Participants
We recruited a total of 7 patients with breast cancer between September 2017 to 2019. The selection criteria for the subjects were: 1) aged 40-80 years, 2) with at least one measurable lesion, 3) with histopathologically diagnosed breast cancer with HER2 expression, 4) an Eastern Cooperative Oncology Group score of 2 or lower.
This study was approved by the 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 individual participants included in the study. 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 as described previously [2]. 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 incubation at 4°C overnight and 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 hour. Radiolabeling efficiency was more than 95%. The reaction mixtures formulated with saline were sterilized by filtration through a 0.22-μm Millex GV filter (Merck Millipore, Billerica, MA, USA).
PET protocol
The 64Cu-NOTA-Trastuzumab PET images were acquired using a GE Discovery 710 PET/ computed tomography (CT) (GE Healthcare, Milwaukee, WI, USA). After intravenous injection of 45mg of Trastuzumab for at least 15 mins, subjects 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). There were no adverse or clinically detectable pharmacologic effects in any of the seven subjects. No significant changes in vital signs or the results of laboratory studies. PET images were obtained at 60 minutes after intravenous injection of 64Cu-NOTA-Trastuzumab. Delayed PET images were obtained between 20 to 25 hours and 46 to 49 hours after injection of 64Cu-NOTA-Trastuzumab. All subjects were scanned from the mid-thigh to the vertex of the skull.
18F-fluorodeoxyglucose (FDG) PET/CT was performed one day before 64Cu-NOTA-Trastuzumab PET. After 6 hours 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 hour after injection, PET images were acquired using a GE Discovery 710 PET/CT (GE Healthcare, Milwaukee, WI, USA).
PET images were reconstructed using a conventional iterative algorithm, 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.
Radiation dosimetry
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) were acquired each organ for calculating residence time. The time activity curve was expressed by three time points at 1, 24, and 48 hours. The residence times were calculated by accumulated radioactivity divided by subject administered activity. S-value of source to target region energy deposited per unit mass was calculated using OLINDA/EXM version 1.1 software with an adult female as the model. The organ absorbed doses were calculated as the self-dose and cross-dose from each organ region.
Biodistribution
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). For normal-organ distribution, blood, liver, kidney, muscle, spleen, bladder, lung, and bone were analyzed. With tumors, the primary tumor, metastatic lymph nodes (LNs), and metastatic bone lesions were also evaluated. One 2–3 cm sized 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 of the liver or blood was used as the background. The SUVmax of the tumors in the breast, metastatic LNs, and metastatic bone lesions were used to assess lesions.
Safety
Safety was assessed before and after the administration of 64Cu-NOTA-Trastuzumab. Feedback such as adverse reactions and other safety-related issues was gathered within 1 month after 64Cu-NOTA-Trastuzumab administration. Adverse events, vital signs, physical examination, and laboratory tests were all considered in the safety evaluation.