Isolation of Circulating Tumor Cells from Peripheral Blood

描述(申请人提供)：本申请的目的是开发一种从血液中分离循环肿瘤细胞(CTCs)的新技术。在过去的十年中，有许多出版物描述了癌症患者血液中CTCs的存在。然而，这些方法一直不够灵敏，不足以作为癌症筛查测试来检测其他隐蔽的实体肿瘤。唯一获得FDA批准的CTCS测试仅被批准用于临床分期，用于识别患有许多CTC的患者。因此，需要一种更灵敏的CTCs样品制备技术。我们开发了唯一的抗原依赖、阴性选择技术，用于从外周血中分离CTCs。我们的技术与现有方法的不同之处在于独特的高CTCs回收率，通常为&gt；90%，以及5-6个对数量级的浓缩。我们通过几项将CTCs损失降至最低的样品制备设计创新实现了这些高比率。例如，我们不引入可能干扰下游分析的外表面，如顺磁珠。通过将我们的方法设计为负选择技术，我们已经克服了与正选择细胞分离方法相关的CTC上有限的EpCAM表达的问题。我们的技术也不依赖于CTCs和血细胞之间的任意物理差异，如细胞密度或对化学裂解的敏感性。这项技术也不同于流动分选，因为只需要基本的实验室仪器。我们的方法还避免了离心法，在我们手中，离心法一直导致不可接受的CTCs损失。在这个第一阶段的项目中，我们将完成我们技术的开发，并用模型血液系统和患者样本进行测试。在特定的目标1，我们将合成新的试剂结合物，用于去除全血中的白细胞和红细胞。我们还将表征CTCs浓缩的参数，并评估用于扩大临床试验规模的全新试剂结合物形成方法(在第二阶段)。在目标2中，我们将表征新型细胞防腐剂在标本运输过程中最大限度地提高CTCs活性的有效性。在已发表的文献中，关于血液样本中CTCs保存的信息很少。在目标3中，我们将用前列腺癌患者的临床样本验证该分析，并表征它是否显著好于现有方法。如果成功，这项技术将建立一个在更早的时间点检测癌症的平台，届时手术干预可能会治愈。实体肿瘤治疗的最好前景是手术切除，前提是肿瘤可以在扩散之前及早发现。不幸的是，太多的癌症只有在扩散后才能在临床上被发现。我们建议开发一种新的技术来检测血液中极其罕见的循环肿瘤细胞，以便于早期发现和根治性手术治疗。

DESCRIPTION (provided by applicant): The aim of this application is to develop a new enabling technology for isolating circulating tumor cells (CTCs) from blood. During the last decade, there have been numerous publications describing the presence of CTCs in the blood of cancer patients. However, the methods have been insufficiently sensitive to serve as a cancer screening test to detect otherwise occult solid tumors. The only FDA- cleared CTCs test is approved solely for clinical staging, in identifying patients with many CTCs. Therefore, there is a need for a more sensitive CTCs sample preparation technology. We have developed the only antigen-dependent, negative selection technology for isolating CTCs from peripheral blood. Our technology is distinguished from existing methods by a uniquely high CTCs recovery rate, generally >90%, and a 5-6 log order enrichment. We attain these high rates through several sample preparation design innovations that minimize CTCs losses. For example, we do not introduce an external surface, such as a paramagnetic bead, that may interfere with downstream analysis. We have overcome the problem of limited EpCAM expression on CTCs associated with positive selection cell separation methods by designing ours as a negative selection technology. Our technology also is not dependent upon arbitrary physical distinctions between CTCs and blood cells, such as cellular density or sensitivity to chemical lysis. The technology is also unlike flow sorting, in that only basic laboratory instrumentation is required. Our method also avoids centrifugation, a procedure which, in our hands, consistently resulted in unacceptable CTCs losses. In this Phase I project, we will complete the development of our technology and test it with both model blood systems and patient samples. In Specific Aim 1 we will synthesize new reagent conjugates for removing leukocytes and erythrocytes from whole blood. We will also characterize the parameters of CTCs enrichment and evaluate entirely new methods of forming the reagent conjugates for scale-up to clinical trials (in Phase II). In Aim 2, we will characterize the efficacy of new cellular preservatives for maximizing CTCs viability during specimen transport. There is a dearth of information in the published literature on CTCs preservation in blood samples. In Aim 3, we will validate the assay with clinical samples from patients with prostate cancer and characterize whether it is substantially better than existing methods. If successful, this technology will establish a platform for detecting cancer at an earlier point in time, when surgical intervention may be curative. The best prospect for curative treatment of solid tumors is surgical excision, provided the tumor can be detected early, before it spreads. Unfortunately, all too many cancers are detected clinically only after they have spread. We propose to develop a novel technology for detecting extremely rare circulating tumor cells in blood, so as to facilitate early detection and curative surgical treatment.