A Prospective Comparison of [ 18 F]FAPI-42 and [ 68 Ga]Ga-FAPI-04 PET/CT in Patients With Various Cancers

Purpose: [18F]FAPI-42 is a new �broblast activation protein (FAP) speci�c tracer used for cancer imaging. Here, we describe the in vivo evaluation of [18F]FAPI-42 and compared intra-individual biodistribution, tumor uptake, and detection ability to [68Ga]Ga-FAPI-04. Methods: A total of 22 patients with various types of cancer received [18F]FAPI-42 whole-body positron emission tomography/computed tomography (PET/CT). Among them, 4 patients underwent PET/CT scans, including an early dynamic 20-min, static 1-hour and static 2-hours. The in vivo biodistribution in normal organs and tumor uptake were semi-quantitatively evaluated using the standardized uptake value (SUV) and tumor-to-background ratio (TBR). Furthermore, both [18F]FAPI-42 and [68Ga]Ga-FAPI-04 PET/CT were performed in 12 patients to compare biodistribution, tumor uptake, and tumor detection ability. Results: [18F]FAPI-42 uptake in the tumors was rapid and reached a high level with an average SUVmax of 15.8 at 18 minutes, which stayed at a similarly high level to 2 hours. The optimal image acquisition time for [18F]FAPI-42 was determined to be 1 hour post injection. Compared to [68Ga]Ga-FAPI-04, [18F]FAPI-42 had a higher uptake in the parotid, salivary gland, thyroid, and pancreas (P < 0.05). For tumor detection, [18F]FAPI-42 had a high uptake and could be clearly visualized in the lesions. [18F]FAPI-42 and [68Ga]Ga-FAPI-04 showed the same detectability for 144 positive lesions. In addition, [18F]FAPI-42 had a higher SUVmax in liver and bone lesions (P < 0.05) and higher TBRs in liver, bone, lymph node, pleura and peritonea lesions (all P < 0.05). Conclusion: The present study demonstrates that [18F]FAPI-42 is a good tracer for imaging malignant tumors and exhibited comparable lesion detectability to [68Ga]Ga-FAPI-04. The 1-hour scan is an appropriate time for tumor detection and is superior to the early 10-min scan for the detection of small lesions.


Introduction
Fibroblast activation protein (FAP) is a transmembrane serine protease that is expressed in stromal broblasts in more than 90% of epithelial cancers as well as in malignant cells in glioblastoma and pancreatic, breast, colorectal, cervical, and oral squamous cell carcinomas [1,2].Overexpression of FAP is associated with high local tumor motility and invasiveness, decreased survival, and poor prognosis in cancer patients [2][3][4].It has been shown that FAP is also upregulated in lesions associated with wound healing, atherosclerotic plaques, rheumatoid arthritis, idiopathic pulmonary brosis, and hepatic brosis [5][6][7][8][9], but is expressed at a negligible or non-detectable level in most normal adult tissues under physiological conditions.Therefore, FAP is considered a good target for diagnosis and treatment of tumors and non-malignant diseases associated with FAP expression.Accordingly, FAP-targeted positron emission tomography/computed tomography (PET/CT) has emerged as a new technique for imaging cancers.The most common FAP tracer is a quinoline-based FAP-ligand radiolabeled with the generator radionuclide 68 Ga (T 1/2 = 67.7 min, 88.9 % β + ) ([ 68 Ga]Ga-FAPI-04).[ 68 Ga]Ga-FAPI-04 PET/CT has been demonstrated to be a good imaging modality for 28 types of cancer and offers higher tumor to non-tumor (T/NT) contrast and higher tumor detectability compared to [ 18 F]FDG PET/CT [10][11][12][13][14][15][16][17][18].However, [ 68 Ga]Ga-FAPI-04 is limited by the relatively short half-life of 68 Ga and availability from the 68 Ge/ 68 Gagenerator, which allows only one or two elution per day and batch production of approximately 1-3 patient doses per elution.In this context, 18 F-labeled FAP tracers may represent a promising alternative for FAP imaging. 18F-ourine, as is well-known, is a positron emitter that is commonly available due to extensively equipped cyclotron worldwide and can be produced in a large amount to meet the requirements of a large number of patients. 18F-labeled radiopharmaceuticals have a longer half-life (T 1/2 = 109.8min) and can thus be delivered over longer distances.Furthermore, the low positron energy of   ]FAPI-42 had a good performance for depicting malignant tumors [20].However, it is not precisely clear whether [ 18 F]FAPI-42 performs as well as [ 68 Ga]Ga-FAPI-04 in the detection of tumors.Furthermore, it is also necessary to determine the optimal time for image acquisition.Thus, we performed a prospective study to compare the tumor detectability of [

