3021 Background: In non-small cell lung cancer (NSCLC), sensitivity to tyrosine kinase inhibitors (TKIs) is associated with activating mutation and genomic gains of the epidermal growth factor receptor (EGFR). HER2 and HER3 overexpression increases sensitivity to TKI in pre-clinical settings. Mutations in the HER2 gene have recently been described, but their role in TKI sensitivity is unclear. Methods: One-hundred one NSCLC patients treated with gefitinib (250mg), and previously evaluated for EGFR status by fluorescence in situ hybridization (FISH) and DNA sequencing, were investigated for HER2 and HER3 genomic gains by FISH, and in frame insertions and duplications in HER2 exon 20 by DNA sequencing. Patients with high polysomy and gene amplification were considered as FISH positive (+). Results: FISH HER2+ and HER3+ patterns were significantly associated with female gender (p=0.03 and 0.02) and never smoking history (p=0.04 and 0.02). HER2+ patients (23%) had significantly better objective response (OR: 35% versus 6%, p=0.001), disease control rate (DCR: 56% versus 33%, p=0.04), time to progression (TTP: 9.05 versus 2.7 months, p=0.02), and a non significant trend toward longer survival (OS: 20.8 versus 8.4 months, p=0.056) than HER2-. EGFR+/HER2+ patients had a significantly better OR, DCR, TTP, and OS than patients EGFR-and/or HER2-. Patients EGFR+/HER2+ had better OR than EGFR+/HER2- (54% versus 21%, p=0.07), while EGFR-/HER2+ had same poor outcome than patients negative for both receptors. Patients HER3+ (26%) had a significant longer TTP (3.7 versus 2.7, p=0.04) than HER3-, but no better response or survival. Patients EGFR+/HER3+ had higher OR (36.4% versus 9.9%, p=0.03) and TTP (7.7 versus 2.7 months, p=0.03) than EGFR- and/or HER3-, while patients EGFR-/HER3- had lower response rate (18.2% versus 81.8%, p=0.02) and TTP (2.6 versus 6.0 months, p=0.002) than EGFR+ and/or HER3+. Mutation was not detected in exon 20 of the HER2 gene in 74 sequenced patients. Conclusions: HER2 and HER3 FISH analyses add to EGFR tests for prediction of clinical benefit to TKI therapy in advanced NSCLC patients. HER2 mutations seem to be infrequent and not clinically relevant. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca, Bristol-Myers Squibb, Eli Lilly, Genentech, ImClone, Ligand, Roche AstraZeneca, Bristol-Myers Squibb, Genentech, ImClone, OSI, Roche