A Microdose Clinical Trial to Evaluate 64Cu-NOTA-Trastuzumab as a Positron Emission Tomography Imaging Agent in Patients with Breast Cancer

Background: The purpose of this study was to evaluate the biodistribution and safety of 64 Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-Trastuzumab, a novel 64 Cu-labelled 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 hours after injection of 296 MBq of 64 Cu-NOTA-Trastuzumab were obtained on seven patients with breast cancer. The maximum standardized uptake value (SUV max ) was evaluated in the tumors, including the primary tumor and metastatic lesions. The mean SUV max (SUV mean ) was evaluated in the normal organs including the blood pool, liver, kidney, muscle, spleen, bladder, lung, and bone. In addition, the internal radiation dosimetry was calculated using the OLINDA/EXM software. Safety was assessed by gathering the feedback of adverse reactions and safety-related issues within 1 month after 64 Cu-NOTA-Trastuzumab administration. Results: The overall values of SUV mean in each normal organ decreased with time on 64 Cu-NOTA-Trastuzumab PET images. The average values of SUV mean of the liver were measured at 5.3 ± 0.7, 4.8 ± 0.6, and 4.4 ± 0.5 on 1 hour, 24 hours, and 48 hours after injection. The average values of SUV mean of the blood were evaluated as 13.1 ± 0.9, 9.1 ± 1.2, and 7.1 ± 1.9 on 1 hour, 24 hours, and 48 hours after injection. The SUV max of HER2-positive tumors showed relatively higher than that of HER2-negative tumors (8.6 ± 5.1 and 5.2 ± 2.8 on 48 hours after injection, respectively). Tumor to background ratios were calculated higher in the HER2-positive tumors than those of HER2-negative tumors. No adverse events related to 64 Cu-NOTA-Trastuzumab were reported. The calculated effective dose with a 296 MBq injection of 64 Cu-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: 64 Cu-NOTA-Trastuzumab showed specic uptake at the HER2-expressing tumors. It suggests that 64 Cu-NOTA-Trastuzumab can be a feasible monitoring tool for HER2 tumor status in the patients with breast cancer with safe.


Background
The speci c receptors that are expressed in cancer cells have been studied as targets for the treatment of tumors, resulting in the improved therapeutic performance of cancer patients [1]. Among them, 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]. It is known that the overexpression of HER, an intrinsic protein tyrosine kinase, is closely related to rapidprogress tumors [3]. A member of the HER receptor family, HER2/neu (HER2) is overexpressed in breast cancer, ovarian cancer, bladder cancer, prostate cancer, and non-small cell lung cancer [3]. Recently, several therapeutic agents targeting HER2 have been developed to improve the patient's treatment outcomes, which include trastuzumab, lapatinib, and pertuzumab [4].
The expression of HER2 is evaluated with tumor tissue and to obtain tumor tissue is inevitably invasive [2,5,6]. It has been reported that the discordance rate of HER2 expression between primary tumors and distant metastatic lesions is 4.9-17.7% [7]. For this reason, it is necessary to re-evaluate HER2 expression in metastatic tumors. It is also reported that HER2 expression may change over time after cancer develops [8]. Therefore, continuous HER2 evaluation is necessary. However, repeated biopsies are di cult for the patient as it causes discomfort. In order to overcome this limitation, the need for a non-invasive evaluation of HER2 expression is growing, and one of the proposed methods is the non-invasive evaluation of the expression of HER2 using radioisotopes [2,5,6,9].
Various attempts have been made to non-invasively evaluate the expression of HER2 using radioisotopes. One of them is evaluating HER2 expression using the single-photon emission computerized tomography (SPECT) with 111 In-Trastuzumab [10,11]. This study showed the possibility of evaluating HER2 expression, but had 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 isotopes, such as 124 I and 89 Zr that were labeled with antibodies such as trastuzumab, have been conducted [9,11,12]. The trials were able to demonstrate that they can quantify HER2 expression of lesions in patients with HER2 expressing tumors [9,11,12]. In addition, the use of HER2 targeted PET imaging using 64 Cu-tetra-azacyclododecanetetra-acetic acid (DOTA)-Trastuzumab has been attempted [5,6,11,13,14]. Clinical trials using this agent in the United States and Japan showed effective identi cation of HER2 expression in breast cancer patients [5,6,13,14].
We have previously developed 64 Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-Trastuzumab targeting HER2expressing tumor and investigated in in vitro and in vivo experiments, which showed that 64 Cu-NOTA-Trastuzumab can be used as a PET-diagnostic application for HER2-positive breast cancer [2]. In this study, we evaluated the safety and pharmacokinetics of 64 Cu-NOTA-Trastuzumab in the breast cancer patients.

