A preliminary clinical trial to evaluate ⁶⁴Cu-NOTA-Trastuzumab as a positron emission tomography imaging agent in patients with breast cancer

Abstract

Background

The purpose of this study was to evaluate both the biodistribution and safety of ⁶⁴Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-Trastuzumab, a novel ⁶⁴Cu-labeled positron emission tomography (PET) tracer for human epidermal growth factor receptor 2 (HER2) in patients with breast cancer.

Methods

PET images at 1, 24, and 48 h after 296 MBq of ⁶⁴Cu-NOTA-Trastuzumab injection were obtained from seven patients with breast cancer. Both the primary tumors’ and metastatic lesions’ maximum standardized uptake value (SUV_max) was evaluated. The mean SUV_max (SUV_mean) was evaluated in the other organs, including the blood pool, liver, kidney, muscle, spleen, bladder, and the lungs, as well as the bones. Moreover, the internal radiation dosimetry was calculated using the OLINDA/EXM software. Safety was assessed based on feedback regarding adverse reactions and safety-related issues within 1 month after ⁶⁴Cu-NOTA-Trastuzumab administration.

Results

⁶⁴Cu-NOTA-Trastuzumab PET images showed that the overall SUV_mean values in each organ negatively correlated with time. The liver’s average SUV_mean values were measured at 5.3 ± 0.7, 4.8 ± 0.6, and 4.4 ± 0.5 on 1 h, 24 h, and 48 h after injection, respectively. The average SUV_mean blood values were measured at 13.1 ± 0.9, 9.1 ± 1.2, and 7.1 ± 1.9 on 1 h, 24 h, and 48 h after injection, respectively. The SUV_max of HER2-positive tumors was relatively higher than HER2-negative tumors (8.6 ± 5.1 and 5.2 ± 2.8 on 48 h after injection, respectively). Tumor-to-background ratios were higher in the HER2-positive tumors than in the HER2-negative tumors. No adverse events related to ⁶⁴Cu-NOTA-Trastuzumab were reported. The calculated effective dose with a 296 MBq injection of ⁶⁴Cu-NOTA-Trastuzumab was 2.96 mSv. The highest absorbed dose was observed in the liver (0.076 mGy/MBq), followed by the spleen (0.063 mGy/MBq), kidney (0.044 mGy/MBq), and heart wall (0.044 mGy/MBq).

Conclusions

⁶⁴Cu-NOTA-Trastuzumab showed a specific uptake at the HER2-expressing tumors, thus making it a feasible and safe monitoring tool of HER2 tumor status in patients with breast cancer.

Trial registration

CRIS, KCT0002790. Registered 02 February 2018, https://cris.nih.go.kr

Background

The specific receptors that are expressed in cancer cells have been considered as targets for the treatment of tumors, resulting in an improved therapeutic performance [1]. Among them, the human epidermal growth factor receptor (HER)—which is involved in the growth of cancer cells—is a target of a representative molecular therapeutic agent [1, 2]. The overexpression of HER—an intrinsic protein tyrosine kinase—is closely related to rapid-progress tumors [3]. A member of the HER receptor family, HER2/neu (HER2), is overexpressed in breast, ovarian, bladder, prostate, and non-small cell lung cancer [3]. Recently, several therapeutic agents targeting HER2 have been developed to improve treatment outcomes, which include Trastuzumab, Lapatinib, and Pertuzumab [4].

The expression of HER2 is evaluated by tumor tissue acquisition, which entails an inevitably invasive procedure [2, 5, 6]. The discordance rate of HER2 expression between primary tumors and distant metastatic lesions is 4.9–17.7% [7]; therefore, it is necessary to re-evaluate HER2 expression in metastatic tumors. Moreover, HER2 expression may change over time after cancer develops [8], thus necessitating continuous HER2 evaluation. However, repeated biopsies are discomforting for the patient. Overcoming this limitation requires a noninvasive evaluation of HER2 expression of using radioisotopes [2, 5, 6, 9].

