Head-to-head comparison of [68 Ga]Ga-P16-093 and [68 Ga]Ga-PSMA-617 in dynamic PET/CT evaluation of the same group of recurrent prostate cancer patients

This study was prospectively designed to evaluate the early dynamic organ distribution and tumor detection capability of [68 Ga]Ga-P16-093, which was compared with [68 Ga]Ga-PSMA-617 in the same group of recurrent prostate cancer patients. Twenty patients with recurrent prostate cancer were enrolled. In 2 consecutive days, each patient underwent a 60-min dynamic PET/CT scan after intravenous administration of 148–185 MBq (4–5 mCi) [68 Ga]Ga-P16-093 and [68 Ga]Ga-PSMA-617, respectively. Following a low-dose CT scan, serial dynamic PET scans were performed from head to proximal thigh at 9 time points (30 s/bed at 4, 7, 10, 13, and 16 min; 1 min/bed at 20, 30, and 45 min; and 2 min/bed at 60 min). Standardized uptake values were measured for semi-quantitative comparison. [68 Ga]Ga-P16-093 PET/CT revealed a significantly higher tumor uptake at 4 min (SUVmax 7.88 ± 5.26 vs. 6.01 ± 3.88, P < 0.001), less blood pool retention at 4 min (SUVmean 5.12 ± 1.16 vs. 6.14 ± 0.98, P < 0.001), and lower bladder accumulation at 60 min (SUVmean 31.33 ± 27.47 vs. 48.74 ± 34.01, P = 0.042) than [68 Ga]Ga-PSMA-617 scan. Significantly higher [68 Ga]Ga-P16-093 uptakes were also observed in the parotid gland, liver, spleen, and kidney. Besides, [68 Ga]Ga-P16-093 exhibited a better detectability of tumor than [68 Ga]Ga-PSMA-617 (366 vs. 321, P = 0.009). [68 Ga]Ga-P16-093 showed advantages over [68 Ga]Ga-PSMA-617 with higher tumor uptakes, tumor-to-blood pool ratio and detection capability, less blood pool, and bladder accumulation in recurrent prostate cancer patients. Trial registration: [68 Ga]Ga-P16-093 and [68 Ga]Ga-PSMA-617 PET/CT Imaging in the Same Group of Prostate Cancer Patients (NCT04796467, Registered 12 March 2021, retrospectively registered) URL of registry:https://clinicaltrials.gov/ct2/show/NCT04796467


Introduction
Prostate cancer (PCa) is one of the most common male malignancies and a leading cause of death [1]. Early diagnosis and accurate staging are of paramount significances to the treatment and prognosis of PCa, yet there are persistent major challenges for all detection modalities. Blood prostate-specific antigen (PSA) test, digital rectal examination (DRE), and transrectal ultrasound (TRUS) are classic methods to monitor PCa, which are also the most common approaches for clinical screening of PCa by far. Abnormal DRE is an indication for biopsy, but as an independent variable, it is reported that PSA value is a better predictor of PCa than either DRE or TRUS [2]. Nonetheless, all the above methods have unsurmountable limitations in tumor exact localization, staging of disease, distinguishing between chronic prostatitis and PCa, and so on. Over the past years, multi-parametric MR imaging (mpMRI) has proven to be a valuable imaging tool to avoid unnecessary prostate biopsies, which can better confirm the integrity of the prostate capsule and tumor invasion into tissues and organs around the prostate, through T1, T2-weighted images, DWI, DCE, and other functional sequences [3]. However, mpMRI also exhibited some drawbacks as its specificity for high-grade PCa is only moderate (37%) and limited tumor staging, which are critical for clinical decision-making [4,5].
In the past few years, PET imaging on prostate cancer patients has been a revolutionary advance due to the development of multitudinous radiopharmaceuticals. Prostate-specific membrane antigen (PSMA), as known as folate hydrolase I or glutamate carboxypeptidase II, is regarded as the best-established target antigen in PCa, as it is highly and specifically expressed on the cells of prostatic adenocarcinoma. Besides, the level of PSMA expression on PCa cells further increases with tumor dedifferentiation and in metastatic castration-resistant prostate cancer (mCRPC) [6][7][8][9]. In the current decade, PSMA-targeted PET/CT has proven to be a promising imaging modality with high sensitivity and specificity in detecting PCa and its metastases, even at a very low serum PSA level [10][11][12]. Currently, quite a few of radiolabeled PSMA probes have been developed, including [ 68 Ga]Ga-PSMA-11 and [ 68 Ga] Ga-PSMA-617, which are the most widely used as imaging agents [13]. [ 68 Ga]Ga-P16-093, a modified radiotracer based on [ 68 Ga]Ga-PSMA-11, targets cellular PSMA with the urea fragment of a conjugate that employs the HBED-CC chelator for labeling with 68 Ga(III) [14,15]. The HBEDbased chelating ligand binds the 68 Ga 3+ ion with high affinity (K a nearly 10 39 ) in a pseudo-octahedral N 2 O 4 coordination sphere by its two phenolate O, two amino-acetate carboxylate O, and two amino N donor atoms [15][16][17][18][19], as shown in Fig. 1

