Background: Preclinical models suggested synergistic antineoplastic activity of anti-estrogen therapy with HER2 and mTOR inhibitors. Methods: We designed a 3+3 dose escalation phase I study of the aromatase inhibitor letrozole 2.5mg PO daily, mTOR inhibitor everolimus 2.5-10mg PO daily and HER2 antibody trastuzumab 4-8mg loading dose followed by 2-4mg maintenance dose IV on day 1 of 21-day cycle in patients with hormone-receptor positive, HER2-positive/amplified or mutant advanced cancers (confirmed by immunohistochemistry and/or FISH and/or next-generation sequencing). The primary objectives were to determine maximum tolerated dose (MTD), dose limiting toxicities (DLT), overall safety and response. Results: A total of 18 patients (men, 1; women, 17; HER2 amplification, 14; HER2 mutation, 4; breast cancer, 15; ovarian cancer, 1; cervical cancer, 1; gastroesophageal junction cancer, 1), median age 56 years, median of 6 prior therapies (including letrozole [9] or other aromatase inhibitor [8]; everolimus [3]; trastuzumab [14] or other HER2 targeted therapy [1]) were enrolled in the planned 6 dose levels. The MTD has not been reached and grade 3 (G3) mucositis at the dose level 4 was the only DLT. Other G3 or G4 drug-related toxicities included G4 hyperglycemia in 1 patient, G3 hyperglycemia in 3 patients, G3 thrombocytopenia in 1 patient, G3 anemia in 1 patient and G3 headache in 1 patient. Of 18 patients, 3 (17%) had a partial response (all with heavily-pretreated breast cancer with HER2 amplification [2] or HER2^{A775_G776insYVMA} mutation [1]), 11 (61%) stable disease (SD) including 7 (39%) patients with SD > 6 months (all with heavily-pretreated breast cancer), 3 (17%) progressed and 1 had pending evaluation. The median change in size of target lesions per RECIST 1.1. was -11% (-68% to +47%). Median progression-free survival was 9 months (95% CI 5.8-12.2). Conclusions: The combination of letrozole, everolimus and trastuzumab is well tolerated with encouraging activity in heavily-pretreated patients with HER2-amplified or mutant advanced breast cancer. Clinical trial information: NCT02152943