The established method in prognosis of breast cancer includes detection of molecular markers, such as the estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu. These markers are routinely checked via immunohistochemistry (IHC). HER-2/neu is also detected by fluorescent in situ hybridization (FISH). Flow cytometric analysis has not yet been used for detection of such markers. Flow cytometry was performed on four established breast cancer cell lines: MCF7, T47D, BT474, MDA-MB-231, and on one normal breast epithelial cell line: MCF10A. Flow cytometric analysis was used for the detection of ER, PR, HER-2/neu, epidermal growth factor receptor (EGFR), and E-cadherin. Currently, EGFR and E-cadherin are not standard predictive factors in determining survival of breast cancer patients, but both may be beneficial to prognosis. Cells undergoing flow cytometric analysis lost marker expression with increasing passage number. The highest expression was found at cells passaged 0-1 times. MCF7, T47D, and BT474 all had similar marker expression patterns. E-cadherin demonstrated a strongly positive pattern with marker expression of 85-92% among the three cell lines. ER, PR, and HER-2/neu demonstrated a weakly positive expression pattern when compared with E-cadherin. Marker expression ranged from 15 to 61%. These three cell lines were almost negative for expression of EGFR where expression ranged from 0 to 6%. MDA-MB-231 had almost no expression of all 5 markers, with positive values ranging from 0 to 5%. MCF10A had weak positive to almost negative expression values of ER, PR, HER-2/neu, and E-cadherin, which ranged from 3 to 13%. EGFR, both surface and cytoplasmic markers, again were not expressed in MCF10A cells with an expression value of <1%. We found that ER, PR, and HER-2/neu marker expressions in 5 out of 5 cell lines were consistent with established expression patterns. EGFR and E-cadherin expression in 4 out of 5 cell lines were also consistent with established expression patterns. We have shown that flow cytometry provides quantitative data on expression patterns of important prognostic markers in breast cancer.

10057 Methods of determining prognosis in breast, ovary, and colon cancer includes detection of molecular markers such as the estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu. These markers are routinely assessed via immunohistochemistry (IHC) and Fluorescent In-situ hybridization (FISH). Flow cytometry (FC) has not yet been established for routine detection of these markers in tumors. We performed FC on cells from fourteen ovary, five breast, and four colon cancer tumors. FC was used to detect the presence of ER, PR, HER-2/neu, Epidermal Growth Factor Receptor (EGFR), E-cadherin, RAS, ERK and TUJ1. Cells obtained directly from tumoral tissue were simultaneous analysized by cell cycle stage (G0, G2M, and > G2M) and presence of antigenic markers. In ovarian tumors, 7 of 14 samples demonstrated >50% of cells with her-2/neu expression in aneuploid fractions (>G2M), 9 of 14 in G2M phase, and only 2 of 14 in G0 phase. With respect to ER, 7 of 14 ovarian tumors exhibited >19% of cells in G2M and >G2M fractions, while only 1 of 14 samples had >19% at G0. The PR receptor was expressed in >19% of cells in 8 of 14 (>G2M), 7 of 14 (G2M), and 2 of 14 (G0). In breast tumors, 4 of 5 tumors demonstrated >50% expression of HER-2/neu in G2M and >G2M cell phases, and only 2 of 5 demonstrated >50% her2neu expression in G0. Three of five tumors had >19% cellular expression of ER in the >G2M and G2M phases, while only one had >19% in G0. With PR, 3 of 5 breast tumors had >19% cellular expression in G0, G2M, and >G2M phases. In colon tumors, 2 of 4 had >50% cellular expression of her2neu in >G2M fractions, 4 of 4 in G2M, and only 1 of 4 in G0. Interestingly, cell fractions containing G2M and > G2M DNA had > 50% cellular expression for RAS and ERK in 2 of 4 colon tumors. No colon tumors expressed RAS and ERK significantly in G0 cells. We found that ER, PR, and HER-2/neu marker expression were consistent with established expression patterns. Additionally, these data point to specific expression patterns that are associated with the cell cycle stage. In particular, we note high expression levels of Her-2/nue, ER, and PR in aneuploid (>G2M) cell populations. These results suggest that patterns of differential expression can be readily assessed in tumor tissues using flow cytometry, and has the potential for broad application to the study of solid tumors. No significant financial relationships to disclose.