Pertuzumab is a HER2 dimerization inhibitor that binds to an epitope unique from that of trastuzumab. Our objective was to determine whether SPECT with 111 In-diethylenetriaminepentaacetic acid-pertuzumab ( 111 In-DTPA-pertuzumab) could sensitively detect an early molecular response to trastuzumab manifested by HER2 downregulation and a later tumor response revealed by a decreased number of HER2-positive viable tumor cells. Methods: Changes in HER2 density in SKBr-3 and MDA-MB-361 BC cells exposed to trastuzumab (14 mg/mL) in vitro were measured by saturation binding assays using 111 In-DTPA-pertuzumab and by confocal immunofluorescence microscopy and flow cytometry with fluorescein isothiocyanate-labeled HER2/neu antibodies. Imaging of HER2 downregulation was studied in vivo in athymic mice with subcutaneous MDA-MB-361 tumors treated for 3 d with trastuzumab (4 mg/kg) or nonspecific human IgG (hIgG) or phosphate-buffered saline (PBS). Imaging of tumor response to trastuzumab was studied in mice bearing subcutaneous MDA-MB-361 xenografts treated with trastuzumab (4 mg/kg), followed by weekly doses of nonspecific hIgG or rituximab or PBS (2 mg/ kg). Mice were imaged on a micro-SPECT/CT system at 72 h after injection of 111 In-DTPA-pertuzumab. Tumor and normal-tissue biodistribution was determined. Results: 111 In-DTPA-pertuzumab saturation binding to SKBr-3 and MDA-MB-361 cells was significantly decreased at 72 h after exposure in vitro to trastuzumab (14 mg/mL), compared with untreated controls (62% 6 2%, P , 0.0001; 32% 6 9%, P , 0.0002, respectively). After 3 d of trastuzumab, in vivo tumor uptake of 111 In-DTPA-pertuzumab decreased 2-fold in trastuzumab-versus PBS-treated mice (13.5 6 2.6 percentage injected dose per gram [%ID/g] vs. 28.5 6 9.1 %ID/g, respectively; P , 0.05). There was also a 2-fold decreased tumor uptake in trastuzumab-versus PBS-treated mice by image volume-of-interest analysis (P 5 0.05), suggesting trastuzumab-mediated HER2 downregulation. After 3 wk of trastuzumab, tumor uptake of 111 In-DTPA-pertuzumab decreased 4.5-fold, compared with PBS-treated mice (7.6 6 0.4 vs. 34.6 6 9.9 %ID/g, respectively; P , 0.001); this decrease was associated with an almost-completed eradication of HER2-positive tumor cells determined immunohistochemically. Mol ecular imaging is a powerful new tool that has great potential for aiding in the optimal use of novel targeted cancer therapies by revealing the expression of target receptors in situ on lesions throughout the body; probing downstream treatment-induced molecular events, thus providing early mechanistic evidence of tumor response; and monitoring the prior existence or emergence of resistance pathways implicated in treatment failure (1). Trastuzumab (Herceptin; Roche Pharmaceuticals) is a humanized IgG 1 monoclonal antibody (mAb) approved for the treatment of early and advanced breast cancer (BC) which overexpresses the HER2 transmembrane tyrosine kinase (2,3). HER2 positivity is evaluated in a primary BC biopsy by immunohistochemical staining for HER...