Automated Systems for Detection and Molecular Characterization of Circulating Tum

项目总结： 循环肿瘤细胞(CTCs)的检测和计数有可能成为一种非常重要的 癌症诊断和治疗的有力工具。然而，目前用于CTC检测的技术是 尚未在常规临床实践中确立。其中一个原因是血液中存在CTCs。 极低的浓度，每1000万个白细胞中就有一个。现有的技术缺乏 可靠地检测这些罕见的CTC所需的敏感性和/或特异性。另一个原因是 目前的技术没有提供关于CTCs分子状态的信息，这可以用来 选择合适的靶向治疗，监测治疗反应。 这项提议的总体目标是开发一种名为CTCcope的自动化系统来进行检测 血液中循环肿瘤细胞(CTCs)的分子表型。这个自动化系统的基础是 关于ACD RNAScope“技术和原型CTCcope”系统的成功开发， 这两项计划都得到了SBIR第二阶段拨款R44CA122444的部分支持。我们在这项建议中的目的是 为了开发和验证自动CTCcope系统，以实现灵敏、准确的检测 四氯化碳的计数和分子表征。这一努力的长期目标是利用 开发一套高价值的癌症诊断产品。这些产品将满足 癌症管理方面许多重大的未得到满足的需求，特别是在早期发现领域， 治疗方法的选择和指导，以及复发监测。 在SBIR第二阶段拨款的支持下，我们成功地开发了RNAScope“分析 能够对单分子敏感的RNA标记进行原位多重检测和定量 和精致的专一性。我们还开发了一个手动原型系统，用于CTC检测和 分析。人工系统包括三个组成部分：1)CTC保存：CTC保存 和稳定在血液样本中的RNA；2)CTC标记：CTC被标记为多个RNA标记 3)CTC检测和分析：CTCs被检测、计数 并根据在荧光显微镜下观察到的RNA标记信号进行表征。vbl.使用 这个系统，我们展示了对循环肿瘤细胞的高度特异性检测，并表征了 乳腺癌患者外周血中EGFR的表达状况。这些数据表明，CTCcope“ 具有几个独特的功能，这些功能可能会提供相对于现有CTC的显著优势 检测方法。 在这项提案中，我们将扩展我们目前的工作，以开发和验证自动CTCcope。 用于CTCs的可靠检测和计数以及RNA标记的定量分析的系统 反映了CTCs的分子表型。此外，作为CTCcope系统验证的一部分， CTC中HER2和EGFR的状态及其与其在小学中的地位的一致性/不一致性 肿瘤将会被确定。具体目标包括：1)开发一个自动化的CTCcope系统，用于 CTC检测和分子表征；2)通过比较CTC Scope的性能来验证其有效性 使用CellSearch(R)系统采集约230名转移性乳腺癌患者的血液样本； 检测CTCs上HER2和EGFR的状态，并比较它们在原发肿瘤中的状态。具体的 这项研究的成果将包括一个成功的自动化CTCcope平台 开发和验证，使一套高价值的诊断产品能够以极大的 改善癌症管理。这项研究也将为有价值的诊断奠定基础 可指导转移性乳腺肿瘤靶向治疗选择和监测治疗反应的产品 癌症患者。随着CTCcope平台的成功开发和验证，一套高性能的 可以开发有价值的诊断产品来极大地改善癌症管理。

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.