Discussion The epidermal growth factor receptor family is of tremen dous biological and clinical importance for many solid epithelial tumors

The supportive data for this hypothesis includes the in vitro observations that these are genes induced when an EGFR Discussion The epidermal growth factor receptor family is of tremen dous biological and clinical importance for many solid epithelial tumors dependent cell line is freed from growth inhibition via EGFR inhibitors and the in vivo associations between the high expression of these signa tures and genes including HRAS, KRAS and EGFR itself. Regardless of the classical markers of activation of the Discussion The epidermal growth factor receptor family is of tremen dous biological and clinical importance for many solid epithelial tumors EGFR RAS MEK pathway, the strong associations between these expression profiles and patient outcomes in two dif ferent data sets suggest that they are important profiles. Currently, we have chosen only to validate our profiles using additional microarray Discussion The epidermal growth factor receptor family is of tremen dous biological and clinical importance for many solid epithelial tumors data sets, as opposed to using western blots or quantitative PCR of the training set, since each of these signatures represents a large number of genesproteins. U0126 was purchased from Cell Signaling. The inhib itory concentration that caused a 50% reduction in MTT dye conversion dose was determined as previously described. Drug combination interactions were analyzed using methods developed by Chou and Talalay. Using cell lines plated as described above, seven treatment combina tions consisting of constant ratios of IC50 doses were applied to cells and growth compared to untreated controls using the MTT assay. Four treatment schedules were tested 72 h concurrent, 72 h inhibitor followed by 72 h chemotherapeutic, 72 h chemotherapeutic followed by 72 h inhibitor, and a 144 h concurrent dose with a media change at 72 h. CalcuSyn was used to determine the combination index, which is a measure ment of the type of drug interactions. A combination index of one indicates an additive response, less than one indicates a synergistic response, and greater than one indicates an antagonistic response. Collection of mRNA for cell line experiments For each treatment, the SUM102 cells were grown in 15 cm dishes until 5060% confluence.

SUM102 cells were treated for 48 h with a dose equivalent to two times the 72h IC50 dose of each inhibitor. To identify EGFR, MEK, and PI3K activation signatures, the medium was removed after 48 h of inhibitor treatment and replaced with fresh medium without inhibitor. mRNA was harvested at 4 h, 8 h, and 24 h. Cells were harvested by scraping, quickly placed into RNA lysis buffer, and mRNA was isolated using the Micro FastTrack kit. Collection of RNA for human tumor samples 248 breast tissue samples represented by 241 fresh frozen breast tumor samples and 7 normal breast tissue samples were obtained from four different sources using IRB approved protocols from each participating institution the University of North Carolina at Chapel Hill, The Uni versity of Utah, Thomas Jefferson University and the Uni versity of Chicago. many of these samples have appeared in previous publications, and 117 are new to this study. The patients were heteroge neously treated in accordance with the standard of care dictated by their disease stage, ER, and HER2 status. Tumor sequence analysis Tumor genomic DNA samples were isolated from 96 tumors using Qiagen DNeasy Kits accord ing to the manufacturers protocol. Gene sequencing anal yses were performed at Polymorphic DNA Technologies using an ABI 3730xl DNA sequencer and cycle sequencing, according to the manufacturers proto col.