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. 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 64 Cu-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 64 Cu-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). 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 64 Cu-NOTA-Trastuzumab uptake at the lesion sites. The SUV mean of the liver or blood was used as the background. The SUV max 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 64 Cu-NOTA-Trastuzumab. Feedback such as adverse reactions and other safety-related issues was gathered within 1 month after 64 Cu-NOTA-Trastuzumab administration.
Adverse events, vital signs, physical examination, and laboratory tests were all considered in the safety evaluation.

Participant characteristics
Seven patients with breast cancer were recruited. One screened participant was excluded for failure of the radioisotope production. Six subjects were evaluated in total.
At the initial diagnosis, IHC results from core needle biopsy showed 3 patients with HER2-positive tumors and 3 patients with HER2-negative tumors. However, after neo-adjuvant chemotherapy, the nal IHC results from tumor excision showed 2 patients with HER2-positive tumors and 4 patients with HER2-negative tumors. One subject with IHC score 3+ from core needle biopsy changed the result into IHC score 1+ from excision after neo-adjuvant chemotherapy and this patient was HER2-negative.
The cancer staging of patients were checked from IIA to IV. The period from evaluation of the histology to 64 Cu-NOTA-Trastuzumab imaging was 1 to 3 months. All subjects except for subject 1 underwent neo-adjuvant chemotherapy with adriamycin and cyclophosphamide before 64 Cu-NOTA-Trastuzumab PET/CT scan. The tumor size at the time of 64 Cu-NOTA-Trastuzumab PET/CT scan was measured from 1.7 cm to 14.0 cm. Detailed subject characteristics are described in Table 1.

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

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

Normal-organ biodistribution and tumor uptake
The uptakes of 64 Cu-NOTA-Trastuzumab in normal organs, including blood, liver, kidney, muscle, spleen, bladder, lung, and bone are presented in Fig. 2 as SUV mean . Maximum intensity projection (MIP) images of subject 1 shows the whole-body distribution of 64 Cu-NOTA-Trastuzumab in Fig. 3.
The uptake of 64 Cu-NOTA-Trastuzumab in the blood showed a high value at 1 hour after injection and a decreasing pattern over time. The SUV mean of the liver also showed a high value at 1 hour after injection and a gradual decrease over time. It was evaluated that the SUV mean of the bladder was maintained at a low value from 1 to 48 hour after injection. Overall, it was con rmed that the uptakes of 64 Cu-NOTA-Trastuzumab in blood, liver, kidney, and spleen were relatively high.
The average values of SUV max for HER2 positive tumors were: 1.9 ± 0.8 at 1 hour; 6.3 ± 2.5 at 24 hour; 8.6 ± 5.1 at 48 hour after 64 Cu-NOTA-Trastuzumab injection. In case of HER2 negative tumors, the average values of SUV max were: 2.1 ± 1.5 at 1 hour; 5.1 ± 3.4 at 24 hour; 5.2 ± 2.8 at 48 hour after 64 Cu-NOTA-Trastuzumab injection. The lesion to liver ratios at 48 hour 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 hours were 1.6 ± 0.9 and 0.7 ± 0.4 HER2 positive and negative tumors, respectively. Figure 4 shows changes in SUV max of the tumors and tumor-to-background ratios depending on time. Overall, the values of SUV max and tumor-to-background ratios were measured higher in HER2-positive tumors than that of HER2-negative  (Fig. 5a upper row), and the uptakes of 64 Cu-NOTA-Trastuzumab increase over time in the same lesion with the FDG PET/CT images (Fig. 5b-5d upper row). The SUV max of the metastatic lymph node are: 1.2 at 1 hour; 6.5 at 24 hour; 11.6 at 48 hour after injection. The lower row of Fig. 5 shows the primary tumor in the left breast. 18 F-FDG uptake can be seen in the left breast cancer (Fig. 5a, lower row, arrow head). The uptakes of 64 Cu-NOTA-Trastuzumab can also be observed in the same lesion with increases over time ( Fig. 5b-5d, lower row, arrowhead). The SUV max of the primary tumor is as follows: 2.2 at 1 hour; 5.8 at 24 hour; 9.7 at 48 hour after injection.

