Background: Trastuzumab, approved by the FDA, has been the standard of care for pts with HER2-positive EG cancer. Resistance to trastuzumab is now emerging in this population. Afatinib, an oral inhibitor of EGFR, HER2, and HER4 has potent single agent activity in HER2-positive EG cancer NCI-N87 and MSKCC patient derived xenografts (Janjigian JNM 2013). We report the initial results of a phase II study of afatinib in patients with trastuzumab refractory EG cancer. Methods: Pts with HER2 positive (IHC 3+ or FISH>2.0) metastatic EG adenocarcinoma, following failure of at least one trastuzumab/chemotherapy regimen received afatinib 40 mg daily. Archival tissue from pre-trastuzumab biopsy, a new biopsy prior to the start of therapy and after 1 wk of afatinib are mandated. The primary endpoint-overall clinical benefit at 4 months: stable disease (SD), complete response (CR), or partial response (PR) by RECIST 1.1. Results: Nine patients treated with afatinib; median follow up 2.7mos (0.5 to 5.3mos). Median age 62, KPS 80, 6 males, 56% IHC3+; 44% IHC1+/2+/FISH>2.0. Number of prior trastuzumab containing regimens: median 2 (1 to 4). The following treatment emergent adverse events were observed: nausea/vomiting (Grade 1/2: 22%), diarrhea (Grade 1/2:67%), fatigue (Grade1/2: 33%), rash (Grade 1/2:44%), anorexia (Grade 1/2:33%,Grade 3:11%), mucositis (Grade1/2: 11%), paronychia/nail loss (Grade 1/2:11%). To date, 7 pts evaluable for response, 3 of 7 patients (43%) derived clinical benefit; 1 pt (14%) with ongoing RECIST 1.1 confirmed PR - a durable 50% regression of biopsy proven metastases in lung and lymph nodes at 5.3 mo. A second patient had 20% tumor regression in biopsy proven liver metastasis, 3.6 mos disease stabilization. A third patient experienced 4.7 mos disease stabilization and regression of biopsy proven skin metastasis. Conclusions: Single agent afatinib shows clinical efficacy in patients with trastuzumab refractory EG cancer. The study has been expanded to accrue additional patients. Efforts to elucidate the mechanisms of trastuzumab resistance in EG cancer are ongoing. Updated molecular and clinical data will be presented. Clinical trial information: NCT01522768.