Abstract Background: Trastuzumab (T) combined with chemotherapy has been the standard of care for pts with HER2+ EG cancer. Resistance to T is now emerging in this population. Afatinib (A), a potent ErbB Family Blocker, induced nearly complete tumor regression in MSKCC HER2+ patient derived xenografts (PDX). We report the initial results of a phase II study of afatinib in patients with T refractory EG cancer. Methods: Pts with HER2+ (IHC 3+ or FISH>2.0) EG cancer -progressive on trastuzumab -received A 40 mg. Archival pre-T tissue, tumor biopsy after progression on T and after 1 week on A mandated on protocol. The primary endpoint-overall disease control: stable disease (SD) or partial response (PR). Results: 14 pts treated with A; median duration 5.1 mos (1.7 to 12.1 mos), median 2 (1 to 4) prior T regimens, 64% of tumors IHC3+; 36% IHC2+/FISH>2.2. Adverse events included: diarrhea (Grade 1/2:69%), fatigue (Grade1/2: 54%), rash (Grade 1/2:54%), mucositis (Grade1: 23%), paronychia (Grade 1/2:15%). 4 of 14 pts (28%) with disease control (PR or SD); including 1 pt with confirmed PR - a durable 75% regression of biopsy proven metastases. Median OS 6.6 mos (1.9 to NR). PDXs established from biopsies of T refractory tumors of 5 pts. EGFR amplification was detected in the tumor of the patient with PR on afatinib, suggesting EGFR amplification as a predictor of response to afatinib. High level of HER2 amplification was retained in post-trastuzumab resistance sample suggesting ongoing dependence on HER2. PIK3CA mutations were observed in tumors of 33% (2 of 6) patients, ERBB3 mutations in 33% cases (2 of 6), and MTOR or FBXW7 mutations in 50% (3 of 6) patients. Conclusions: Afatinib shows clinical efficacy in patients with T refractory EG cancer. The study met the criteria for expansion of the accrual based on original Simon's minimax two-stage design. Efforts to elucidate the mechanisms of T resistance including validation of potential drivers of T resistance using HER2+ PDXs are ongoing. Updated molecular and clinical data will be presented. Citation Format: Yelena Y. Janjigian, Tooba Imtiaz, David H. Ilson, David B, Solit, Michael F. Berger, Efsevia Vakiani. A phase II study of afatinib (A) in patients (pts) with metastatic human epidermal growth factor receptor (HER2)-positive trastuzumab (T) refractory esophagogastric (EG) cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT228. doi:10.1158/1538-7445.AM2014-CT228