4336.0) approved by the Animal Care Committee at the University Health Network in accordance with Canadian Council on Animal Care guidelines. MicroPET/CT imaging studies For microPET/CT imaging, groups of 3-4 mice with s.c. tumors. Image analysis of mice treated with trastuzumab showed 2-fold reduced uptake of 64Cu-NOTA-pertuzumab F(ab)2 in BT-474 tumors after 1 week of trastuzumab normalized to baseline, and 1.9-fold increased uptake in SK-OV-3 tumors after 3 weeks of trastuzumab, consistent with tumor response and resistance, respectively. We conclude that PET/CT imaging with 64Cu-NOTA-pertuzumab F(ab)2 detected changes in HER2 expression in response to trastuzumab while delivering a lower total body radiation dose compared to 111In-labeled pertuzumab. KEYWORDS: Copper-64, HER2, molecular imaging, pertuzumab, PET, radiation dosimetry, trastuzumab Introduction The human epidermal growth factor receptor-2 (HER2) is overexpressed in 15C20% of breast cancers (BC) and confers a poor prognosis.1-3 Treatment with trastuzumab (Herceptin, Roche), a humanized IgG1 anti-HER2 monoclonal antibody Pungiolide A (mAb) combined with chemotherapy improves patient outcome in the adjuvant and metastatic settings in patients who have BC that is defined as HER2-positive either by immunohistochemistry (IHC) or hybridization (ISH) analyses.4-6 Guidelines have been established to define tumor HER2 positivity using these techniques.7 Despite the establishment of Pungiolide A trastuzumab as the standard-of-care for treatment of HER2-positive BC, clinical trials revealed that only 1 1 in 2 patients with HER2-positive tumors responded to trastuzumab combined with chemotherapy4 and most responding patients acquire resistance within a year.8 It has also been proposed that some patients with BC classified as HER2-negative may also receive benefit from trastuzumab.9 Molecular imaging which includes single photon emission computed tomography (SPECT) and positron emission tomography (PET) provides a sensitive tool to non-invasively assess tumor phenotype at any location in the body and monitor response to targeted cancer therapies.10 One proposed mechanism of action of trastuzumab involves the induction of HER2 internalization, which reduces the Eledoisin Acetate density of HER2 on tumor cells available for receptor dimerization and oncogenic signaling.11 Probing changes in HER2 expression in tumors could be a promising strategy to discriminate responders from non-responders to trastuzumab treatment. Pertuzumab is a humanized IgG1 mAb that binds domain II on HER2 and hinders receptor dimerization.12 Because the HER2 binding website of pertuzumab is distinct from that of trastuzumab (website IV) and pertuzumab has a different mechanism of action than trastuzumab,13 these mAbs have been administered in combination to improve patient end result.14,15 We previously reported that microSPECT/CT imaging with 111In-labeled Pungiolide A pertuzumab sensitively recognized changes in HER2 expression in MDA-MB-361 human BC xenografts in athymic mice following treatment with trastuzumab, based on our finding that the binding of the imaging probe to HER2 is not affected by trastuzumab binding.16 Decreased HER2 expression was recognized by imaging as early as 3?days after commencing trastuzumab treatment, and images at 21?days demonstrated significantly lower tumor uptake of 111In-labeled pertuzumab was associated with almost complete tumor eradication. Our group offers launched a Phase 1/2 medical trial (PETRA trial; ClinicalTrials.gov identifier NCT01805908) investigating SPECT/CT imaging with 111In-labeled pertuzumab to detect changes in tumor HER2 manifestation in individuals with metastatic BC treated with trastuzumab and chemotherapy. The medical formulation and translational preclinical studies that were required to advance this imaging agent to medical trial are reported elsewhere.17,18 In the study reported here, our goal was to develop an analogous positron-emitting imaging probe based on pertuzumab to detect trastuzumab-mediated HER2 internalization that would extend these promising findings to PET, and potentially Pungiolide A reduce the radiation dose associated with the 3 administrations of 111In-labeled pertuzumab required in the PETRA clinical trial protocol. The predicted combined total body radiation dose for these 3 imaging studies performed at baseline, 1 week and 4 weeks after commencing treatment with trastuzumab and chemotherapy was 17 mSv, based on an given amount of 111 MBq of 111In-labeled pertuzumab for each study (0.05 mSv/MBq).18 PET is 100-fold more sensitive than SPECT and yields high resolution images that are more accurately quantitated.19 64Cu is an attractive positron-emitter for labeling pertuzumab because it is produced in a biomedical cyclotron,20 emits a moderate energy positron [0.7 MeV (19%)] that provides good intrinsic spatial resolution (0.7?mm), and is strongly complexed by macrocyclic chelators such as 1,4,7-triazacyclononane-1,4,7-triacetate (NOTA) that are easily conjugated to antibodies.21 Due to the short half-life of.