Discussion
The present study demonstrated that a novel HER2-targeted PET ligand, 64 Cu-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 64 Cu-NOTA-Trastuzumab was observed in the HER2-expressing tumors including primary breast cancer, metastatic lymph nodes, and metastatic bones.
Due to the heterogeneity of the tumor, HER2 expression may be different between the primary lesion and the metastatic lesions, and may vary depending on the progression of the disease [7,8]. For this reason, it is important to evaluate HER2 expression before HER2-targeted therapy in order to enhance the treatment e cacy of the patients. 64 Cu-DOTA-Trastuzumab PET, one of the methods for evaluating HER2 expression in a non-invasive method, was reported as a feasible modality with clinical trials [5,6,13,14]. Compared to other PET agents, such as 89 Zr and 124 I, which have been used for evaluating HER2 expression, 64 Cu has the bene t of reducing radiation exposure with a relatively short half-life [2], and also has the advantage of being able to perform PET/CT in an outpatient setting.
However, previous studies reported that 64 Cu-DOTA-Trastuzumab PET has di culty in distinguishing metastatic lesions or tumors in liver and around blood vessels due to high physiologic uptakes of 64 Cu-DOTA-Trastuzumab [6]. It is also well known that the high uptakes of 64 Cu-containing agents in the liver and blood [15]. Therefore, we developed 64 Cu-NOTA-Trastuzumab using NOTA as a chelator to make a more stable ligand than 64 Cu-DOTA-Trastuzumab [2]. Our study showed that relatively higher uptake of 64 Cu-NOTA-Trastuzumab in HER2 positive tumors than in HER2 negative tumors. We determined the HER2 speci c uptake of 64 Cu-NOTA-Trastuzumab from comparing the uptake at HER2 negative tumor. In contrast, previous studies of 64 Cu-DOTA-Trastuzumab assessed only HER2 positive tumors without HER2 negative tumors [6,16]. With in vitro and in vivo studies, it was reported that 64 Cu-NOTA-Trastuzumab shows e cient targeting ability to HER2-expressing tumors [2].
The biodistribution of 64 Cu-NOTA-Trastuzumab in the normal organs showed high uptakes in the blood and liver, as was seen in the in vivo study. However, 64 Cu-NOTA-Trastuzumab shows an advantage in the relative low uptake in the liver compared to 64 Cu-DOTA-Trastuzumab. On the other hand, the uptake in the blood is relatively higher than that of to of NOTA than that of DOTA, which can reduce the accumulation of free copper. Furthermore, 64 Cu-NOTA-Trastuzumab shows a relatively low effective dose (0.014 mSv/MBq) compared to radiolabeled trastuzumab that has been studied so far ( 64 Cu-DOTA-Trastuzumab, 0.036 mSv/MBq; 89 Zr-Trastuumab, 0.61 mSv/MBq), which can reduce radiation exposure in patients [6,9].
The uptake of 64 Cu-DOTA-Trastuzumab in HER2-experssing tumors was not observed at 1 hour after injection. However, the speci c uptake increased at 24 hours after injection, and further increase of speci c uptake of 64 Cu-DOTA-Trastuzumab in HER2-expressing tumors could be observed after 48 hours after injection, showing a distinctive feature from the background. It is suggested that 64 Cu-DOTA-Trastuzumab PET at 48 hours 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. This is because HER2 expression is not all-or-none as determined by the immunohistochemistry methods [5,17]. In other words, even if HER2 expression of the tumor is negative, the SUV max of the tumor increases in proportion to the IHC score of the tumor [5,17]. Therefore, it is important to set the cut-off values of SUV max or SUV ratio carefully between tumor and background for determining HER2 expression using 64 Cu-NOTA-trastuzumab PET image.
This study has some limitations. First, the number of enrolled subjects was relatively small. In addition, since all the patients except for subject 1 underwent neoadjuvant chemotherapy, the SUV of tumors after neo-adjuvant chemotherapy might re ect tumor cell suppression after chemotherapy. Therefore, the SUV might be less than expectation. Especially, in the case of subject 2 and 3 with HER2 positive tumors, it was con rmed that the size and

Consent for publication
Not applicable.

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

Competing interests
The authors declare that they have no competing interests.      The changes of SUVmax (a), tumor-to-liver ratio (b), and tumor-to-blood pool ratio (c) of HER2-positive and HER2negative tumors over time.