Human epidermal growth factor receptor 2 (HER2) is a member of the HER family of 
proteins containing four receptor tyrosine kinases. It plays an important role 
in the pathogenesis of certain human cancers. In non-small-cell lung cancer 
(NSCLC), HER2 amplification or mutations have been reported. However, little is 
known about the benefit of HER2-targeted therapy for NSCLCs harboring HER2 
alterations. In this study, we investigated the antitumor effect of afatinib, an 
irreversible epidermal growth factor receptor (EGFR)-HER2 dual inhibitor, in 
lung cancers harboring HER2 oncogene alterations, including novel HER2 mutations 
in the transmembrane domain, which we recently identified. Normal bronchial 
epithelial cells, BEAS-2B, ectopically overexpressing wild-type HER2 or mutants 
(A775insYVMA, G776VC, G776LC, P780insGSP, V659E, and G660D) showed constitutive 
autophosphorylation of HER2 and activation of downstream signaling. They were 
sensitive to afatinib, but insensitive to gefitinib. Furthermore, we examined 
the antitumor activity of afatinib and gefitinib in several NSCLC cell lines, 
and investigated the association between their genetic alterations and 
sensitivity to afatinib treatment. In HER2-altered NSCLC cells (H2170, Calu-3, 
and H1781), afatinib downregulated the phosphorylation of HER2 and EGFR as well 
as their downstream signaling, and induced an antiproliferative effect through 
G1 arrest and apoptotic cell death. In contrast, HER2- or EGFR-non-dependent 
NSCLC cells were insensitive to afatinib. In addition, these effects were 
confirmed in vivo by using a xenograft mouse model of HER2-altered lung cancer 
cells. Our results suggest that afatinib is a therapeutic option as a 
HER2-targeted therapy for NSCLC harboring HER2 amplification or mutations.