Study design and patient population
This study was approved by the institutional review board of the Ethics Committee of Nanfang Hospital (No. NFEC-2020-205) and registered at Chinese Clinical Trial Registry (ChiCTR2100045757).All patients signed a written informed consent before participation and all procedures were conducted in accordance with the Declaration of Helsinki.
Patients were consecutively recruited for enrollment in this study from April 2021 to July 2021.The inclusion criteria were as follows: (1) patients with newly diagnosed cancer who had not received any previous treatment or patients with recurrent or post-treatment metastatic cancer who had not received any treatment within the last three months; (2) primary malignancies con rmed by pathology; and (3) patients who agreed to undergo the two above-described PET/CT scans.The exclusion criteria were: (1) patients with another primary malignancy at the time of examination; (2) patients who suffered from severe hepatic and renal insu ciency; and (3) patients who refused to undergo the scans.
The primary malignancy was diagnosed by biopsy and histopathological examination.Diagnosis of lymph node metastasis and distant metastasis was established by multiple imaging modalities, including magnetic resonance imaging (MRI) of the brain, chest CT, abdominal ultrasound, whole-body bone scan, and whole-body PET/CT.

Radiopharmaceuticals
The radiolabeling precursors were obtained from Nanchang TanzhenBio Co., Ltd.(Nanchang, China) with high chemical purity (>95%).[ 18 F]FAPI-42 was produced using a fully automated process as previously reported [22].In brief, [ 18 F]F − (37-74 GBq) was transferred into the module and trapped on a Sep-Pak Plus QMA (Waters Corporation, preconditioned with 5 mL of 0.5 M sodium acetate buffer pH 3.9 and 10 mL of water) and eluted using 0.35 mL of 0.5 M sodium acetate buffer pH 3.9 to a mixture of AlCl 3 (40.0nmol, 20.0 μL, 2.0 mM in 0.2 M sodium acetate buffer pH 4.0) and precursor NOTA-FAPI-42 (80.0 nmol) in 300 μL dimethyl sulfoxide (DMSO).After heating for 15 min at 105 ºC, the mixture was cooled, diluted with 5 mL of water, and transferred over an activated HLB cartridge (Waters Corporation, preconditioned with 5 mL ethanol and 10 mL water).Next, the HLB cartridge was washed with 20 mL of water, eluted with 1 mL of ethanol/water (1/1, v/v), and ushed using 10 mL of NaCl 0.9%.The eluate was then passed through a sterile Millipore filter (0.22 μm) into a sterile vial.

Imaging procedures
All patients underwent both [ 18 F]FAPI-42 and [ 68 Ga]Ga-FAPI-04 PET/CT scans on two consecutive days.PET/CT imaging was performed using a total-body PET/CT scanner (uEXPLORER, United Imaging Healthcare; Shanghai, China).For each patient, both examinations were performed on the same scanner.
Before FAPI administration, patients were instructed to fast for at least 2 hours in order to reduce hepatobiliary excretion.The PET/CT scan was performed approximately at 1 hour after intravenous injection of [ 18 F]FAPI-42/[ 68 Ga]Ga-FAPI-04 (1.85-3.7 MBq/kg, 0.05-0.1 mCi/kg).The PET scan was performed with a 5-min single bed position 3D acquisition.Low-dose CT (80 mA, 120 kVp) was acquired for attenuation correction and all corrections applied to the reconstructed images, and for anatomic localization of lesions.All data were reconstructed using list-mode OSEM-PSF-TOF [24,25].

