DETECTION OF RARE DISSEMINATED TUMOR CELLS IN BLOOD AND BONE MARROW

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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.