MICROFLUIDIC DIAGNOSTICS FOR MONITORING OF BRAF INHIBITOR RESISTANCE IN MELANOMA

本PPG中项目3的目标是利用我们建立的微流体诊断工具箱。 研究小组对来自细针抽吸细胞的多种信号传导事件和基因组病变进行定量 (FNA)活组织检查或循环黑素瘤细胞（CMC）。我们将研究最低限度适用于 侵入性采样技术（即，用于CMC富集的FNA活检和外周血抽取）， 在BRAF抑制剂（BRAFi）治疗的过程中重复取样黑素瘤细胞。肿瘤细胞 从FNA活检和CMC中分离的细胞然后将进行单细胞信号分析技术 包括用于多种信号传导的定量蛋白质组学分析的微流控图像细胞术（MIC）， 分子，以及用于逆转录酶聚合酶链反应的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可以实现 系统病理学方法，能够解剖肿瘤异质性和监测颞叶疾病 进化我们的长期目标是早期临床检测耐药机制，并“在患者治疗中” 基于信号传导反应的肿瘤对关节激酶抑制剂的反应性预测。 激活BRAFV 600 E激酶突变发生在50%的人类黑色素瘤中。临床经验 新型突变BRAF选择性抑制剂维罗非尼发现前所未有的60-80%的抗肿瘤反应率 BRAFV 600 E阳性黑色素瘤患者中。然而，获得性耐药性经常发展 在几乎所有接受治疗的患者中，我们的联合团队最近的研究发现， 对BRAF抑制的获得性抗性包括MAPK途径的再活化（例如，通过NRAS 突变）或通过RTK/AKT途径激活替代信号（例如，通过PDGFRP 过表达）。为了克服BRAFi抗性，我们需要更好地了解，监测和研究进化 在BRAFi治疗过程中的耐药机制。项目3旨在展示微流体诊断， 动态监测BRAFi耐药性的临床演变。 随着联合研究奋进的展开，我们的微流衍生单细胞蛋白质组学和基因组学检测 将应用于检测项目1和项目2中与耐药相关的基因组和磷酸谱结果， 2在临床患者样本中，以帮助指导治疗选择。我们还设想，所提出的微流体 诊断学可用于评估BRAF抑制剂对免疫疗法的影响（项目4）。 相关性（参见说明）： 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， 在治疗过程中表征进展性肿瘤。本PPG中项目3的目标是 利用微流控诊断工具箱定量多种信号事件和基因组损伤 来自细针抽吸（FNA）活组织检查或循环黑素瘤细胞（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).