Imaging interpretation
The acquired CT and PET images were sent to a MedEx workstation for registration, fusion, and measurement.Two experienced nuclear physicians with more than 10 years of certi cated experience for each scan independently performed image interpretation and any disagreements were resolved by consensus.Any focal accumulations of [ 18 F]FAPI-42 or [ 68 Ga]Ga-FAPI-04 that was higher than the neighboring tissue was interpreted as a positive lesion.Tumor tracer uptake was quanti ed by the maximal standardized uptake value (SUVmax) and target-to-background ratio (TBR).The region of interest (ROI) was drawn along the margins of the lesion on the axial PET image and automatically adapted to a three-dimensional volume-of-interest at a 60% isocontour, which was used to measure the SUVmax.TBR was calculated by dividing the SUVmax of the lesion by the SUVmean of the organs (brain background for brain lesions, lung background for lung and pleura lesions, aortic lumen background for lymph nodes, liver background for liver lesions, adrenal glands background for adrenal glands lesions, L5 background for bone lesions, gluteal muscle background for all other lesions).Tumors with a diameter larger than 1 cm were de ned as large lesions, while tumors with a diameter smaller than 1 cm were de ned as small lesions.
Any drug-related side effects were recorded and the vital parameters of the patients were observed for one week.

Statistical analysis
Normally distributed variables are reported as the means ± standard deviations while skewed variables are reported as the medians (range).The differences in SUVmax and TBR between [  1).Among them, 12 patients were newly diagnosed and the other 10 patients had relapse tumors after single or multiple modality treatments.Twelve patients had staged diseases, 17 patients had local advanced staged diseases, and 3 patients had early staged diseases (Table 1).

Safety
No drug-related side effects were reported during or after [ 18 F]FAPI-42 or [ 68 Ga]Ga-FAPI-04 PET/CT.PET imaging was well tolerated by all patients.Vital parameters remained stable and no patient reported any new symptoms during the observation period.

Dynamic scans of [ 18 F]FAPI-42
The maximum intensity projection (MIP) and biodistribution data assessed by SUV kinetics for one patient (male) are shown in Fig. 1.The images and SUV kinetics for the other three patients are available in Supplemental Fig. 1-3.The pooled SUVmax and tumor SUVmax/organ SUVmean for all four patients that underwent this scan are summarized in Fig. 2a and 2b.The highest average normal organ SUVmax at all time points was observed in the kidneys, decreasing from an average SUVmax of 238 at 10 min to 27.1 by 2 hours (decline of 89%).Tracer uptake in the tumor was rapid and showed excellent retention with an average SUVmax of 15.8 at 18 min, and 15.3 at 2 hours (decrease of 3%).The SUVmax of the organs decreased in all patients 5 min post-injection (p.i.) to the last time point, while TBRs increased with time (with the exception of the gallbladder TBR).The highest TBR among all timepoints was observed in the lungs, small intestine, and bone, with a ratio of 54.1, 40.3 and 26.9.
The SUVmax and TBR (blood) for large and small lesions are summarized in Fig. 2c and 2d.Although some small lesions could be visualized at the early 10-min scan, other lesions were still indistinct due to low TBR (blood) (2.6 ± 0.64).At the 1-hour scan, although the increase in SUVmax was not signi cantly different than the 10-min scan (11.3 ± 4.1 vs. 7.7 ± 1.5, P = 0.05), the TBR (blood) increased signi cantly to a much higher level (8.1 ± 2.7 vs. 2.6 ± 0.64, P = 0.0076) and the small lesions were all clearly visualized (Fig. 1).The TBR further increased from 1 hour to 2 hours; however, this did not result in more lesions detected.For detection of large tumors, the lesions could be clearly visualized at the early 10-min scan, although they were clearer at the 1-hour and 2-hour scans.
Biodistribution comparison between [ 18 F]FAPI-42 and [ 68 Ga]Ga-FAPI-04 PET/CT In the total 22 patients, [ 18 F]FAPI-42 PET/CT demonstrated intense radioactivity in the urinary tract, indicating that the kidneys were the main excretory organs.Intense uptake of the radioactivity was also observed in the gallbladder and common bile duct, implying that some [ 18 F]FAPI-42 was also eliminated via the hepatobiliary system.Moderate uptake of radioactivity was seen in other organs in some patients, including the submandibular gland (14/22), thyroid (12/22), and pancreas (19/22), similar to the ndings of the dynamic scan.Only minimal or mild physiological uptake was observed in other organs and tissue, including the brain, parotid, oral mucosa, lung, myocardium, liver, intestine, fat, spine, and muscle.Radioactivity in bones was low, suggesting that no de uorination occurred in vivo (Fig. 3).

