Preparation and Preclinical Evaluation of Al18F-HER2-BCH
Al18F-HER2-BCH was prepared with a nondecayed corrected radiochemical yield of 45.2% ± 10.5% (n=20). The radiochemical purity reached 98.2% ± 0.2% with a specific activity of 15.6-24.2 GBq/µmol (n>20). The results of quality control were shown in Figure S1. Radio-HPLC analysis of the radiotracers revealed no aggregates, fragments, or radioactive purities. In vitro stability, Al18F-HER2-BCH was stable in both saline and 5% HSA within 6 h. In vivo stability, after the mice were injected with the radiotracer for 30 min, the radio-HPLC results of blood showed the main peak (>90%) at retention time of 10.26 min, which was close to that of Al18F-HER2-BCH (T=9.85), indicating that Al18F-HER2-BCH was stable without significant decomposition in 30 min.
Cell uptake and Kd study
As shown in Figure 1A, Al18F-HER2-BCH showed significantly higher uptake in NCI-N87 cells than in MDA-MB-231 cells (1.95±0.24% vs 0.54±0.02 % IA/106 cells, p< 0.0001, 2 h) within 4 h. The uptake of Al18F-HER2-BCH in NCI-N87 cells decreased sharply when co-injection of 50 µg HER-affibody-BCH (0.41±0.13 %IA/106 cells, p< 0.0001, 2 h), indicating that Al18F-HER2-BCH had good specificity. The Kd value of Al18F-HER2-BCH to HER2 in NCI-N87 cells was determined to be 25.53±2.29 nM (Figure 1B).
Bio-distribution study The uptake of Al18F-HER2-BCH in the NCI-N87 tumors was obviously higher than in MDA-MB-231 tumors (12.2±1.08% ID/g vs 1.46 ± 0.57 %ID/g, p < 0.0001) at 2 h, and the tumor uptake was blocked to 1.46 ± 0.57 %ID/g by coinjection of 500 µg HER-affibody-BCH (Figure 1D).
Micro-PET imaging study As showed in Figure 1E, Al18F-HER2-BCH depicted clearly tumor uptake at 1 h, maintain high uptake until 4 h. Compared with MDA-MB-231 models, Al18F-HER2-BCH showed significantly higher uptake in the NCI-N87 models during whole imaging. When 500 µg HER2-affibody-BCH was coinjected, the uptake in the N87 models markedly decreased further confirmed the specific uptake. The tumor-to-muscle (T/M) ratios of NCI-N87 was higher than MDA-MB-231 and NCI-N87 block, with T/M values of 8.03, 1.27, 1.41 at 2 h, respectively (Figure 1C).
Pathological examination Figure 1F described the HE staining and IHC results of NCI-N87 tumors and MDA-MB-231 tumors. IHC results showed high expression in NCI-N87 tumors and low expression in MDA-MB-231 tumors, which consistent with the results of bio-distribution and micro-PET imaging of Al18F-HER2-BCH.
No significant differences in weight, hematologic markers, liver function tests or renal function tests were observed between the mice injected with Al18F-HER2-BCH (1.48 GBq/kg) and those injected with saline within 1 week of growing (p>0.05, Figure S2 and Figure S3). In addition, no lethal or chronic toxicity, hematologic effects, or biochemical effects were observed.
Clinical study in BC Patients
Patient characteristics Between June 2020 and February 2021, 14 HER2-positive breast cancer patients and one HER2-negative breast cancer patient were completed the study protocol. Among them, 7 patients were newly diagnosed and had not received any treatment. Totally 9 primary lesions in 7 newly diagnosed patients were pathologically confirmed. The other 7 patients relapsed during follow-up according to increased tumor markers or other features on imaging examinations.
The optimal time point for Al18F-HER2-BCH PET/CT imaging
Dynamic imaging showed blood pool activity was initially high, decreased rapidly in the first 8 min, and was then followed by a slower decrease until 32 min. High uptake was noticeable in both the liver and spleen in the first 8 min. Uptake in the glands (lacrimal gland, parotid gland, submandibular gland and thyroid gland) and other tissues increased gradually. The tumor could be visualized at 8 min, exceeded the blood pool activity after 30 min, and maintained high accumulation throughout the examination. Static images were collected for 2 h and 4 h. The high uptake of Al18F-HER2-BCH was observed in the glands, liver, kidney, and tumor at 2 h, and slightly changed at 4 h (Figure 2 and Figure S4).
