Automated Systems for Detection and Molecular Characterization of Circulating Tum

项目摘要： 循环肿瘤细胞（CTC）的检测和计数有可能成为一个非常重要的工具。 癌症诊断和治疗的有力工具。然而，目前的CTC检测技术 尚未在常规临床实践中确立。一个原因是CTC以约20%的浓度存在于血液中。 极低浓度的一千万分之一的白色血细胞。现有技术缺乏 可靠检测这些罕见CTC所需的灵敏度和/或特异性。另一个原因是 目前的技术不能提供关于CTC分子状态的信息，这些信息可用于 选择合适的靶向治疗并监测治疗反应。 该提案的总体目标是开发一种名为"CTCscope”的自动化检测系统 和血液中循环肿瘤细胞（CTC）的分子表型。该自动化系统基于 在ACD的RNAscope”技术和原型CTCscope“系统的成功开发之后， 这两个项目都得到了SBIR第二阶段资助R44 CA 122444的部分支持。我们提出这项建议的目的是 开发和验证自动化CTCscope”系统的灵敏检测，准确 CTC的计数和分子表征。这项工作的长期目标是利用 CTCscope”开发一套高价值的癌症诊断产品。这些产品将满足 在癌症管理方面，特别是在早期发现领域， 治疗选择和指导以及复发监测。 在SBIR第二阶段拨款的支持下，我们成功开发了RNAscope“检测方法， 能够以单分子灵敏度原位多重检测和定量RNA标志物 和精确的特异性。我们还开发了用于CTC检测的手动原型系统， 分析.手动系统包括三个组成部分：1）CTC保存：CTC保存 2）CTC标记：CTC标记多种RNA标志物 3）CTC检测和分析：检测、计数CTC， 并基于在荧光显微镜下观察到的RNA标记信号进行表征。使用 该系统，我们已经证明了循环肿瘤细胞的高度特异性检测和表征 乳腺癌患者EGFR的表达状况。这些数据表明，CTCscope” 有几个独特的能力，可能提供显着的优势，比现有的CTC 检测方法 在本提案中，我们将扩展目前的工作，以开发和验证自动化CTC示波器。 用于CTC的可靠检测和计数以及用于RNA标志物的定量分析的系统 反映CTC的分子表型。此外，作为CTCscope系统验证的一部分， CTC的HER 2和EGFR状态及其与其在原发性肿瘤中的状态的一致性/不一致性 肿瘤将被确定。具体目标包括：1）开发一个自动化的CTCscope系统， CTC检测和分子表征; 2）通过比较其性能， 使用来自约230名转移性乳腺癌患者的血液样本的CellSearch（R）系统;以及3） 确定CTC上的HER 2和EGFR状态，并比较它们在原发性肿瘤上的状态。具体 这项研究的成果将包括一个自动化的CTCscope”平台， 开发和验证，使一套高价值的诊断产品，以大大开发 改善癌症管理。这项研究也将为一项有价值的诊断奠定基础。 可指导转移性乳腺癌靶向治疗选择并监测治疗反应的产品 癌症患者。随着CTCscope”平台的成功开发和验证， 可以开发有价值的诊断产品，以大大改善癌症管理。

Project Summary: The detection and enumeration of circulating tumor cells (CTCs) has the potential to become a very powerful tool in cancer diagnosis and treatment. However, current technologies for CTC detection are not yet established in routine clinical practice. One reason is that CTCs are present in blood at an extremely low concentration of one in 10 million white blood cells. Existing technologies lack the sensitivity and/or specificity required for reliable detection of these rare CTCs. Another reason is that current technologies do not offer information about the molecular status of CTCs, which can be used to select appropriate targeted therapy and monitor therapy response. The overall goal of this proposal is to develop an automated system called CTCscope" for detection and molecular phenotyping of circulating tumor cells (CTCs) in blood. This automated system is based on the successful development of ACD's RNAscope" technology and prototype CTCscope"system, both of which are supported in part by a SBIR Phase II grant R44CA122444. Our aim in this proposal is to develop and validate the automated CTCscope" system for sensitive detection, accurate enumeration, and molecular characterization of CTCs. The long term goal of this effort is to leverage CTCscope" to develop a suite of high value cancer diagnostic products. These products would satisfy many significant unmet needs in cancer management, especially in the areas of early detection, therapy selection and guidance, and recurrence monitoring. With the support of the SBIR phase II grant, we have successfully developed the RNAscope"assay capable of in situ multiplex detection and quantification of RNA markers with single molecule sensitivity and exquisite specificity. We have also developed a manual prototype system for CTC detection and analysis. The manual system includes three components: 1) CTC preservation: CTCs are preserved and RNA stabilized in the blood sample; 2) CTC labeling: CTCs are labeled for multiple RNA markers in situ by the RNAscope"assay; and 3) CTC detection and analysis: CTCs are detected, enumerated and characterized based on RNA marker signals observed under the fluorescent microscope. Using this system, we have demonstrated highly specific detection of circulating tumor cells and characterized their EGFR expression status in breast cancer patients. These data demonstrated that CTCscope" has several unique capabilities which could potentially offer significant advantages over existing CTC detection methods. In this proposal, we will extend our current work to develop and validate an automated CTCscope" system for reliable detection and enumeration of CTCs, and for quantitative analysis of RNA markers reflective of CTCs' molecular phenotype. In addition, as part of the CTCscope" system validation, the HER2 and EGFR status of the CTCs and their concordance/discordance with their status in primary tumor will be determined. The specific aims include: 1) Develop an automated CTCscope" system for CTC detection and molecular characterization; 2) Validate CTCscope" by comparing its performance with the CellSearch(R) system using blood samples from ~230 metastatic breast cancer patients; and 3) Determine HER2 and EGFR status on CTCs and compare their status on primary tumors. The specific outcomes of this research will include an automated CTCscope" platform that is successfully developed and validated, enabling a suite of high value diagnostic products to be developed for greatly improved cancer management. This research will also lay the foundation for a valuable diagnostic product that can guide targeted therapy selection and monitor therapy response for metastatic breast cancer patients. With the CTCscope" platform successfully developed and validated, a suite of high value diagnostic products can be developed for greatly improved cancer management.