DETECTION OF RARE DISSEMINATED TUMOR CELLS IN BLOOD AND BONE MARROW

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，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到播散的肿瘤细胞提供了重要的预后信息。（Braun，Pantel等人，2000）检测稀有细胞的方法存在许多技术挑战，包括1）处理样本以减少正常造血细胞的巨大背景，2）候选肿瘤细胞的免疫组织化学染色，和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性选择（Funke and Schraut 1998）。 保留在所选材料中的候选肿瘤细胞用与共同上皮抗原结合的抗体标记。由于上皮细胞通常不存在于血液或骨髓中，任何上皮抗原染色阳性的细胞都增加了成为播散性上皮癌细胞的可能性。最后一步是将标记的细胞评分为良性上皮细胞或恶性肿瘤细胞。这涉及解释细胞类型的专门细胞病理学技能。 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学相关的几个参数。例如，我们已经评估了用于处理标本以减少背景造血细胞（HC）的所有主要方法。在我们手中，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。我们还评估了用于候选肿瘤细胞的抗体标记的几种策略。 迄今为止工作的最重要结果是，所有报告的抗体导致HC的不期望的标记。标记HC的频率不同，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，以克服与上皮抗体的不良标记的问题是使用额外的抗体标记HC。用上皮抗体标记的造血细胞也将用造血抗体标记。因此，双重标记的细胞将提示该细胞不是癌细胞，而实际上是造血细胞。 我们已经使用传统的荧光显微镜，通过使用在不同颜色光谱中发荧光的试剂，对不同抗体组进行了合理的简便评价。例如，上皮抗体将标记的细胞染成亮绿色，HC抗体将细胞染成亮红色。显微镜上的滤光片允许我们选择性地只看到绿色，或红色，或绿色和红色的组合。在实践中，候选绿色单元首先被容易地可视化。然后改变滤光器，如果相同的细胞是红色的，则它是造血细胞。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HC。 我们评价的试验条件有几种排列。我们一直在寻求通过评估其他排列来改进该测定。例如，我们尚未鉴定出标记100%骨髓穿刺造血细胞的抗体混合物。这是一个重要的目标，我们将继续寻找额外的HC标记抗体，以增加标记的百分比。 本研究的假设认为，可以开发上皮细胞和造血细胞标志物抗体的组合，从而有效区分罕见的播散性上皮癌细胞和背景非恶性造血细胞。 具体目标： 1. 优化血液和骨髓样本处理，以减少背景造血细胞相对于靶上皮细胞的数量。 2. 鉴定一种造血抗体混合物，可有效标记骨髓和血液中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。

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.