Materials and methods
This matched-pair study was approved by the Institutional Review Board of Peking Union Medical College Hospital and registered at clinicaltrials.gov (NCT04796467). Written informed consent was obtained from all subjects. Patients with recurrent prostate cancer were prospectively recruited to this study.

Synthesis of [ 68 Ga]Ga-P16-093 and [ 68 Ga] Ga-PSMA-617
The P16-093 molecule was synthesized as previously described [14]. 68 GaCl 3 was eluted from a 68 Ge/ 68 Ga generator produced by Eckert & Ziegler using 5 mL of 0.1 M ultrapure hydrochloride acid. The eluted 68 GaCl 3 solution was added to a reaction vial, which contained P16-093 (15 μg) and NaOAc·3H 2 O (68 mg) as a lyophilized powder. The reaction mixture was heated for 5 min at 95℃. Subsequently, the final product was diluted with saline and sterilized by filtered through a 0.2-μm sterile vented PVDF filter into a sterile septum-capped vial.
Preparation of PSMA-617 and 68 Ga labeling was performed as described previously [20].
The quality control of the labeled product was conducted by ITLC assessment. Final radiochemical purities over 95% for both [ 68 Ga]Ga-P16-093 and [ 68 Ga]Ga-PSMA-617 were accepted for intravenous injection.

PET/CT imaging acquisition and analysis
In 2 consecutive days, patients were instructed to drink 500 mL of water within 2 h prior to acquisition and to void immediately before the start of scan. Each patient underwent a 60-min dynamic PET/CT (dPET/CT) acquisition by using a Biograph 64 Truepoint TrueV system (Siemens Medical Solutions, Erlangen, Germany) after intravenous injection of 148-185 MBq (4-5 mCi) of [ 68 Ga]Ga-P16-093 and [ 68 Ga] Ga-PSMA-617, respectively. The dPET/CT scan started with a low-dose CT scan (120 kV, 35 mA, 512 × 512 matrix, 3-mm layer, and 70 cm field of view) for attenuation correction and anatomical localization from head to proximal thigh. Then PET scan of each patient was performed at 9 time points (30 s/bed at 4, 7, 10, 13, and 16 min; 1 min/bed at 20, 30, and 45 min; and 2 min/bed at 60 min), with the arms placed on the sides of body. The acquired data were reconstructed using ordered-subset expectation maximization (Siemens Biograph 64: 2 iterations, 8 subsets, gaussian filter of 5 mm in full width at half maximum, 168 × 168 image size).
The mean standardized uptake value (SUVmean) of selected normal tissues was established by placing 3D volumes of interest (VOIs) on the right side of the parotid gland, blood pool (at the arcus aortae level), the right liver lobe, the spleen, the left kidney, and the bladder, respectively. As for prostate cancer lesions, [ 68 Ga]Ga-P16-093 and [ 68 Ga]Ga-PSMA-617 scans were interpreted independently by 2 experienced nuclear medicine physicians blinded to all relevant clinical information; any focal accumulation that was higher than surrounding background activity and did not match for physiologic tracer uptake was interpreted as PCa lesion. The calculation of maximum standardized uptake value (SUVmax) in PCa lesion was based on body weight, using a spheric VOI that can cover the tumor and avoided high physiological uptakes of surrounding tissues.

Statistical analysis
All statistical analyses were performed using SPSS (version 26.0; IBM Corp, for Windows [Microsoft]). All quantitative data were expressed as mean ± standard deviation (SD). For comparison of uptake values, two-sided paired t-test and Spearman correlation coefficient analysis were used. The results were considered significant when P < 0.05.

