AZD9291 inhibits EGFR phosphorylation and downstream signallng in murine models of EGFR T790M resistant lung cancer. A, Subcutaneous H1975 (L858R/T790M) xenografts, treated with a single 5 mg/kg dose of AZD9291 for the indicated times, were examined for phospho-EGFR, -ERK, -S6, and -PRAS40 status by immunohistochemistry. Representative images were taken from scans at 20× magnification and then size adjusted to fill the screen. B, Lungs from representative transgenic mice treated with control, AZD9291, or afatinib were harvested 6 hours after dose administration. Formalin-fixed paraffin-embedded sections were stained with the indicated antibodies. Representative images were taken from Aperio scans at 100× magnification. C. Lungs were harvested from either untreated tumor-bearing transgenic mice (as confirmed by MRI) (control) or from tumor-bearing mice 8 hours after a single treatment with AZD9291 5 mg/kg. Corresponding lysates from individual animals were immunoblotted with the indicated antibodies. Anti-SP-C (surfactant protein C) antibody was used as a surrogate marker for tumor burden, as tumors express the protein.