Various attempts have been made to noninvasively evaluate the expression of HER2 using radioisotopes, including the evaluation of the HER2 expression using single-photon emission computerized tomography (SPECT) with ¹¹¹In-Trastuzumab [10, 11]. We investigated the possibility of evaluating HER2 expression; however, it presented both low sensitivity and limited spatial resolution [10]. To overcome these limitations, a diagnostic method using positron emission tomography (PET) has been studied. Clinical trials of antibodies such as Trastuzumab labeled with radioisotopes, including ¹²⁴I and ⁸⁹Zr, have been conducted [9, 11, 12], demonstrating HER2 expression capability of lesions in patients with HER2 expressing tumors [9, 11, 12]. Moreover, the use of HER2-targeted PET imaging, using ⁶⁴Cu-tetra-azacyclododecanetetra-acetic acid (DOTA)-Trastuzumab, has been attempted [5, 6, 11, 13, 14]; related clinical trials in the USA and Japan showed effective identification of HER2 expression in breast cancer patients [5, 6, 13, 14].

We have previously developed the ⁶⁴Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-Trastuzumab, targeting HER2-expressing tumors and conducted both in vitro and in vivo experiments, showing that ⁶⁴Cu-NOTA-Trastuzumab can be used as a PET-diagnostic application for HER2-positive breast cancer [2]. Here, we evaluated the safety and pharmacokinetics of ⁶⁴Cu-NOTA-Trastuzumab in breast cancer patients.

Methods

Participants

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, and (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 Declaration of Helsinki 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 [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 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 5 mg 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).

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 a 45 mg 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.

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) was acquired from each organ to calculate residence time. The time-activity curve was expressed by three time points at 1, 24, and 48 h. 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.

Biodistribution

The biodistribution of ⁶⁴Cu-NOTA-Trastuzumab was evaluated using the maximum standardized uptake value (SUV_max) and the mean standardized uptake value (SUV_mean) 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 SUV_mean either of the liver or blood was used as the background. The SUV_max of the breast tumors, metastatic LNs, and metastatic bone lesions was used for lesion assessment.

Safety

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.

Results

Participant characteristics

Seven patients with breast cancer were recruited. One screened participant was excluded, because of a failure to produce radioisotopes, thus evaluating a total of six participants.

During the initial diagnosis, the immunohistochemistry (IHC) results from core needle biopsy found three patients with HER2-positive tumors and another three with HER2-negative tumors; however, after neo-adjuvant chemotherapy, the final IHC results from tumor excision showed two patients with HER2-positive tumors and four with HER2-negative tumors. One patient with an IHC score of 3+ from core needle biopsy had the result changed into IHC score 1+ from excision after neoadjuvant chemotherapy, thus classifying this patient as HER2-negative.

Patient cancer staging was checked from IIA to IV. The period between histology evaluation and ⁶⁴Cu-NOTA-Trastuzumab imaging was 1–3 months. All participants—except for participant 1—underwent neoadjuvant chemotherapy with adriamycin and cyclophosphamide before the ⁶⁴Cu-NOTA-Trastuzumab PET/CT scan. The tumor size at the time of ⁶⁴Cu-NOTA-Trastuzumab PET/CT scan was measured at 1.7–4.0 cm.

Detailed participant characteristics are described in Table 1.

Table 1.

Participant characteristics

Subject no	Age (years)	Histology	Stage	IHC score* (CNB)	SISH	IHC score† (excision)	Neoadjuvant chemotherapy	History of Trastuzumab treatment	Interval from CNB to 64Cu-NOTA-Trastuzumab (months)	Tumor size (cm)
1	45	IDC	IV	3+	N/A	N/A	–	–	1	7.3
2	42	IDC	IIIB	3+	N/A	3+	AC #3	–	2	2.4
3	46	IDC	IIIA	3+	N/A	1+	AC #4	–	3	1.7
4	45	IDC	IIA	–	N/A	–	AC #4	–	3	2.2
5	56	IDC	IIIB	2+	–	1+	AC #4	–	3	14.0
6	54	IDC	IIB	–	N/A	–	AC #3	–	2	4.6

IHC immunohistochemistry, SISH silver-enhanced in situ hybridization, CNB core needle biopsy, IDC invasive ductal carcinoma, N/A not applicable, AC anthracycline, and cyclophosphamide

^*IHC score with core needle biopsy at the initial diagnosis

^†IHC score with tumor excision after neoadjuvant chemotherapy

Safety

No adverse events related to the use of ⁶⁴Cu-NOTA-Trastuzumab were observed.

