Response to trastuzumab, erlotinib, and bevacizumab, alone and in combination, is correlated with the level of human epidermal growth factor receptor-2 expression in human breast cancer cell lines.

Human epidermal growth factor receptor-2 (HER2) and epidermal growth factor receptor (EGFR) heterodimerize to activate mitogenic signaling pathways. We have shown previously, using MCF7 subcloned cell lines with graded levels of HER2 expression, that responsiveness to trastuzumab and AG1478 (an anti-EGFR agent), varied directly with levels of HER2 expression. HER2 and EGFR up-regulate vascular endothelial growth factor (VEGF), a growth factor that promotes angiogenesis and participates in autocrine growth-stimulatory pathways that might be active in vitro. Here, we show that trastuzumab, erlotinib, and bevacizumab, individually and in combination, inhibit cell proliferation in a panel of unrelated human breast cancer cell lines, in proportion to their levels of HER2 expression. The combination of all three drugs provided a greater suppression of growth than any single drug or two-drug combination in the high HER2-expressing cell lines (P < 0.001). Combination index analysis suggested that the effects of these drugs in combination were additive. The pretreatment net level of VEGF production in each cell line was correlated with the level of HER2 expression (r = 0.883, P = 0.016). Trastuzumab and erlotinib each reduced total net VEGF production in all cell lines. Multiparameter flow cytometry studies indicated that erlotinib alone and the triple drug combination produced a prolonged but reversible blockade of cells in G1, but did not increase apoptosis substantially. These studies suggest that the effects of two and three-drug combinations of trastuzumab, erlotinib, and bevacizumab might offer potential therapeutic advantages in HER2-overexpressing breast cancers, although these effects are of low magnitude, and are likely to be transient.

Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.

BACKGROUND: Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements. METHODS AND RESULTS: We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided. CONCLUSIONS: Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications.