The supportive data for this hypothesis includes the in vitro observations that these are genes induced when an EGFR Wnt signaling pathway 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 http://www.selleckchem.com/products/dorsomorphin-2hcl.html 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 selleck chemicals 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. 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. A two step boostnested PCR strategy was used where first a PCR reaction is performed to generate a larger DNA fragment, which is then used as a template for the nested reaction with a second set of PCR primers. Double stranded sequencing was performed on the nested prod uct using the nested PCR primers as the sequencing prim ers. Exons 19 and 21 of EGFR were sequenced across all 96 patients, while exons 1 and 2 of KRAS2, 1 and 2 of HRAS, and 11 and 15 of BRAF were sequenced across 54 patients. No somatic alterations were detected. Microarray experiments For the human tumor samples, the total RNA isolation and microarray protocols were performed as described in Hu et al. in this study, a number of tumor samples from previous studies were retested using a new custom Agilent microarray enriched for breast cancer genes. For cell lines experiments, labeled cRNA was generated from the mRNA using Agilents Low RNA Input Linear Amplifi cation Kit as described in Hu et al. For the cell line studies, the 48 h inhibitor treated samples were compared to an untreated cell line reference to look for effects of an inhibitor, and for the post treatment samples, to identify an activation signature for that drugpathway. Labeled experimental sample and reference were mixed and co hybridized overnight on the same Custom 22K Agilent Human Whole Genome Oligonucle otide Microarray described above. Two to four microar rays per experimental cell line condition were performed, including a dye flip replicate for gefitinib and cetuximab treated samples. Microarrays were scanned on an Axon GenePix 4000B microarray scanner and analyzed using GenePix Pro 5. 1 software. Microarray raw data were uploaded into the UNC Microarray Database and Lowess normalization was performed on the Cy3 and Cy5 chan nels.