Radiation dosimetry

The estimated radiation-absorbed dose for each organ is described in Table 2. The organ with the highest absorbed doses was the liver, with 0.076 ± 0.007 mGy/MBq. The effective dose was calculated as 0.010 ± 0.001 mSv/MBq. Therefore, when injected with 296 MBq of ⁶⁴Cu-NOTA-Trastuzumab, the effective dose was 2.96 mSv. Figure 1 shows the residence time for each organ.

Table 2.

Dosimetry of ⁶⁴Cu-NOTA-Trastuzumab (OLINDA)

Organ	Absorbed dose (mGv/MBq)
	64Cu-NOTA-Trastuzumab	64Cu-DOTA-Trastuzumab [6]	[89Zr]Trastuzumab [9]
Adrenals	0.005 ± 0.001	0.031 ± 0.004	0.80
Brain	0.009 ± 0.002	0.015 ± 0.003	0.39
Breasts	0.002 ± 0.000	0.020 ± 0.001	0.42
Gallbladder wall	0.006 ± 0.001	0.035 ± 0.008	0.86
LLI wall	0.000 ± 0.000	0.018 ± 0.002	0.58
Small intestine	0.001 ± 0.000	0.019 ± 0.001	0.57
Stomach wall	0.008 ± 0.002	0.024 ± 0.002	0.63
ULI wall	0.002 ± 0.000	0.022 ± 0.002	0.65
Heart wall	0.042 ± 0.008	0.340 ± 0.046	1.11
Kidneys	0.044 ± 0.009	0.103 ± 0.034	1.23
Liver	0.076 ± 0.007	0.237 ± 0.117	1.63
Lungs	0.034 ± 0.004	0.057 ± 0.070	0.59
Muscle	0.001 ± 0.000	0.023 ± 0.006	0.49
Ovaries	0.001 ± 0.000	0.018 ± 0.002	0.59
Pancreas	0.005 ± 0.001	0.032 ± 0.003	0.78
Red marrow	0.001 ± 0.000	0.017 ± 0.001	0.69
Osteogenic cells	0.001 ± 0.000	0.035 ± 0.001	0.79
Skin	0.001 ± 0.000	0.015 ± 0.001	0.34
Spleen	0.063 ± 0.010	0.142 ± 0.040	0.86
Thymus	0.003 ± 0.000	0.030 ± 0.002	0.57
Thyroid	0.000 ± 0.000	0.016 ± 0.001	0.43
Urinary	0.003 ± 0.001	0.023 ± 0.006	0.42
Uterus	0.001 ± 0.000	0.018 ± 0.002	0.58
Total body	0.004 ± 0.000	0.029 ± 0.004	0.55
Effective dose (mSv/MBq)	0.010 ± 0.001	0.036 ± 0.009	0.61

Fig. 1.

Residence time derived from serial positron emission tomography images (PET). Mean organ residence times (± standard deviation) for ⁶⁴Cu-NOTA-Trastuzumab

Normal-organ biodistribution and tumor uptake

The uptakes of ⁶⁴Cu-NOTA-Trastuzumab in normal organs, including blood pool, liver, kidneys, muscles, spleen, bladder, lungs, and bones, are presented in Fig. 2 as SUV_mean. Maximum intensity projection (MIP) images of participant 1 show the whole-body distribution of ⁶⁴Cu-NOTA-Trastuzumab in Fig. 3.

Fig. 2.

