Clinical Evaluation of 68Ga-FAPI-RGD for Imaging of Fibroblast Activation Protein and Integrin αvβ3 in Various Cancer Types

Visual Abstract

Tumor receptor imaging is an important component of oncologic molecular imaging and plays a key role in cancer diagnosis and management. It is made possible by the intense expression of specific biomarkers in the cellular membrane. Integrin a v b 3 is a heterodimeric transmembrane glycoprotein that is highly expressed in activated endothelial cells, newly formed blood vessels, and several types of tumor cells. In contrast, it exhibits low or no expression in normal cells (1). Therefore, integrin a v b 3 is a promising target for tumor imaging and therapy (2). The tripeptide Arg-Gly-Asp (RGD) moiety exhibits a high binding affinity and specificity for integrin a v b 3 . Various RGD-based cyclic peptides have been labeled with radionuclides and extensively evaluated for PET or SPECT imaging of cancers (2). However, the rapid blood clearance of RGD peptides is one of the reasons that their application is limited in radionuclide therapy (3). The need to improve the pharmacokinetics of RGD peptides has led to multimeric strategies or conjugation with albuminbinding moieties (3,4). These approaches allow the transformation of a drug into a theranostic agent for use as both an imaging agent and a therapeutic agent.
The importance of the tumor microenvironment in cancer development and clinical prognosis is now widely appreciated (5). Besides tumor cells and tumor angiogenesis, cancer-associated fibroblasts of the tumor microenvironment are the major components of solid tumors (6). Fibroblast activation protein (FAP)-a is highly expressed in cancer-associated fibroblasts in most epithelial cancers but is weakly expressed in normal tissues, making it an attractive target for cancer imaging and therapy (7). In the past few years, quinolinebased FAP inhibitor (FAPI) PET/CT has become an active field in nuclear oncology (7,8). Various studies have demonstrated that FAPI-based radiopharmaceuticals are promising radiotracers for cancer diagnosis, staging, and restaging. They may be better alternatives for cancer types that exhibit low to moderate uptake of 18 F-FDG, including gastric, pancreatic, and liver cancers (9). Recently, a heterodimeric peptide, denoted as FAPI-RGD, was synthesized from FAPI-02 and cyclo-RGD-D-Phe-Lys (c[RGDfK]) for targeting both FAP and integrin a v b 3 receptors (10). It was radiolabeled with 68 Ga for preclinical evaluation and was further assessed in a pilot clinical study. The tumor uptake and retention of 68 Ga-FAPI-RGD were significantly greater than those of 68 Ga-FAPI-46 and 68 Ga-c(RGDfK) in mouse xenografts (10). Furthermore, this pilot clinical study on 6 patients demonstrated that 68 Ga-FAPI-RGD PET/CT enabled visualization of tumor lesions with favorable imaging contrast. These results encouraged us to further explore the role of 68 Ga-FAPI-RGD PET/CT for cancer imaging.
The aim of this study was to evaluate, in patients with various types of cancer, the radiotracer uptake and clinical feasibility of 68 Ga-FAPI-RGD PET/CT compared with those of 18 F-FDG and 68 Ga-FAPI-46 PET/CT.

Chemistry and Radiochemistry
The detailed synthesis procedure for FAPI-RGD was reported recently (10). Information on the chemicals and reagents is briefly presented in the supplemental materials (available at http://jnm.snmjournals. org). 68 Ga-radiolabeling of FAPI-02, FAPI-46, c(RGDfK), and FAPI-RGD variants was performed as previously described (11)(12)(13). In brief, 25 nmol of FAPI-RGD in 1 mL of sodium acetate buffer (0.25 M) were reacted with 4 mL of 68 Ga-solution (1.1 GBq in 0.6 M HCl) at 100 C for 15 min. For clinical imaging, the final product was passed through a 0.22-mm Millipore filter for sterilization of each preparation of 68 Ga-FAPI-RGD. Quality control was performed using ultraviolet and radio-high-performance liquid chromatography (supplemental materials). The stability of the radiolabeled compound was determined by incubating the product in phosphate-buffered saline at 37 C and analyzing it via radio-high-performance liquid chromatography after 1, 2, and 4 h of incubation.

