Background: Human epidermal growth factor receptor 2 (HER2, ERBB2) aberrations have been identified as oncogenic drivers and potential therapeutic targets in lung cancers. The molecular associations of HER2 protein overexpression, HER2 gene amplification and HER2mutation in lung cancers have not been distinctly defined. To explore these associations, Memorial Sloan Kettering and the University of Colorado combined their data on HER2 in lung cancers. Methods: Tumor specimens from 175 patients with lung adenocarcinomas with no prior targeted therapy were evaluated for the presence of HER2 overexpression, HER2 amplification and mutation. Overexpression was assessed by immunohistochemistry (IHC) using the 4B5 Ventana antibody. Amplification was assessed by fluorescence in-situ hybridization (FISH) using FDA approved probe sets (PathVysion, Abbott and HER2 IQFISH pharmDx, Dako) and defined as HER2/CEP17 ratio ≥ 2.0. Mutation was assessed by fragment analysis and mass spectrometry genotyping for indels and recurrent point mutations in exon 20, respectively. The frequencies of HER2 overexpression, HER2 amplification and mutation were calculated and their concordance examined. Results: HER2 amplification by FISH was detected in 5 of 175 (3%) cases, and 46 (26%) showed polysomy (HER2 copy ≥ 4 but HER2/CEP17 ratio < 2). HER2 overexpression (2+, 3+) on IHC was not detected in the 25 specimens tested to date and negative IHC correlated with negative results on FISH. HER2 mutation was detected in 4 of 145 (3%) specimens, including 3 identical 12bp insertions [(p.A775_G776insYVMA (c.2324_2325ins12)] and a 9bp insertion, all in exon 20. None of the HER2 mutant cases were amplified and 3 had polysomy. Conclusions: HER2 mutations are not associated with HER2 amplification or HER2 protein overexpression suggesting a distinct entity and therapeutic target. “HER2-positive lung cancers” may not be an adequate term and patient cohorts for the study of HER2 targeted agents should be defined by the specific HER2 aberrations present. Funded in part by Boehringer-Ingelheim, NCI 1 RC2 CA148394-01, NCI P50CA058187, and NCI CCSG P30CA046934.