20025 Background: Within the last years, protein microarrays have been developed to quantify a large number of parameters present in a given sample simultaneously. Such miniaturised and parallelised sandwich immunoassays are of general interest for all proteomic and diagnostic approaches in which several parameters have to be determined from small samples. We describe the development of a bead-based flow cytometry that represents a convenient approach for rapid multiplex detection of functional target molecules from breast cancer biopsies. Methods: Briefly, sonographically guided core needle biopsies (CNB) were performed from 120 breast cancer masses from different patients. All biopsies were carried out with an automated core biopsy device fitted with 14-gauge (22 mm excursion) needles. Tumor samples were frozen on at −70 Celsius and pulverized. Proteins were extracted, allowing the simultaneous quantification of more than hundred proteins by a bead-based multiplex sandwich immunoassay. Results: The total amount of extracted protein from tumor tissues (mean weight of 20 mg) ranged from 300 to 1000 μg. We demonstrate appropriate sensitivity, reproducibility, and robustness for this protein microarray technology to characterize clinical samples and generate reliable data sets. A set of multiplexed sandwich immunoassays based on Luminex beads were developed that identify marker proteins indicative of prognosis or response to therapeutic options. Up to almost hundred parameters were identified and quantified simultaneously of which estrogen, progesterone and HER2 status in the multiplex assay was highly significant correlated to results obtained by immunohistochemistry (IHC) (p < 0.01). Conclusions: Our results demonstrate that this new bead-based protein microarray technology rapidly and reliably characterizes tissue samples to generate data sets of molecular marker. Therefore, this technology may once replace traditional IHC in determining marker proteins indicative of prognosis or response to therapeutic options. No significant financial relationships to disclose.
Discovery of 830 candidate therapeutic targets and diagnostic markers for breast cancer using oligonucleotide microarray technology