Analysis of scant cancer cells in fine needle aspirates

 描述(由申请人提供)：对临床样本中的关键癌症蛋白质和途径标记物的全面分析仍然具有挑战性，但对于在临床试验中评估分子靶向药物的疗效和了解复杂的肿瘤生物学是至关重要的。目前，正在研究的标记物的数量通常是有限的(10英镑)，并且需要对大量核心活检获得的组织切片进行耗时的分析，这带来了不小的发病率。我们开发了一种技术，可以同时分析从细针抽吸物(FNA)中获取的癌细胞中的数百种蛋白质。该方法利用DNA条形码抗体传感，其中条形码被光解并在没有任何放大步骤的情况下进行数字检测。在最近的一项概念验证研究中(Sci Transl Med 2014；6：219ra9)，该方法显示出高度的重复性，实现了单细胞敏感性，并能够识别对分子靶向药物的途径反应，即使在单细胞中也是如此。与现有技术(免疫组织化学、细胞学和质谱学)相比，它：i)允许同时检测数百个标记物；ii)在单个细胞或少量细胞中工作良好；iii)不破坏有价值的样本；iv)快速且廉价；v)可以与mRNA和DNA分析技术相结合。这项R33应用的目标是通过将其与i)用于癌细胞扩增和单个/大量细胞采集的芯片上微流体以及ii)蛋白质、信使核糖核酸和DNA分析相结合来进一步发展这项技术。然后，我们将在两项临床研究中扩展和严格测试这种用于单细胞护理点分析的新一代设备，以广泛展示其广泛的抗癌功效：i)在临床诊断研究中，比较乳腺癌患者的癌细胞蛋白质谱；以及ii)在药物试验中抑制PI3K，以确定随时间推移的治疗反应/失败。所提出的综合分析方法具有改变癌症研究和临床实践的潜力。它将在临床试验的稀缺和有价值的材料中对细胞标志物进行廉价、更广泛和更强大的剖析。

 DESCRIPTION (provided by applicant): Comprehensive analysis of key cancer proteins and pathway markers in clinical samples remains challenging yet is critical in assessing efficacy of molecularly targeted drugs in clinical trial and in understanding complex tumor biology. Currently, the number of markers being studies is often limited (<10) and requires time- consuming analyses of tissue sections harvested by large core biopsies which carry a not insignificant morbidity. We developed a technology that allows simultaneous analysis of hundreds of proteins in cancer cells harvested from fine needle aspirates (FNA). The method capitalizes on DNA-barcoded antibody sensing where barcodes are photo-cleaved and digitally detected without any amplification steps. In a recent proof-of- concept study (Sci Transl Med 2014;6: 219ra9) this method showed high reproducibility, achieved single cell sensitivity and was able to identify pathway responses to molecularly targeted drugs, even in single cells. Compared to existing technology (immunohistochemistry, cytometry and mass spectrometry) it: i) allows hundreds of markers to be detected simultaneously, ii) works well in single cells or small numbers of cells, iii) does not destroy valuable samples, iv) is fast and inexpensive and v) can be combined with mRNA and DNA analytical techniques. The goal of this R33 application is to further the technology by integrating it with i) on-chip microfluidics for cancer cell enrichmen and single/bulk cell harvesting and ii) combined protein, mRNA and DNA analysis. We will then expand and rigorously test this next generation device for point-of-care analyses of single cells in two clinical studies to broadly demonstrate its broad cancer utility: i) in a clinical diagnosti study to compare cancer cell protein profiles in breast cancer patients and ii) in a drug trial of PI3K inhibition to determine treatment response/failure over time. The proposed integrated profiling method has the potential to transform cancer research and clinical practice. It will allo inexpensive, more extensive and robust profiling of cellular markers in scant and valuable materials from clinical trials.