Comparison of tumor detection and tumor uptake between [ 18 F]FAPI-42 and [ 68 Ga]Ga-FAPI-04 PET/CT
On 18 F-FAPI -42 PET/CT images, the uptake of [ 18 F]FAPI-42 in tumors was intense and the lesions could be clearly visualized (Fig. 4).[ 18 F]FAPI-42 PET/CT was positive in all 22 enrolled patients.[ 18 F]FAPI-42 PET/CT depicted primary and relapse tumors in 16 patients, suspected lymph node metastases in 14 patients and suspected distant metastases in 14 patients, including lung lesions in 4 patients, pleural lesions in 5 patients, liver lesions in 6 patients, bone lesions in 4 patients, peritoneal lesions in 2 patients and other suspected metastases in 3 patients.In the 12 patients who received dual-tracer scans, both PET/CT demonstrated the same positive detection for 144 lesions (Table 2).In the visual analysis, [ 18 F]FAPI-42 and [ 68 Ga]Ga-FAPI-04 PET/CT showed comparable results for primary tumors, lymph node lesions, pleural lesions, and peritoneal lesions in terms of both patient-based and lesion-based comparison (Table 2).However, liver and bone lesions were more clearly visualized by [ 18 F]FAPI-42 PET/CT than [ 68 Ga]Ga-FAPI-04 PET/CT (P 0.05; Fig. 5).