Clinical PET/CT Imaging in Healthy Volunteers and Patients with Cancer
The prospective clinical study protocol was approved by the institutional review board of the First Affiliated Hospital of Xiamen University and was registered at ClinicalTrials.gov (NCT05543317). Written informed consent was obtained from all healthy volunteers and patients. The dose of intravenously injected 68 Ga-FAPI-RGD was calculated according to the participants' body weight (3.0-3.7 MBq/kg), which corresponds to approximately 7-8 nmol per subject. Safety data (blood pressure, heart rate, and temperature) and adverse events were recorded before and 4 h after injection of 68 Ga-FAPI-RGD. The PET/CT scanning and reconstruction protocols are presented in the supplemental materials. For dosimetry evaluation, 68 Ga-FAPI-RGD PET imaging was performed at 30, 60, and 180 min after tracer injection. Time-activity curve fitting and subsequent dose calculations were performed using OLINDA/EXM software, version 1.1 (14).
All patients underwent paired 68 Ga-FAPI-RGD and 18 F-FDG PET/ CT scans. An additional 68 Ga-FAPI-46 PET/CT scan was performed for comparative purposes depending on the patient's willingness. We evaluated the in vivo distribution pattern of 68 Ga-FAPI-RGD at later time points, by performing 3-h-delayed 68 Ga-FAPI-RGD PET/CT scans for all patients (because of the relatively short half-life [68 min] of 68 Ga). All PET images were evaluated by 2 board-certified and experienced nuclear medicine physicians. Disagreements were resolved via consensus.
For quantitative analysis, the SUV max was used to quantify radiopharmaceutical uptake by normal organs and tumor tissues. Tracer uptake in normal organs (background) was quantified by SUV mean , which was delineated with a sphere that had a diameter of 1 cm (for small organs, including thyroid, salivary gland, and pancreas) to 2 cm (for other organs, including brain, heart, liver, kidney, spleen, muscle, and bone marrow) placed inside the organ parenchyma. The tumor-tobackground ratio (TBR) was calculated as tumor SUV max /background SUV mean .
To evaluate the diagnostic performance of 68 Ga-FAPI-RGD and 18 F-FDG PET imaging, the results of the visually interpreted PET images were compared with the histopathologic results (via surgery or biopsy), which were used as the gold standard for the final diagnosis. For patients for whom tissue diagnosis was not applicable, clinical and radiographic follow-up data were used as the reference standard to validate the PET/CT findings. Lesions were considered malignant on the basis of any of the following follow-up criteria: typical malignant features confirmed by multimodal medical imaging, significant progression on follow-up imaging, or a significant decrease in posttreatment tumor size. The minimum follow-up period was 3 mo. Histopathologic staining of surgical and biopsy samples was performed as previously described (15).

Statistical Analysis
All statistical analyses were conducted using Prism (version 8.0; GraphPad Software Inc.) and SPSS Statistics (version 22.0; IBM Corp.). All quantitative data are expressed as the mean. The Wilcoxon matched-pairs signed-rank test was used to compare SUVs derived from 18 F-FDG, 68 Ga-FAPI-RGD, and 68 Ga-FAPI-46 PET/CT. The McNemar test was used to compare the lesion detectability of the different PET/CT scans. Statistical significance was defined as a P value of less than 0.05.

