Background: Targeting a single node in the HER2/PI3K pathway has been associated with benefit but frequently results in acquired resistance in the metastatic setting. Direct inhibition of HER2 even with combinations may be limited by mutational activation of the downstream PI3K pathway. Conversely, inhibition downstream results in feedback upregulation of receptor tyrosine kinase signaling which can diminish efficacy. We hypothesize that dual targeting of HER2 and mTOR will be tolerable while overcoming these limitations. We conducted a phase I/II trial examining the tolerability and efficacy of temsirolimus (T), an mTOR inhibitor, and neratinib (N), a HER1/2 kinase inhibitor, in patients with HER2+ metastatic breast cancer (MBC). Tumor biopsies were obtained to ascertain if PI3K pathway activation is frequent and to identify biomarkers of response. Methods: The phase I study utilized a 3+3 dose escalation design to determine the maximum tolerated dose (MTD) of T (IV weekly) with N (fixed dose 240 mg oral daily) in patients with HER2+ trastuzumab-refractory MBC. Loperamide prophylaxis (4 mg daily) was initiated at Day 1 and then left to patient/physician discretion. The phase II study utilized a Simon two-stage design to assess the overall response rate by RECIST in HER2+ trastuzumab-refractory MBC. An expansion cohort has been subsequently initiated to investigate the benefit of more aggressive loperamide prophylaxis (16 mg daily) and temsirolimus dose escalation. All patients on the initial Ph I/II study underwent biopsy of metastatic disease for biomarker assessment. Activating mutations in the PI3K pathway were assayed using the Sequenom MassARRAY system or as part of Next Generation Sequencing of panel of ∼250 cancer related genes along with PTEN immunohistochemistry. Results: Eight patients enrolled in the phase I trial and the MTD was determined to be T at 8 mg IV weekly with neratinib at 240 mg daily with grade 3 diarrhea as the dose limiting toxicity. The phase II trial enrolled 34 patients. Seventy percent of patients on the Ph I/II studies had progression of disease on prior lapatinib, pertuzumab, or T-DM1. The most frequent treatment-related grade 2/3 events at the MTD (n=40) were diarrhea (gr 2 35%/gr 3 25%), mucositis (23%/10%), and leukopenia (28%/5%). Thirty-five patients treated at the MTD are evaluable for response; 13 patients had PR (9 cPRs) and 2 patients had SD ≥6 months. Most of the 15 responders had prior T-DM1 (6), pertuzumab (2), or lapatinib (8). Molecular analyses of pretreatment biopsies is ongoing and thus far has revealed PI3K pathway activation (PIK3CA or AKT mutation or PTEN low) in >70% of the analyzed tumors (24/33). Responses were frequent in this cohort but not in a group of tumors that had lost HER2 overexpression. Conclusions: Temsirolimus and neratinib has clinical activity in the setting of HER2+ MBC with mutational activation of PI3K pathway and among patients exposed to multiple prior HER2 targeted agents. Final results of the efficacy, safety, and biomarker data will be presented along with preliminary data from the ongoing expansion study. Citation Format: Devika Gajria, Shanu Modi, Cristina Saura, Rita Sakr, Karl Solano, Helen Won, Harpreet Pannu, Sujata Patil, Diana Lake, Tiffany Traina, Tari King, Michael Berger, Jose Baselga, Neal Rosen, Cliff Hudis, Sarat Chandarlapaty. A phase I/II study of neratinib plus temsirolimus in HER2+ metastatic breast cancer reveals ongoing HER2 pathway dependence in many patients despite several lines of HER2 targeted therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-19-04.