Micro-SPECT/CT with ¹¹¹In-DTPA-Pertuzumab Sensitively Detects Trastuzumab-Mediated HER2 Downregulation and Tumor Response in Athymic Mice Bearing MDA-MB-361 Human Breast Cancer Xenografts

Abstract

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...

18F-FDG Small-Animal PET/CT Differentiates Trastuzumab-Responsive from Unresponsive Human Breast Cancer Xenografts in Athymic Mice

“…Furthermore, other molecular imaging agents may also be useful for monitoring response to trastuzumab. In a previous study, we have shown that HER2 downregulation-one of the proposed mechanisms of action of trastuzumab-could be sensitively detected by micro-SPECT/CT using 111 In-DTPA-pertuzumab after only 3 d of trastuzumab therapy in MDA-MB-361 xenografts (22). Furthermore, changes in tumor uptake of 111 In-DTPA-pertuzumab were informative on the decreased viability of tumor cells after 3 wk of trastuzumab therapy.…”

“…In this study, the relative HER2 expression level of MDA-MB-231 and MDA-MB-361 cells was verified by flow cytometry using fluorescein isothiocyanate-conjugated anti-HER2/neu IgG 1 (Clone Neu 24.7; BD Biosciences) as previously reported (22). Negative controls consisting of cells without immunofluorescence staining or cells incubated with the corresponding IgG isotype controls were included.…”

Imaging of Human Epidermal Growth Factor Receptor Type 2 Expression with 18F-Labeled Affibody Molecule ZHER2:2395 in a Mouse Model for Ovarian Cancer

“…For example, radiolabeled trastuzumab and pertuzumab have been used for SPECT and PET of HER2 expression (13,14,16,31). However, these large proteins are characterized by slow tumor penetration and clearance from the circulation.…”

“…Several approaches have been used for PET and SPECT of HER2 expression, including radiolabeled antibodies trastuzumab and pertuzumab (HER2 dimerization inhibitor) and radiolabeled trastuzumab fragments (13)(14)(15)(16). These radiotracers were able to visualize HER2 expression, but their large size resulted in slow tumor accumulation and slow clearance from the circulation.…”

Imaging Diagnostic and Therapeutic Targets: Human Epidermal Growth Factor Receptor 2

“…An anti-HER2 mAb introduced later-pertuzumab-was also radiolabeled with 111 In or 89 Zr and was used to successfully image HER2-positive tumor xenografts (15,16).…”