18 F-FDG and Al 18 F-HER2-BCH PET/CT imaging in newly diagnosed patients
Al18F-HER2-BCH detected more primary lesions than 18F-FDG (9/9 vs 6/9). The background of normal breast tissue on 18F-FDG and Al18F-HER2-BCH were both low (1.02±0.4 and 0.6±0.2). Al18F-HER2-BCH showed a higher tumor-to-background contrast and clearer tumor delineation than 18F-FDG, with tumor/breast (T/B) ratios of 17.9±13.2 vs 5.4±3.4 (p<0.05) (Table 1). The primary breast cancer included 5 nodular lesions and 4 non-nodular lesions. 18F-FDG uptake in the nodular lesions was higher than non-nodular lesions( 9.7±0.60 vs 2.9±0.36, p<0.0001), while Al18F-HER2-BCH uptake showed no difference between two groups (12.23±6.67 vs 8.63±0.49, p=0.165). (Figure 3). In addition, as showed in Figure S5, Al18F-HER2-BCH could also detect the other HER2-positive small lesion of the patient, which was HER2 3+.
For metastatic lymph nodes detection, Al18F-HER2-BCH detected more lesions than 18F-FDG (20 vs 7), and the uptake was higher (SUVmax, 6.42±3.84 vs 5.1±1.06). The average diameter of lymph nodes detected by Al18F-HER2-BCH was 0.72 cm, and the smallest one was only 0.29 cm. As depicted in Figure 4, Al18F-HER2-BCH more clearly showed the primary tumor and the involved axillary lymph nodes than 18F-FDG in a HER2-positive primary diffuse breast cancer patient. Puncture pathology showed that the primary focus was HER2 3+, proving that the patient was a HER2-positive patient.
Both images revealed no distant metastasis in the 7 patients.
Moreover, Al18F-HER2-BCH changed TNM stages classified by 18F-FDG. One patient changed from T0 to T1, one patient changed from T2 to T3, two patients changed from N1 to N2, and one patient changed from N1 to N3.
Diagnostic performance of 18F-FDG and Al18F-HER2-BCH PET/CT in relapsed patients
Al18F-HER2-BCH detected visceral metastases in 7 of 7 patients (100%), while 18F-FDG detected them in 5 of 7 patients (71.4%), missed 1 patients with liver and bone metastases and 1 patient with liver metastases. In addition, Al18F-HER2-BCH detected more bone metastases in 2/5 patients (Figure 5). Among 7 patients, 93 lesions (19 lymph node metastases, 39 bone metastases, 30 liver metastases, and 5 lung metastases) were selected for analyses of two images. The number of lesions and the semi-quantitative parameters were presented in Table 2 and Figure S6.
Al18F-HER2-BCH detected more positive lymph nodes (19 vs 14), with higher uptake than 18F-FDG (9.54±7.75 vs 5.78±1.86). The diameter of lymph nodes ranged from 0.42 cm-3.88 cm, with an average value of 1.01 cm. For bone metastasis, Al18F-HER2-BCH detected more lesions (39 vs 21), with higher uptake than 18F-FDG (13.57±6.90 vs 4.62±3.68, p<0.0001).Al18F-HER2-BCH also detected more liver metastases (30 vs 3), with higher uptake than 18F-FDG (8.7±4.17 vs 3.2±0.61). The smallest liver metastatic lesion was only 0.61 cm, For lung metastases, the uptake of 18F-FDG was higher than that of Al18F-HER2-BCH (5.50±2.25 vs 4.26±1.89, p=0.41), and lesions detected by two images was same (5 vs 5) (Table 2).
Diagnostic performance of 18F-FDG and Al18F-HER2-BCH PET/CT in HER2-negative patient
As described in Figure 6, 18F-FDG showed high uptake in the bone metastases in a HER2-negative breast cancer patient with recurrence, while there was relatively low uptake in the same lesion sites of Al18F-HER2-BCH. The pathological results of the breast lesions showed HER2 2+, and FISH test showed no amplification, proving that the patient was a HER2-negative patient. The HE staining results could be seen in Figure S7.