Mean standardized uptake value (SUV_mean) with a standard error of ⁶⁴Cu-NOTA-Trastuzumab in normal organs and maximum standardized uptake value (SUV_max) with a standard error of ⁶⁴Cu-NOTA-Trastuzumab in tumors

Fig. 3.

Maximum intensity projection images of ⁶⁴Cu-NOTA-Trastuzumab PET at 1, 24, and 48 h after injection

The uptake of ⁶⁴Cu-NOTA-Trastuzumab in the blood showed a high value at 1 h after injection and a decreasing pattern over time. The SUV_mean of the liver also showed a high value at 1 h after injection and a gradual decrease over time. The bladder’s SUV_mean was maintained at a low value at 1–48 h after injection. Overall, the uptakes of ⁶⁴Cu-NOTA-Trastuzumab in the blood, liver, kidneys, and spleen were relatively high.

The average values of SUV_max for HER2 positive tumors were: 1.9 ± 0.8 at 1 h; 6.3 ± 2.5 at 24 h; and 8.6 ± 5.1 at 48 h after ⁶⁴Cu-NOTA-Trastuzumab injection. In the case of HER2 negative tumors, the average values of SUV_max were: 2.1 ± 1.5 at 1 h; 5.1 ± 3.4 at 24 h; and 5.2 ± 2.8 at 48 h after ⁶⁴Cu-NOTA-Trastuzumab injection. The lesion-to-liver ratios at 48 h after injection were 1.8 ± 1.0 and 1.3 ± 0.8 for HER2 positive and negative tumors, respectively. The lesion-to-blood ratios at 48 h were 1.6 ± 0.9 and 0.7 ± 0.4 for HER2 positive and negative tumors, respectively. Figure 4 shows changes in both the SUV_max of the tumors and tumor-to-background ratios depending on time. Overall, the values of both SUV_max and tumor-to-background ratios were higher in HER2-positive tumors than HER2-negative tumors. The values of HER2-positive tumors increased over time up to 48 h after injection; however, HER2-negative tumors did not show the same increase in uptake values observed for the HER2-positive tumors.

Fig. 4.

The changes of SUV_max (a), tumor-to-liver ratio (b), and tumor-to-blood pool ratio (c) of HER2-positive and HER2-negative tumors over time

Participant 1’s images of ⁶⁴Cu-NOTA-Trastuzumab PET/CT are shown in Fig. 5; Fig. 5a (first column) is the ¹⁸F-FDG PET/CT image and Fig. 5b–d (second to fourth columns) shows ⁶⁴Cu-NOTA-Trastuzumab PET/CT images regarding time (on 1, 24, and 48 h, respectively). Participant 1 was a 45-year-old, left breast cancer patient with multiple metastatic lymph nodes and bones. The tumor showed a HER2-positive expression (HER2 3 +). The upper-row arrows show the metastatic lymph node on the left side of the neck. High ¹⁸F-FDG uptake was shown in the ¹⁸F-FDG PET/CT (Fig. 5a upper row), and the uptakes of ⁶⁴Cu-NOTA-Trastuzumab increased over time in the same lesion with the FDG PET/CT images (Fig. 5b–d upper row). The SUV_max of the metastatic lymph node was: 1.2 at 1 h; 6.5 at 24 h; and 11.6 at 48 h after injection. The lower row of Fig. 5 shows the primary tumor in the left breast. ¹⁸F-FDG uptake appears in the left breast cancer (Fig. 5a, lower row, arrowhead). The uptakes of ⁶⁴Cu-NOTA-Trastuzumab were also found in the same lesion with increases over time (Fig. 5b–d, lower row, arrowhead). The SUV_max of the primary tumor was: 2.2 at 1 h; 5.8 at 24 h; 9.7 at 48 h after injection.

Fig. 5.

