Abstract Overexpression of HER2, a transmembrane tyrosine kinase receptor, is correlated with poor prognosis in patients with breast cancer. HER2 expression may vary between primary tumors and metastatic lesions and also change during the treatment. Therefore, there is a need for methods that can assess global HER2 expression non-invasively and reproducibly. PET imaging is well suited to this task based on its high sensitivity. Fluorine-18 (18F) is the most commonly used PET emitter for in vivo imaging since it is relatively short lived radionuclide (109 min). However, its production requires a cyclotron. Gallium-68 (68Ga) is an attractive alternative to 18F. It has a similarly short half-life (68 min) but can be obtained from a generator. In this work, we sought to monitor HER2 expression in vivo with 68Ga-labeled DOTA-ZHER2:2891 Affibody in a panel of breast cancer xenografts with different levels of HER2 expression. ZHER2:2891 Affibody molecules were conjugated with the chelator, DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid), and labeled with 68Ga. The in vitro binding characteristic of the tracer was evaluated using saturation cell-binding assay. In vivo studies were conducted in athymic nude mice bearing subcutaneous human breast cancer tumors with four different levels of HER2 expression. PET/CT scans were performed 1, 2, 3 hour post tracer injection. After imaging, the animals were sacrificed and selected organs were dissected to characterize the relationship between the signal detected by PET, ex vivo assessment of the tracer uptake, and the HER2 expression (in tumors). Nonspecific uptake was analyzed 1 hour post His6-ZTaq-GS-Cys-DOTA-68Ga (non-HER2-specific Affibody molecules) injection. In vitro assays showed high binding affinity of 68Ga-DOTA-ZHER2:2891 Affibody probe to MDA-MB-361 cells (Kd = 1.4±0.19 nM). In vivo biodistribution and PET imaging studies demonstrated high radioactivity uptake in the HER2 overexpressing tumors. Tracer was eliminated quickly from the blood and normal tissues, providing high tumor-to-blood ratios. The highest normal tissue concentration of radioactivity was seen in the kidneys. High-contrast PET images were recorded in BT474 and MDA-MB361 tumors 1 h post tracer injection. Low, but still detectable, uptake was observed for MCF7 tumors. Very good correlation was observed between PET and ex vivo estimates of tracer concentration and the receptor expression in the tumor tissue, as assessed by, respectively, radioactivity accumulation and enzyme-linked immunosorbent assay (ELISA). Stability tests of 68Ga-DOTA- ZHER2:2891 Affibody in vivo revealed that the tracer is stable in blood. These results suggest that PET imaging using 68Ga-DOTA-ZHER2:2891 Affibody is sensitive enough to detect different levels of HER2 expression in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5284. doi:10.1158/1538-7445.AM2011-5284