Automated Systems for Detection and Molecular Characterization of Circulating Tum

描述（由申请人提供）：循环肿瘤细胞（CTCs）的检测和计数有可能成为癌症诊断和治疗的一个非常强大的工具。然而，目前的CTC检测技术尚未建立在常规临床实践中。原因之一是ctc在血液中的浓度极低，仅为千万分之一。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有关ctc分子状态的信息，这些信息可用于选择合适的靶向治疗和监测治疗反应。该提案的总体目标是开发一种称为CTCscope的自动化系统，用于血液中循环肿瘤细胞（CTCs）的检测和分子表型分析。该自动化系统基于ACD的RNAscope“技术”和原型CTCscope系统的成功开发，两者都得到了SBIR二期拨款R44CA122444的部分支持。我们的目标是开发和验证自动CTCscope系统，用于CTCs的灵敏检测，准确枚举和分子表征。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。这些产品将满足癌症管理中许多未被满足的重要需求，特别是在早期发现、治疗选择和指导以及复发监测方面。在SBIR二期资助的支持下，我们已经成功开发了RNAscope”检测，能够原位多重检测和定量RNA标记物，具有单分子敏感性和精致的特异性。我们还开发了一个用于CTC检测和分析的手动原型系统。手工系统包括三个部分：1)CTC保存：保存CTC并稳定血液样品中的RNA；2) CTC标记：CTC通过RNAscope原位标记多种RNA标记物；3) CTC检测与分析：基于荧光显微镜下观察到的RNA标记信号对CTC进行检测、枚举和表征。使用该系统，我们已经证明了对乳腺癌患者循环肿瘤细胞的高度特异性检测，并表征了其EGFR表达状态。这些数据表明，CTCscope具有几种独特的功能，可能比现有的CTC检测方法具有显著的优势。在本提案中，我们将扩展我们目前的工作，开发和验证一个自动化的CTCscope系统，用于可靠地检测和枚举CTCs，并用于定量分析反映CTCs分子表型的RNA标记。此外，作为CTCscope系统验证的一部分，将确定CTCs的HER2和EGFR状态及其与原发肿瘤状态的一致性/不一致性。具体目标包括：1)开发用于CTC检测和分子表征的自动化CTCscope系统；2)通过将CTCscope与CellSearch(R)系统的性能进行比较，验证CTCscope的有效性，该系统使用了约230例转移性乳腺癌患者的血液样本；3)检测CTCs中HER2和EGFR的状态，比较其在原发肿瘤中的状态。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CTCscope平台，使一套高价值的诊断产品能够开发出来，大大改善癌症管理。该研究也将为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CTCscope平台的成功开发和验证，一套高价值的诊断产品可以大大改善癌症管理。

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.