We recruited a total of seven patients with breast cancer between September 2017 and September 2019 with the following selection criteria: 1) aged 40–80 years 2) at least one measurable lesion 3) a histopathologically diagnosed breast cancer with a HER2 expression 4) an Eastern Cooperative Oncology Group score of 2 or lower.
This study was approved by the Korean Ministry of Food and Drug Safety (MFDS), and the Institutional Review Board of KIRAMS (IRB No.: KIRAMS 2017-09-006-020). All procedures were performed following the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all participants. This preliminary clinical trial is registered with the Clinical Research Information Service (https://cris.nih.go.kr), registration number KCT0002790.
Preparation of 64Cu-NOTA-Trastuzumab
64Cu-NOTA-Trastuzumab was produced from the immunoconjugate NOTA-Trastuzumab, radiolabeled with 64Cu from 50-MeV cyclotron irradiation . Briefly, Trastuzumab (Herceptin®; F. Hoffmann-La Roche, Basel, Switzerland) was dissolved in 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 8.5) at a concentration of 10 mg/ml and mixed with a 20-fold molar excess of p–SCN-Bn-NOTA in 100% ethanol. The immunoconjugate (NOTA-Trastuzumab) was purified after an overnight incubation at 4°C and was concentrated to 5 mg/mL with 0.1 M ammonium acetate buffer (pH 6). For radiolabeling, 370 MBq of 64CuCl2 was added to 5mg of NOTA-Trastuzumab. The reaction mixtures were incubated at room temperature for 1 h, with a radiolabeling efficiency of >95%. The reaction mixtures formulated with saline were sterilized by filtration through a 0.22 μm Millex GV filter (Merck Millipore, Billerica, MA, USA).
The 64Cu-NOTA-Trastuzumab PET images were acquired using a GE Discovery 710 PET/ computed tomography (CT) (GE Healthcare, Milwaukee, WI, USA). After a 45mg Trastuzumab intravenous injection for at least 15 min, participants were intravenously injected with 64Cu-NOTA-Trastuzumab (296 MBq). The mean administered activity was 278.4 ± 13.0 MBq (range: 259.0–297.0 MBq). No adverse or clinically detectable pharmacologic effects were found in any of the seven participants; moreover, no significant changes either in vital signs or the results of laboratory studies were reported. PET images were obtained at 60 min after intravenous injection of 64Cu-NOTA-Trastuzumab. Delayed PET images were obtained between 20 and 25 h, and 46 and 49 h after 64Cu-NOTA-Trastuzumab injection. All participants were scanned from the mid-thigh to the vertex of the skull.
18F-Fluorodeoxyglucose (FDG) PET/CT was performed 1 day before the 64Cu-NOTA-Trastuzumab PET. After 6 h of fasting, 370 MBq of 18F-FDG was intravenously injected. The blood glucose level before 18F-FDG injection did not exceed 7.2 mmol/L. One h after injection, PET images were acquired using GE Discovery 710 PET/CT (GE Healthcare, Milwaukee, WI, USA).
PET images were reconstructed using a conventional iterative algorithm and ordered-subsets expectation-maximization with parameters of four iterations and eight subsets. For attenuation correction, CT scans were obtained (130 kVp, 30 mA, 0.6 s/CT rotation, and 6 pitch), after voiding the bladder.
The internal dosimetry of 64Cu-NOTA-Trastuzumab was evaluated using accumulated radioactivity in PET images. The organ time-activity curve of radioactivity in the target region (ID) divided by target mass (g) was acquired from each organ to calculate residence time. The time-activity curve was expressed by three time points at 1, 24, and 48 hr. The residence times were calculated by accumulated radioactivity divided by subject administered activity. The S-value of the source-to-target region energy, deposited per unit mass, was calculated using OLINDA/EXM version 1.1 software, using an adult female as the model. The organ-absorbed doses were calculated as the self-dose and cross-dose from each organ region.
The biodistribution of 64Cu-NOTA-Trastuzumab was evaluated using the maximum standardized uptake value (SUVmax) and the mean standardized uptake value (SUVmean) from the three sequential PET images using GE AW software (GE Healthcare, Milwaukee, WI, USA). Regarding normal-organ distribution, the blood, liver, kidney, muscle, spleen, bladder, lung, and bone were analyzed. Regarding tumors, the primary tumor, metastatic lymph nodes (LNs), and metastatic bone lesions were also evaluated. A 2–3 cm ellipsoidal volume of interest was drawn inside the organ on the PET images to calculate the SUV.
The lesion-to-background ratios were calculated to test the degree of 64Cu-NOTA-Trastuzumab uptake at the lesion sites. The SUVmean either of the liver or blood was used as the background. The SUVmax of the breast tumors, metastatic LNs, and metastatic bone lesions were used for lesion assessment.
Safety was assessed both before and after 64Cu-NOTA-Trastuzumab administration, acquiring feedback—including adverse reactions and other safety-related issues—1 month later. Adverse events, vital signs, physical examination data, and laboratory test results were all considered in the safety evaluation.