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The aim of this study was to determine if imaging with 89Zr-trastuzumab, a human epidermal growth factor receptor 2 (HER2)–targeting PET tracer, can detect HER2-positive metastases in patients with HER2-negative primary breast cancer.As part of an institutional review board–approved, prospective clinical trial of 89Zr-trastuzumab PET/CT (ClinicalTrials.gov identifier NCT02286843), a second group of 11 patients with HER2-negative primary breast cancer and known metastatic disease were recruited. Patients with confirmed HER2-negative primary breast cancer underwent 89Zr-trastuzumab PET/CT to screen for 89Zr-trastuzumab–avid lesions suggestive of unsuspected HER2-positive metastases. 89Zr-trastuzumab–avid lesions on PET/CT were biopsied and pathologically examined to determine HER2 status.All 11 patients had confirmed HER2-negative primary breast cancer. Four patients demonstrated suspicious foci on 89Zr-trastuzumab PET/CT. Of these 4 patients, 1 patient had biopsy-proven HER2-positive metastases. The other 3 patients with suspicious 89Zr-trastuzumab–avid foci had biopsy demonstrating a metastasis that was HER2-negative and were considered false-positive 89Zr-trastuzumab PET foci. Combined with a published report of the first 9 patients, there have been a total of 20 HER2-negative primary breast cancer patients, with 3 patients (15%) having pathologically confirmed HER2-positive distant metastases and 6 (30%) with suspicious 89Zr-trastuzumab–avid foci that were HER2-negative on pathology, which were thus considered false-positive 89Zr-trastuzumab findings.This second group of patients confirms the proof of concept that 89Zr-trastuzumab PET/CT detects unsuspected HER2-positive metastases in a subset of patients with HER2-negtive primary breast cancer. False-positive 89Zr-trastuzumab–avid foci present a challenge to using this tracer.