Multiplexed Tumor Marker Profiling at Single-Cell Resolution: Quantifying Breast Cancer Molecular Signatures.

Excluding skin cancers, about 1.5 million new cases of cancer and about 450,000 cancer related deaths occur each year in the United States. The validation and molecular profiling of tumor markers are contributing to new paradigms in cancer research. In this presentation, we will discuss the information that can be obtained by using an Intelligent Single-Cell Optical Profiling Engine (I-SCOPE), consisting of hyperspectral imaging, multiplexed biomarker kits, and bioinformatics tools, to markedly expand the molecular profiling of tumor markers on individual carcinoma cells.Using biomarker antibodies conjugated to particular fluorochromes, we developed a multiplex of 10 different fluorochrome conjugates, one for each different tumor marker plus a nuclear dye. Staining and scanning using the I-SCOPE system showed that it can presently distinguish 10 different fluorochromes in a single pass, resulting in the identification of spatial location and relative intensity of each biomarker being interrogated. By using this system, we analyzed thousands of tumor cells from touch preparations of tumor tissue, circulating tumor cells (CTCs) in pleural fluid and blood, and disseminated tumor cells (DTCs) from bone marrow with 2 multiplex kits for a total of 17 different tumor markers. We have utilized clinically-accepted markers such as estrogen receptor (ER), progesterone receptor (PR), and HER2 in addition to biomarkers undergoing investigation such as B-cell lymphoma 2 (Bcl-2) and urokinase-type plasminogen receptor (uPAR). Furthermore, with the ability to simultaneously image 10 markers, each panel can also contain desired positive and negative controls such as CD45 (positive for white blood cells, negative for CTCs) and cytokeratin (positive for epithelial cells).The results of immunophenotyping of individual tumor cells with 17 different tumor markers indicated that the average expression of each tumor marker in 25 tumor cells was a reproducible representation of the tumor. However, comparison of individual CTCs9 expression levels revealed remarkable heterogeneity in primary breast cancer cases.We conclude that hyperspectral microscope imaging together with individual tumor cell analysis represents a major step forward in developing a far more detailed and precise molecular signature of a patient9s cancer. Further studies might demonstrate how it can facilitate treatment tailored to the individual patient9s tumor, an important objective as new oncology drugs with specific molecular targets become more prevalent. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6002.