DETECTION OF RARE DISSEMINATED TUMOR CELLS IN BLOOD AND BONE MARROW

血液和骨髓中罕见播散性肿瘤细胞的检测

• 批准号: 7378599
• 负责人: DAVID N KRAG
• 金额: $ 0.11万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-09 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7378599
• 关键词: antibody; blood; bone marrow; neoplasm /cancer

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. European investigators have reported that detection of disseminated tumor cells in the bone marrow and blood of breast cancer patients provides important prognostic information.(Braun, Pantel et al. 2000) A number of technical challenges exist with the methodology for detection of rare cells including 1) processing of the specimen to decrease the enormous background of normal hematopoietic cells, 2) immunohistochemical staining of candidate tumor cells, and 3) interpretation of candidate cells as cancer cells. The current strategy in most labs to reduce background hematopoietic cells is to use density centrifugation or an immunomagnetic selection (Funke and Schraut 1998). Candidate tumor cells remaining in the selected material are labeled with antibodies that bind to common epithelial antigens. Since epithelial cells are not typically present in blood or marrow any cell that stains positive for an epithelial antigen has increased probability of being a disseminated epithelial cancer cell. The final step is scoring the labeled cell as benign epithelial or a malignant tumor cell. This involves specialized cytopathological skill in interpreting cells types. For the past years our laboratory has evaluated several parameters related to the methodology of rare tumor cell detection in blood and marrow. For example we have evaluated all major methods for processing the specimen to reduce background hematopoietic cells (HCs). In our hands density centrifugation is the preferred method that strikes a balance between background cell reduction, cost, and convenience. We have also evaluated several strategies for antibody labeling of candidate tumor cells. The most important result from work to date is that all of the reported antibodies result in undesirable labeling of HCs. The frequency of labeling HCs varies but in all cases is sufficiently high to be competitive with the frequency of labeling candidate tumor cells. Our strategy to overcome the issue of undesirable labeling with epithelial antibodies is to use additional antibodies that label HCs. A hematopoietic cell that is labeled with an epithelial antibody would also be labeled with the hematopoietic antibodies. Thus a dual labeled cell would be a tip-off that the cell is not a cancer cell but in fact is a hematopoietic cell. We have used conventional fluorescence microscopy to all reasonably facile evaluation of different sets of antibodies by using reagents that fluoresce in different color spectra. For example, epithelial antibodies stain labeled cells bright green and the HC antibodies stain cells bright red. Light filters on the microscope allow us to selectively visualize only green, or red, or a combination of green and red. In practice, a candidate green cell is readily visualized first. Then the light filter is changed and if that same cell is red it is a hematopoietic cell. Using this approach we have determined that a surprisingly high percentage of candidate tumor cells are in fact HCs. There are several permutations of the experimental conditions for the assay that we have evaluated. We continually are seeking to improve this assay by evaluating additional permutations. For example, we have not yet identified a cocktail of antibodies that label 100% of the marrow aspirate hematopoietic cells. This is an important goal and we continue to seek additional HC-labeling antibodies that will increase the percentage labeling. The hypothesis of the current study maintains that a combination of epithelial and hematopoietic cell marker antibodies can be developed that allow effective discrimination of rare disseminated epithelial cancer cells from background nonmalignant hematopoietic cells. Specific Aims: 1. Optimize blood and marrow sample processing to decrease the number of background hematopoietic cells relative to the target epithelial cells. 2. Identify a cocktail of hematopoietic antibodies that effectively label more than 90% of HCs in marrow and blood. 3. Identify optimal exposure duration and concentration of epithelial cancer cell and hematopoietic cell antibodies. 4. Determine most effect strategy for screening optimally stained specimens.

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。欧洲研究人员报告说，在乳腺癌患者的骨髓和血液中检测弥散性肿瘤细胞提供了重要的预后信息。（Braun, Pantel et al. 2000）检测稀有细胞的方法存在许多技术挑战，包括1)处理标本以减少正常造血细胞的大量背景，2)候选肿瘤细胞的免疫组织化学染色，以及3)将候选细胞解释为癌细胞。目前大多数实验室减少背景造血细胞的策略是使用密度离心或免疫磁选择（Funke和Schraut 1998）。残留在选定材料中的候选肿瘤细胞被标记为与常见上皮抗原结合的抗体。由于上皮细胞通常不存在于血液或骨髓中，任何上皮抗原染色呈阳性的细胞都增加了播散性上皮癌细胞的可能性。最后一步是将标记的细胞标记为良性上皮细胞或恶性肿瘤细胞。这涉及到解释细胞类型的特殊细胞病理学技能。在过去的几年里，我们的实验室已经评估了与血液和骨髓中罕见肿瘤细胞检测方法相关的几个参数。例如，我们已经评估了处理标本以减少背景造血细胞（hc）的所有主要方法。在我们看来，密度离心是在背景细胞减少、成本和便利性之间取得平衡的首选方法。我们还评估了几种候选肿瘤细胞的抗体标记策略。迄今为止工作中最重要的结果是，所有报告的抗体都会导致不良的hc标记。标记hcc的频率各不相同，但在所有情况下都足够高，足以与标记候选肿瘤细胞的频率竞争。我们的策略是克服不良标记的上皮抗体的问题是使用额外的抗体来标记hcc。用上皮抗体标记的造血细胞也会被标记为造血抗体。因此，双标记细胞将提示该细胞不是癌细胞，而实际上是一种造血细胞。我们使用传统的荧光显微镜，通过使用不同颜色光谱的荧光试剂，对不同组的抗体进行合理方便的评估。例如，上皮抗体将标记细胞染色为亮绿色，HC抗体将标记细胞染色为亮红色。显微镜上的滤光片允许我们选择性地只看到绿色、红色或绿色和红色的组合。在实践中，候选绿色细胞很容易首先可视化。然后改变滤光器，如果同样的细胞是红色的，那就是造血细胞。使用这种方法，我们已经确定了令人惊讶的高百分比的候选肿瘤细胞实际上是hcc。我们已经评估过的试验条件有几种排列。我们不断寻求通过评估其他排列来改进这一分析。例如，我们还没有发现一种鸡尾酒抗体能够标记100%的骨髓抽取的造血细胞。这是一个重要的目标，我们将继续寻找更多的hc标记抗体，以增加标记百分比。本研究的假设认为，可以开发一种上皮细胞和造血细胞标记抗体的组合，从而有效地区分罕见的弥散性上皮癌细胞和背景的非恶性造血细胞。具体目标：1；优化血液和骨髓样品处理，以减少相对于目标上皮细胞的背景造血细胞的数量。2. 鉴定一种鸡尾酒的造血抗体，它能有效地标记骨髓和血液中90%以上的hc。3. 确定上皮癌细胞和造血细胞抗体的最佳暴露时间和浓度。4. 确定筛选最佳染色标本的最有效策略。