Safety and Radiation Dosimetry in Healthy Volunteers
All observed vital signs (including temperature, heart rate, and blood pressure) remained normal during the injection and 4-h postinjection follow-up. 68 Ga-FAPI-RGD was tolerated well, with no adverse events in any of the healthy volunteers or patients. Representative PET images and biodistribution data of healthy volunteers (n 5 3) are provided in Figure 1. Tracer uptake rapidly decreased in most normal organs from 30 to 180 min, particularly in the thyroid, pancreas, and salivary glands. The 68 Ga-FAPI-RGD effective dose was 1.01 3 10 22 mSv/MBq (Supplemental Table 1), which was comparable to that of 68 Ga-FAPI-02 (1.80 3 10 22 mSv/MBq) (16). The organ with the highest effective dose was the thyroid (3.01 3 10 23 mSv/MBq), followed by the urinary bladder wall (1.37 3 10 23 mSv/MBq), liver (1.10 3 10 23 mSv/ MBq), and lungs (1.09 3 10 23 mSv/MBq).

Patients' Characteristics
From July 1 to September 15, 2022, 22 patients with cancer (15 men; median age, 57 y; range, 34-79 y; 17 for staging and 5 for restaging) who underwent paired 68 Ga-FAPI-RGD and 18 F-FDG PET/CT were enrolled in this study. The median interval between the 2 scans was 4 d (range, 1-7 d). Furthermore, 7 of the 22 patients underwent additional 68 Ga-FAPI-46 PET/CT for comparison. Among the 22 patients, the final diagnosis was based on the histopathologic results for 20 and diagnostic radiology results for 2. Detailed information on the enrolled patients is provided in Supplemental Table 2.

Dual-Time-Point 68 Ga-FAPI-RGD PET/CT Imaging in Patients with Cancer
To evaluate the in vivo distribution pattern of the radiotracer and tumor uptake over time, dual-time-point 68 Ga-FAPI-RGD PET/CT (1 vs. 3 h) was performed for all patients. As demonstrated in Figure 2, most tumor lesions demonstrated increased uptake over time. Specifically, SUV max derived from the delayed scan (3 h) was significantly higher than SUV max derived from routine scans (1 h) in the primary tumors (18.0 vs. 12.6; P , 0.001), lymph node metastases (12.1 vs. 9.3; P , 0.001), lung metastases (median, 8.0 vs. 4.6; P , 0.001), and bone metastases (16.2 vs. 13.2; P , 0.001). Interestingly, background activity decreased greatly over time. Consequently, TBR improved significantly in primary tumors with involved lymph nodes and with lung, liver, peritoneal, and bone metastases. Detailed data are presented in Supplemental Table 3.

Comparison of 68 Ga-FAPI-RGD and 18 F-FDG Uptake in Patients with Cancer
Among the 17 patients who underwent paired 68 Ga-FAPI-RGD and 18 F-FDG PET/CT for initial staging, 68 Ga-FAPI-RGD PET/ CT allowed detection of all primary tumors (19/19) with intense radiotracer uptake, whereas 18 F-FDG PET/CT resulted in 3 missed tumor lesions in 1 patient with multifocal breast cancer. In all primary tumors, SUV max was significantly higher when derived from 68 Ga-FAPI-RGD PET/CT than from 18 F-FDG (18.0 vs. 9.1, P , 0.001). Furthermore, the TBRs of the primary tumors from 68 Ga-FAPI-RGD PET/CT were approximately 3 times greater than those from 18 F-FDG PET/CT (15.2 vs. 5.5, P , 0.001). Detailed data and representative images are presented in Table 1 and      Representative PET images are presented in Figure 6. Interestingly, in 1 patient with small cell lung cancer, 4 bone metastases and 3 liver metastases exhibited no abnormal uptake of 68 Ga-FAPI-46, but uptake of 68 Ga-FAPI-RGD was increased. Immunohistochemical staining of a liver metastasis demonstrated negative FAP expression but positive integrin a v b 3 expression (Fig. 7A). Notably, in another patient with nasopharyngeal carcinoma, both tracers exhibited similar uptake in several metastatic lung lesions, which demonstrated positive FAP expression and negative integrin a v b 3 expression (Fig. 7B).

