Background: Despite initial responses to reversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, (erlotinib or gefitinib), all non-small cell lung cancer (NSCLC) patients with EGFR-sensitizing mutations experience disease progression. This “acquired resistance” (AR) is associated with a second site exon 20 EGFR T790M mutation (M) in >50% of cases. No therapy, including the anti-EGFR antibody cetuximab plus erlotinib, has proven effective in treating AR (Janjigian YY. Clin Cancer Res; Epub Jan 2011). Preclinical data suggest afatinib, a potent ErbB family blocker, is active in T790M M cell lines such as H1975. Combined EGFR targeting with afatinib and cetuximab has induced near complete responses in T790M transgenic murine models. This study assesses safety and preliminary efficacy of this combination in NSCLC. Methods: NSCLC patients with clinically defined AR (Jackman D. J Clin Oncol 2010;28:357) received oral afatinib 40 mg daily with escalating dose cohorts of biweekly cetuximab at 250 and 500 mg/m². Patients receiving the recommended Phase II dose (RP2D) were evaluated for objective response. Acquisition of tumor tissue after emergence of AR was mandated. Results: Of 26 treated patients, 22 received the predefined maximum dose (RP2D: afatinib 40 mg + cetuximab 500 mg/m²). Median time on erlotinib or gefitinib at study entry was 2.4 years. No dose-limiting toxicity was observed. Common adverse events were grade 1/2 rash (35/46%) and diarrhea (50/19%), respectively, and three patients (11.5%) had grade 3 rash. Disease control was observed in all patients enrolled at RP2D (tumor size reduction up to 76%; treatment duration up to 5+ months so far). Confirmed partial responses (PRs) were seen in 8/22 evaluable patients (36%, 95% confidence interval [CI]: 0.17–0.59), including 4/13 (29%) confirmed PRs in T790M+NSCLC. Enrollment has now begun in an 80-patient expansion cohort. Conclusions: Combined EGFR targeting with afatinib and cetuximab is tolerable at RP2D. Owing to encouraging clinical activity in AR to prior erlotinib or gefitinib, the study has been expanded. Elucidating mechanisms underlying regression in T790M+ and T790M– patients is critical.