Automated Systems for Detection and Molecular Characterization of Circulating Tum

描述(申请人提供)：循环肿瘤细胞(CTCs)的检测和计数具有成为癌症诊断和治疗的非常强大的工具的潜力。然而，目前的CTC检测技术还没有建立在常规的临床实践中。其中一个原因是，CTCs在血液中的浓度极低，每1000万个白细胞中就有一个。现有技术缺乏可靠检测这些稀有CTCs所需的灵敏度和/或特异性。另一个原因是，目前的技术没有提供CTCs分子状态的信息，这些信息可以用于选择适当的靶向治疗和监测治疗反应。这项提案的总体目标是开发一种名为CTCcope的自动化系统，用于血液中循环肿瘤细胞(CTCs)的检测和分子表型鉴定。该自动化系统基于ACD的RNAScope“技术和原型CTCcope”系统的成功开发，这两项技术和原型系统都得到了SBIR第二阶段拨款R44CA122444的部分支持。我们在这项提案中的目标是开发和验证用于CTCs的灵敏检测、准确计数和分子表征的自动化CTCcope系统。这项工作的长期目标是利用CTCcope“开发一套高价值的癌症诊断产品。这些产品将满足癌症管理中许多重要的未得到满足的需求，特别是在早期检测、治疗选择和指导以及复发监测方面。在SBIR第二阶段拨款的支持下，我们成功地开发了RNAScope”检测方法，能够以单分子敏感性和极高的特异性对RNA标记进行原位多重检测和定量。我们还开发了一个用于四氯化碳检测和分析的手动原型系统。该手动系统包括三个部分：1)CTC保存：保存CTC并稳定在血液样本中的RNA；2)CTC标记：通过RNAScope检测方法原位标记多个RNA标记物；以及3)CTC检测和分析：根据荧光显微镜下观察到的RNA标记信号检测、计数和鉴定CTC。使用该系统，我们已经展示了对乳腺癌患者循环中肿瘤细胞的高度特异性检测，并表征了它们的EGFR表达状态。这些数据表明，CTCcope具有几个独特的能力，可能会提供潜在的显著优势超过现有的CTC检测方法。在这项提案中，我们将扩展我们目前的工作，开发和验证一个自动化的CTCcope系统，用于可靠地检测和计数CTCs，并对反映CTCs分子表型的RNA标记进行定量分析。此外，作为CTCcope系统验证的一部分，将确定CTCs的HER2和EGFR状态及其与它们在原发肿瘤中的状态的一致性/不一致性。具体目标包括：1)开发一种自动CTCcope“用于CTC检测和分子表征的系统；2)通过使用约230名转移性乳腺癌患者的血液样本与CellSearch(R)系统的性能比较来验证CTCcope；以及3)确定CTC上HER2和EGFR的状态，并比较它们在原发肿瘤上的状态。这项研究的具体成果将包括一个成功开发和验证的自动化CTCcope“平台，从而能够开发一套高价值的诊断产品来极大地改善癌症管理。这项研究还将为一种有价值的诊断产品奠定基础，该产品可以指导转移性乳腺癌患者的靶向治疗选择和监测治疗反应。随着CTCcope”平台的成功开发和验证，可以开发一套高价值的诊断产品来极大地改善癌症管理。 公共卫生相关性：这是SBIR第二阶段过渡性赠款申请。根据这项提议开发的自动化CTCcope“系统将能够对循环中的肿瘤细胞进行可靠的检测、准确的计数和分子分析，从而产生一套高价值的癌症诊断产品，用于早期发现癌症，选择合适的癌症患者进行靶向治疗，确定他们对治疗的反应，以及监测癌症的进展和复发。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: This is a SBIR Phase II Bridge Grant application. The automated CTCscope" system developed under this proposal will enable reliable detection, accurate enumeration, and molecular analysis of circulating tumor cells, which will lead to a suite of high value cancer diagnostic products for early detection of cancer, for selecting right cancer patients for targeted therapies, for determining their response to treatment, and for monitoring cancer progression and recurrence.