Discussion
68 Ga-labeled FAP probes have recently been proven useful for the detection of tumors with high expression of FAP and can be used for imaging various cancers [12,[26][27][28].To take advantage of the favorable properties of uorine-18, a few 18 F-labeled FAPI probes have been introduced for PET imaging [20,21,27,29].However, clinical data comparing 68 Ga-labeled and 18 F-labeled FAP tracers are scarce and the suitable time for the detection of tumors also needs to be identi ed for 18 F-labeled FAP probes.In the present study, we compared biodistribution and lesion detectability between the FAP tracers [ 18 F]FAPI-42 and [ 68 Ga]Ga-FAPI-04 in cancer patients.In addition, the optimal image acquisition time for [ 18  The optimal image acquisition time for [ 18 F]FAPI-42 was determined to be 1 hour p.i. by comparing the ratios of small/large lesions to aortic lumen at different time points, which is consistent with the optimal image acquisition time of [ 18 F]FAPI-74 reported previously [27].Although the uptake of [ 18 F]FAPI-42 in the tumors was rapid and high, the low TBR (blood) at the early 10-min scan made it unfavorable to detect some small lesions.It may not be necessary to postpone the [ 18 F]FAPI-42 scan to 2 hours because it did not show more tumor detection.It was observed that the radioactivity in the pancreas was obviously reduced in three patients after 2 hours, which may be useful for detection of pancreatic carcinoma.A similar phenomenon was reported such that [ 68 Ga]Ga-FAPI-04 detected pancreatic carcinoma using a 3hour delay scan to reduce the physiologic radioactivity in the pancreas [31].
The biodistribution of [ 18 F]FAPI-42 in normal organs and tumor lesions was head-to-head compared with [ 68 Ga]Ga-FAPI-04 in the same twelve patients.The SUVmax analysis of the biodistribution of [ 68 Ga]Ga-FAPI-04 in our cohort was comparable with previously described data for [ 68 Ga]Ga-FAPI-04 [12,30].
Comparison of the biodistribution of [ 18 F]FAPI-42 and [ 68 Ga]Ga-FAPI-04 in normal organs showed somewhat higher tracer uptake for [ 18 F]FAPI-42; however, this did not in uence diagnostic accuracy (Fig. 4).This result might be due to the different lipophilicity of the NOTA-chelator and DOTA-chelator groups.Compared to [ 68 Ga]Ga-FAPI-04, [ 18 F]FAPI-42 showed a higher uptake in the parotid gland, submandibular gland, thyroid, myocardium, gallbladder, biliary tract, and pancreas, which might be caused by the different lipophilicity of the NOTA-chelator and DOTA-chelator groups.This shortcoming might in uence the detection of lesions in these regions, especially for pancreatic, gallbladder and biliary tract tumors.[ 18 F]FAPI-42 accumulation in bone was low on PET/CT image in all patients, which indicated that no de uorination occurred in vivo.Of note, [ 18 F]FAPI-42 showed high bone uptake in preclinical mice models, as reported in our previous work and in the literature [20].
In the present study, we selected various tumors for [ 18 F]FAPI-42 scanning, including [ 18 F]FDG avid tumors, such as lung cancer, colorectal cancer and sarcoma, and some tumors prone to be [ 18 F]FDG nonavid, such as gastric cancer and hepatocellular cancer [32].Our data indicated that all lesions were positive on [ 18 F]FAPI-42 scan, which implied that [ 18 F]FAPI-42 may play a supplementary role to [ 18 F]FDG, similar to recently reported of [ 68 Ga]Ga-FAPI-04 [20].When compared with [ 68 Ga]Ga-FAPI-04, our results showed that [ 18 F]FAPI-42 had high uptake comparable to that of [ 68 Ga]Ga-FAPI-04 in the primary tumors, suspected lymph node metastases, pleural metastases and peritoneal metastases.The visual analysis also showed no signi cant differences between both tracers in the above lesions.Furthermore, [ 18 F]FAPI-42 showed superior detection ability compared to [ 68 Ga]Ga-FAPI-04 in liver and bone lesions based on the patient-based or lesion-based comparison, which was due to higher uptake and TBR in liver and bone tumors for [ 18 F]FAPI-42.
There are some limitations of the present study.Firstly, the dynamic scan was performed at early 20-min, but not 60-min, which could not provide more information concerning dynamic biodistribution of [ 18 F]FAPI-42 in vivo.In addition, our study is limited by the small sample size of the enrolled patients and limited tumor types.Further research is needed to investigate the real clinical value of [ 18 F]FAPI-42 in the diagnosing and staging of various tumors.

Conclusion
The biodistribution of [  * Patient 5 had diffuse uptake of FAP tracers in the peritoneum.We arbitrarily set the number of lesions to 10.

Figures
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Figure 1 a
Figure 1

Figure 4 Maximum
Figure 4

Figure 5 PET
Figure 5
The biodistribution of [ 18 F]FAPI-42 in four cancer patients from 0 to 2 hours after tracer administration demonstrated rapid and high tumor uptake and satisfactory retention, while normal organs showed rapid radioactivity decreases and excretion through the kidneys.Combined with the biodistribution results in another 12 18F]FAPI-42 showed high TBRs with increased over time and 1 hour was a suitable time for image acquisition.[ 18 F]FAPI-42 and [ 68 Ga]Ga-FAPI-04 exhibited comparably high lesion detectability in patients with various cancers.[ 18 F]FAPI-42 with the favorable characteristics is an attractive alternative to [ 68 Ga]Ga-FAPI-04.