DISCUSSION
As pan-cancer biomarkers, FAP with radiolabeled FAPI-based molecules (including FAPI-04/46) have yielded encouraging results for PET imaging of cancer (7,9). However, the relatively short tumor retention time may hamper the use of FAP molecules for radioligand therapy applications. We and others have applied the polyvalence effect to develop homomultimers to enhance tumor uptake and retention, and dimeric FAPI-based tracers have been synthesized and evaluated (17,18). A dual-receptor-targeting approach with a heterodimer is another strategy to improve the tumortargeting efficacy, especially for imaging probes that recognize only 1 receptor (19). Considering that our previous data showed tumor uptake and retention of 68 Ga-FAPI-RGD to be significantly greater than those of 68 Ga-FAPI-46 and 68 Ga-c(RGDfK) in mouse xenografts (10), we speculated that 68 Ga-FAPI-RGD would be a promising radiotracer for imaging tumors expressing either FAP or integrin a v b 3 . In the present study, 68 Ga-FAPI-RGD, a heterodimeric PET tracer that targets both FAP and integrin a v b 3 , was evaluated in 3 healthy volunteers and 22 patients with cancer. Our clinical studies demonstrated 68 Ga-FAPI-RGD to be a promising PET agent that allows imaging of various types of cancer. Its dualreceptor-targeting property results in improved tumor uptake and retention, allowing imaging of tumors with either or both receptor expression patterns. 68 Ga-FAPI-RGD was safe and well tolerated in all healthy volunteers and patients with cancer. The average effective wholebody dose of 68 Ga-FAPI-RGD was 1.  (16). However, intense physiologic 68 Ga-FAPI-RGD uptake was observed in the thyroid and pancreas, with a distribution pattern similar to that of 68 Ga-FAPI dimers previously reported by us and others (17,18). Interestingly, significantly decreased 68 Ga-FAPI-RGD activity was observed in normal organs (particularly in the thyroid, pancreas, and salivary glands), whereas increased uptake was observed in tumor lesions from 0.5 to 3 h after injection, resulting in optimized lesion contrast in delayed scans. Therefore, we speculate that additional delayed 68 Ga-FAPI-RGD PET/CT scans may help improve the lesion  detection rate and offer advantages for discrimination of tumor and nontumor lesions. However, the clinical benefits of delayed scans require further investigation in a larger patient population.
The SUV max and TBR of primary tumors from 68 Ga-FAPI-RGD PET/CT were significantly higher than those from 18 F-FDG PET/ CT, particularly in non-small cell lung cancer (NSCLC) and in esophageal, breast, and pancreatic cancers. The reason for this finding is that all primary tumors (19/19) were satisfactorily visualized via 68 Ga-FAPI-RGD, whereas 3 breast cancer lesions were missed via 18 F-FDG. 68 Ga-FAPI-RGD PET/CT also demonstrated significantly greater radiotracer uptake in lymph node, bone, and visceral metastases and a significantly higher TBR than 18 F-FDG PET/CT, resulting in an improved lesion detection rate, particularly for the diagnosis of lymph node (99% vs. 91%) and bone (100% vs. 80%) metastases. However, in our previous 68 Ga-FAPI-RGD PET/CT study on 6 patients, the tumor uptake of 68 Ga-FAPI-RGD did not differ from that of 18 F-FDG (10). Possible explanations may be the limited number of patients, different cancer types investigated, and different acquisition time after injection (30-120 min vs. 60-180 min). According to the results of the present study, 68 Ga-FAPI-RGD may be more suitable for imaging tumors with both FAP and integrin a v b 3 expression, particularly for NSCLC and esophageal cancer (20,21). First, the radiotracer uptake and TBR derived from 68 Ga-FAPI-RGD PET/ CT were higher than those from 18 F-FDG in these cancer types, resulting in improved lesion