⁶⁴Cu-NOTA-Trastuzumab PET images of HER2-positive breast cancer (arrowheads, lower row) and metastatic lymph node (arrows, upper row). The primary tumor and metastatic lymph nodes were clearly observed by ¹⁸F-FDG PET/CT (a) and ⁶⁴Cu-NOTA-Trastuzumab PET/CT (b–d). In HER2-expressing lesions, the uptakes of NOTA increased over time to 48 h after injection

Discussion

This study demonstrated that a novel HER2-targeted PET ligand, ⁶⁴Cu-NOTA-Trastuzumab, was safe, had no adverse effects, and provided a relatively low exposure to radiation (2.96 mSv from a 296-MBq injection). Moreover, the uptakes of ⁶⁴Cu-NOTA-Trastuzumab were observed in the HER2-expressing tumors including primary breast cancer, metastatic lymph nodes, and metastatic bones.

Due to the tumor heterogeneity, HER2 expression could differ between the primary and the metastatic lesions and may vary depending on disease progression [7, 8]. Therefore, evaluating the HER2 expression before HER2-targeted therapy to enhance the patients’ treatment efficacy is vital.

⁶⁴Cu-DOTA-Trastuzumab PET—an evaluation method of HER2 expression in a noninvasive procedure—was a feasible modality based on clinical trials [5, 6, 13, 14]. Compared to other PET agents—such as ⁸⁹Zr and ¹²⁴I for evaluating HER2 expression—⁶⁴Cu has the benefit of reducing radiation exposure with a relatively short half-life [2]. Moreover, PET/CT can be performed in an outpatient setting, thus making it advantageous. Previous studies have reported that ⁶⁴Cu-DOTA-Trastuzumab PET has difficulty in distinguishing either metastatic lesions or tumors, in the liver and around the blood vessels due to the high physiologic uptakes of ⁶⁴Cu-DOTA-Trastuzumab [6], which are found in both the liver and blood [15]. However, in a chelator comparison study for ⁶⁴Cu labeled compound administration in vivo, a reduction in non-specific uptake was observed for the ⁶⁴Cu-NOTA compound compared to the ⁶⁴Cu-DOTA compound [16]. Further, the ⁶⁴Cu-NOTA compound showed considerably lower accumulation in the liver than the ⁶⁴Cu-DOTA compound [17]. Therefore, we developed ⁶⁴Cu-NOTA-Trastuzumab to achieve improved PET imaging for HER2 expression, compared to ⁶⁴Cu-DOTA-Trastuzumab [2].

Previous studies with ⁶⁴Cu-DOTA-Trastuzumab [6, 18] evaluated breast cancer patients with only HER2-positive tumors; however, despite the limitation of the small sample number, our study included both patients with HER2-positive and negative tumors. In our study, HER2-positive tumors showed a high ⁶⁴Cu-NOTA-Trastuzumab uptake, whereas HER2-negative tumors did not. Adding to the previous preclinical results [2], this study suggested that ⁶⁴Cu-NOTA-Trastuzumab can effectively differentiate HER2-positive from HER2-negative tumors in patients with breast cancer.

The biodistribution of ⁶⁴Cu-NOTA-Trastuzumab in normal organs showed high uptakes in both the blood and liver, in line with the in vivo study [2]. Nevertheless, ⁶⁴Cu-NOTA-Trastuzumab showed that it is advantageous regarding the relatively low uptake in the liver compared to ⁶⁴Cu-DOTA-Trastuzumab. Conversely, blood uptake is relatively higher than ⁶⁴Cu-DOTA-Trastuzumab. The relatively low uptake in the liver could be due to the stable copper-binding ability of NOTA, potentially reducing free copper accumulation; however, a direct comparison of the results is limited, as the liver uptake between ⁶⁴Cu-NOTA-Trastuzumab and ⁶⁴Cu-DOTA-Trastuzumab was compared in different tumor models [19]. ⁶⁴Cu-NOTA-Trastuzumab showed a relatively low effective dose (0.014 mSv/MBq) compared to the latest radiolabeled Trastuzumab studies (⁶⁴Cu-DOTA-Trastuzumab, 0.036 mSv/MBq; ⁸⁹Zr-Trastuzumab, 0.61 mSv/MBq), potentially reducing patient radiation exposure [6, 9].

