Background: Hyperactivation of the PI3K-AKT-mTOR pathway is a postulated mechanism of resistance to anti-HER2 therapies. In laboratory models, inhibition of this pathway induces activation of upstream receptor tyrosine kinases such as HER3. We hypothesize that dual inhibition of the PI3K pathway and induced HER3 may be highly effective in patients (pts) with HER2-amplifed (HER2+) breast cancers (BC). This phase I trial aims to determine the tolerability and efficacy of the mTOR inhibitor temsirolimus (T) plus the HER1/2 inhibitor neratinib (N) in pts with trastuzumab-refractory, HER2+ BC. We further sought to ascertain if mutational activation of the PI3K pathway is present in trastuzumab-refractory tumors and affects the response to the T-N combination. Methods: This 3+3 dose-escalation study investigated T (flat dose IV weekly) plus N (240 mg oral daily) in pts with HER2+ metastatic BC. Cycle length was 4 weeks. End points included definition of maximum tolerated dose (MTD) and response rate (RR) per RECIST. Response was evaluated radiographically every 8 weeks, toxicity assessed every 2 weeks. All pts underwent biopsy of metastatic disease for biomarker assessment. Activating mutations in PIK3CA were assayed using the Sequenom MassARRAY system. Expression of PTEN was assessed by immunohistochemistry utilizing a published scoring system. Results: Eight pts enrolled and received a median of 4 (1-8) cycles of therapy. All pts had received trastuzumab and a median of 5 (2-12) prior lines of therapy. Frequent treatment-related grade 2 events were: hyperglycemia (4/8), elevated CPK (3/8), diarrhea (2/8), and rash (2/8). Grade 3 diarrhea was the dose-limiting toxicity; other grade 3 toxicities and hematologic toxicities were not observed. The MTD of temsirolimus with neratinib is 8 mg IV weekly. Six pts treated at MTD were evaluable for response; 4 pts had PR, 1 had SD for a RR of 67%. Molecular analyses revealed PI3K pathway activation in 4/6 tumors analyzed. Conclusions: Temsirolimus plus neratinib has clinical activity and was well tolerated in pts with trastuzumab-refractory HER2+ BC. Additional efficacy and safety data, and final tumor analyses will be presented.