detectability, particularly of liver, bone, and brain metastases. These results suggest that 68 Ga-FAPI-RGD PET/CT may contribute to the diagnosis of NSCLC and esophageal cancer, especially in detecting  small metastases with low-to-moderate uptake on 18 F-FDG PET/ CT. Second, false-positive findings in the mediastinal lymph nodes often confound interpretation of preoperative 18 F-FDG PET/CT images in NSCLC and esophageal cancer. On the basis of previous publications, nonmetastatic reactive lymph nodes presenting increased 18 F-FDG uptake might be correctly diagnosed either by FAP or by integrin a v b 3 -targeting radiotracers (21)(22)(23)(24). Therefore, 68 Ga-FAPI-RGD PET/CT may be more suitable than 18 F-FDG PET/ CT for determining the preoperative lymph node status in these cancer types. Taken together, the results indicate that NSCLC and esophageal cancer may be potential indications for future clinical use of 68 Ga-FAPI-RGD, and further prospective studies are warranted to confirm this possibility.
PET imaging demonstrated that the radiotracer uptake and TBR of 68 Ga-FAPI-RGD were significantly higher than those of 68 Ga-FAPI-46, especially in the involved lymph node, bone, and visceral metastases. Interestingly, we noted that several lesions from metastatic small cell lung cancer and thyroid cancer exhibited low uptake of 68 Ga-FAPI-46 but higher uptake of 68 Ga-FAPI-RGD. Histopathologic results revealed low expression of FAP but higher expression of integrin a v b 3 . Therefore, 68 Ga-FAPI-RGD PET/CT may be used to image tumors that are either FAP-positive or integrin a v b 3 -positive, whereas 68 Ga-FAPI-46 failed to visualize lesions that were FAP-negative and integrin a v b 3 -positive. Moreover, both tracers showed similar uptake in lesions with FAP-positive and integrin a v b 3 -negative expression, suggesting comparable FAPtargeting ability between 68 Ga-FAPI-RGD and 68 Ga-FAPI-46. On the basis of these findings, we speculate that 68 Ga-FAPI-RGD PET/CT would be superior to 68 Ga-FAPI-46 PET/CT for the diagnosis of cancer, especially when 68 Ga-FAPI-46 PET/CT findings are inconclusive.
With the rather rapid washout from tumors and unsatisfactory therapeutic efficacy, 177 Lu-FAPI-04/46 may not be an optimal targeting vector for radioligand therapy (25,26). Besides the improved tumor uptake of 68 Ga-FAPI-RGD, the prolonged tumor retention was an unexpected finding in this study, as may be explained by the synergistic interaction between the 2 binding motifs in the heterodimer. It is possible that the binding of the first motif, even if only temporary, may first direct 68 Ga-FAPI-RGD to the target surface or reduce the off-rate of 68 Ga-FAPI-RGD, allowing the second binding motif to also attach to the tumor and therefore increasing the overall binding and the probability of adhering to the tumor (27). Improved tumor accumulation and prolonged tumor retention are the potential advantages of FAPI-RGD over the current FAPI variants, making it a suitable targeting vector after labeling with 177 Lu/ 90 Y/ 225 Ac for therapeutic applications. In addition, the rapid clearance of 68 Ga-FAPI-RGD in normal organs, particularly in the thyroid, salivary glands, and blood pool, may decrease the absorbed dose delivered to normal human tissues. This possibility should be extensively studied in nuclear oncology research in the future.
Our study had several limitations. First, because of the relatively short half-life of 68 Ga, the in vivo distribution pattern and tumor retention of FAPI-RGD could not be fully investigated. Second, because few patients underwent paired 68 Ga-FAPI-RGD/ 18 F-FDG (n 5 22) or 68 Ga-FAPI-RGD/ 68 Ga-FAPI-46 PET/CT imaging (n 5 7), only a descriptive comparison was possible. Third, this study focused primarily on lesion detection rates, and the specificity of