The uptake of ⁶⁴Cu-NOTA-Trastuzumab in HER2-expressing tumors was not observed 1 h after injection. Nevertheless, the specific uptake increased 24 h after injection, and a further increase of specific uptake of ⁶⁴Cu- NOTA-Trastuzumab in HER2-expressing tumors could be observed after 48 h following the injection, showing a distinctive feature from the background. It is suggested that ⁶⁴Cu- NOTA-Trastuzumab PET at 48 h after injection can evaluate the HER2 expression in the clinical setting. Mild diffuse uptakes were also observed in the tumors with a negative expression of HER2, as HER2 expression is not all-or-none as determined by the immunohistochemistry methods [5, 20]. Namely, even if the tumor’s HER2 expression is negative, its SUV_max positively correlates to the tumor IHC score [5, 20]. Therefore, it is important to carefully set the cut-off values of SUV_max or SUV ratio between the tumor and the background to determine HER2 expression using a ⁶⁴Cu-NOTA-trastuzumab PET image.

This study has some limitations. First, the number of enrolled participants was relatively small. Second, since all the patients—except for participant 1—underwent neoadjuvant chemotherapy, the SUV of tumors might have reflected tumor cell suppression. Therefore, the SUV might be lower than expected. Namely, regarding participants 2 and 3, with HER2 positive tumors as an initial diagnosis, the ¹⁸FDG PET/CT—performed before and after neoadjuvant chemotherapy—showed both decreased tumor size and metabolic activity after chemotherapy (data not shown). Therefore, the SUV of HER2 positive tumors on ⁶⁴Cu-NOTA-Trastuzumab PET could have been underestimated. Consequently, a further study with a larger sample size and ⁶⁴Cu-NOTA-Trastuzumab PET before neoadjuvant chemotherapy is needed to evaluate the exact efficacy of ⁶⁴Cu-NOTA-Trastuzumab PET imaging.

Conclusion

This preliminary clinical trial showed that ⁶⁴Cu-NOTA-Trastuzumab PET is both safe and feasible. ⁶⁴Cu-NOTA-Trastuzumab showed a specific uptake at the HER2-expressing tumors with a relatively low liver uptake. ⁶⁴Cu-NOTA-Trastuzumab can be used to evaluate radiation dosimetry and prediction of treatment-response in targeted therapy for HER2-positive breast cancer with HER2-targeted therapy.

Abbreviations

⁶⁴Cu-DOTA-Trastuzumab

⁶⁴Cu-tetra-azacyclododecanetetra-acetic acid-Trastuzumab

⁶⁴Cu-NOTA-Trastuzumab

⁶⁴Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid-Trastuzumab PET: Positron emission tomography

CT

Computed tomography

FDG

¹⁸F-Fluorodeoxyglucose

HER2

Human epidermal growth factor receptor 2

SPECT

Single-photon emission computerized tomography

SUV_max

Maximum standardized uptake value

SUV_mean

Mean SUV_max

Authors’ contributions

All authors contributed to the study conception and design of this study. IL, BHB, BIK, CWC, MS, HK, WCN, SML, and ILim designed and performed the clinical trial, analyzed data, and wrote the manuscript; SW, KIK, KCL, and JHK made contributions to conception by analyzing and interpreting data; All authors gave final approval of the final content of the manuscript.

Funding

This study was supported by a grant from the Korea Institute of Radiological and Medical Sciences (KIRAMS), funded by the Ministry of Science and ICT, Republic of Korea (No. 50547-2020) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science and ICT) (Nos. 2020R1A2C2102492, 2019M2D2A1A02057204, and 2017M2A2A7A02070985).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

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 Declaration of Helsinki and its later amendments or comparable ethical standards. This clinical trial has been registered with the Clinical Research Information Service (https://cris.nih.go.kr), registration number KCT0002790. Informed consent was obtained from all individual participants included in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.