team found that mechanisms of acquired resistance to BRAF inhibition include reactivation of the MAPK pathway (e.g., via NRAS mutation) or activation of alternative signaling through the RTK/AKT pathway (e.g., via PDGFRp overexpression). To overcome BRAFi resistance, we need to better understand, monitor and study evolution of resistance mechanisms during BRAFi treatment. Project 3 aims to demonstrate microfluidic diagnostics for dynamic monitoring the clinical evolution of BRAFi resistance. As the joint research endeavor unfolds, our microfluldlcs-derived single-cell proteomic and genomic assays will be applied to detect the resistance-associated genomic and phospho-profile findings from Projects 1 and 2 in clinical patient samples to help guide therapy choices. We also envision that the proposed microfluidic diagnostics can be employed to assess that the Impact of BRAF inhibitors on immune therapies (Project 4). RELEVANCE (See instructions): A key issue In analyzing acquired resistance in melanoma is the limitation of repeated diagnostic measurements of tumors. This can be overcome by applying minimally invasive sampling techniques to characterize the progressive tumors over the course of treatment. The objective of Project 3 in this PPG is to exploit a microfluidic diagnostics toolbox for quantification of multiple signaling events and genomic lesions from fine needle aspirated (FNA) biopsies or circulating melanoma cells (CMCs).