8083 Background: Patients with EGFR-mutant NSCLC often respond initially to EGFR kinase inhibitor therapy but ultimately relapse. The spectrum of clinical resistance mechanisms and mechanistically appropriate therapeutic strategies to enhance EGFR inhibitor treatment responses remains incompletely defined. Methods: We used whole exome and transcriptome deep sequencing analysis of tumors obtained from 16 EGFR-mutant NSCLC patients both prior to EGFR inhibitor therapy and at clinical resistance to identify the spectrum and biological basis of molecular alterations that could drive therapy resistance and be targeted to enhance clinical responses. Results: Tumors with acquired EGFR TKI resistance harbored individual and concurrent established resistance-conferring alterations, including EGFRT790M and upregulation of several receptor kinases, and other molecular alterations not previously associated with this resistance, including activating KRAS mutations. Pathway analysis of the transcriptome data revealed upregulation of ERBB2 and FGFR signaling broadly in the therapy-resistant tumors and that the EGFRT790M positive resistant tumors exhibited therapy-induced upregulation of biological pathways underlying cell cycle progression and genome maintenance. The evolution of EGFRT790M-positive resistance was also associated with increased genetic divergence and genomic instability. Genomic amplification and increased levels of the NF-kB inhibitor NFKBIA correlated with improved EGFR TKI response. Conclusions: This genome-scale characterization of the molecular landscape of EGFR inhibitor resistance uncovered biological programs and molecular events underlying the evolution of resistance, establishes the utility of whole exome and transcriptome deep sequencing in repeat biopsy specimens from NSCLC patients, and provides rationale for serial, comprehensive tumor molecular profiling in individual cancer patients to enhance therapeutic precision and response. Our findings also uncovered new molecular biomarkers of therapy response and provide new insight into the biological basis and complexity underlying the evolution of resistance.