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 adenocarcinoma –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 clinical benefit at 4 months: 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 age 62, KPS 80, median 2 (1 to 4) prior T containing regimens, 64% of tumors IHC3+; 36% IHC2+/FISH>2.2. Adverse events included: diarrhea (Grade 1/2:69%), fatigue (Grade 1/2:54%), rash (Grade 1/2:54%), mucositis (Grade 1:23%), paronychia (Grade 1/2:15%). To date, 13 pts evaluable for response, 3 of 13 pts (23%) had disease stabilization (PR or SD); 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. Next generation sequencing of matched pre-T and post-T progression tumors from 6 pts was performed and results will be reported. Conclusions: Afatinib shows clinical efficacy in patients with T refractory EG cancer. The study has been expanded to accrue additional patients. 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. Clinical trial information: NCT01522768.