Characteristics of enrolled patients
A total of 20 patients with recurrent PCa after therapies were enrolled into the study from October 2020 to May 2021, including 6 cases of BCR and 14 cases of mCRPC; the average age of them was 67.8 ± 6.6 years (range 56-83, median 68.5), with a mean Gleason score of 8.5 ± 1.1 (range 7-10, median 9) and a mean PSA level of 36

Radionuclide distribution of normal tissues
We calculated the SUV values of specific tissues, in which PSMA ligands showed normally high uptakes (parotid gland, blood pool, liver, spleen, kidney, and bladder). The distribution of the two tracers in body was similar in several aspects, but there were also clearly observable differences (Fig. 2) Table 2 and Figs. 4 and 5).

Discussion
The clinical application of PET imaging with the 68 Galabeled PSMA radioligands, such as [ 68 Ga]Ga-PSMA-11 and [ 68 Ga]Ga-PSMA-617, has been regarded as a revolutionary breakthrough in the diagnosis of PCa [21]. In the past few years, PSMA-targeting radiotracers have been structurally modified with the goal to achieve improved specificity and sensitivity for clinical applications [22].
We performed this research by comparing the two agents in the same patients, rather than conducting group analysis. And both PET/CT scans were randomly performed in 2 successive days. Obviously, given the nature of PCa progression, there was no significant variation in PCa lesions within such a short period of time. So, findings of this prospective head-to-head comparison study are credible.
As expected, the biodistribution patterns in normal tissues of [ 68 Ga]Ga-P16-093 were similar to [ 68 Ga]Ga-PSMA-617 and other small molecular weight PSMA-targeting radiotracers [23][24][25][26]. But there were substantial differences between them. We will emphasize and discuss the results of this study in detail to further confirm the advantages of [ 68 Ga]Ga-P16-093 over [ 68 Ga]Ga-PSMA-617 as the following.
First and foremost, National Comprehensive Cancer Network (NCCN) guidelines recommend that confirming metastases was a key factor for PCa patients with BCR and CRPC, as this determined their therapeutic schedules [27]. Nevertheless, accurate detection of lesion is still challenging due to its slowly progressive disease occurring and multiple treatment options. There were a number of prospective and retrospective studies using different imaging agents to compare the detectability of biochemical recurrent and progressive PCa patients, with the purpose of finding a more appropriate imaging modality [28][29][30][31][32]. In our study, statistically significant higher tracer uptakes (higher SUVmax) and a superior satisfactory detectability of tumor lesions by [ 68 Ga]Ga-P16-093 are definite advantages over [ 68 Ga] Ga-PSMA-617, which might benefit the subsequent management of patients. Secondly, the faster blood clearance rate of [ 68 Ga] Ga-P16-093 was an important aspect to enhance the detection capability. In this study, the positive finding, as shown in Fig. 4, had well demonstrated this superiority. Furthermore, the lower bladder accumulation exhibited by [ 68 Ga] Ga-P16-093 was also of paramount importance in the diagnosis of recurrent prostate cancer. Low bladder activity was beneficial to identify localized lesions in close anatomical relation to the urinary bladder as recurrence PCa was most frequently associated with pelvic lymph node, which was previously demonstrated in a prospective study [33]. Although there were no lesions found around the bladder in our study, this showed a special advantage of [ 68 Ga]Ga-P16-093 over [ 68 Ga]Ga-PSMA-617 for detection of lesions in the pelvic area. However, it should be noted that the kidney was still the critical organ, since the slightly higher radioactive retention for [ 68 Ga]Ga-P16-093 than [ 68 Ga]Ga-PSMA-617, which was consistent with previous research [15].
Thirdly, as shown in Fig. 3, the higher hepatobiliary excretion and parotid gland cumulated activity value of [ 68 Ga]Ga-P16-093 than [ 68 Ga]Ga-PSMA-617 were phenomena worthy of attention; in general, we prefer a lower uptake in the liver so as not to affect the detection of liver metastases. High liver uptake could, however, be a less significant factor for using [ 68 Ga]Ga-P16-093 imaging in PCa, because it is uncommon to have liver metastasis in these patients.
As for our dPET/CT studies, we also observed that quite a few of PCa lesions presented with visible contrast at 4 min after the injection of imaging agents. Whereas, the blood pool background accumulation was relatively high at this time point, it was not the optimal time point of imaging. Although previous studies have demonstrated that early dynamic scan in PET/CT can reliably identify 2) and 4R region (blue arrow, SUVmax 12.1), which was ambiguous for lymph node in mediastinal 2R region (green arrow, SUVmax 2.9) on [ 68 Ga] Ga-PSMA-617 PET/CT (b) due to higher radiotracer accumulation in the mediastinum and the blood pool, and tracer accumulation of lymph node in mediastinal 4R region was distinct (blue arrow, SUVmax 7.8) pathologic tracer uptake in PC lesions from physiologic accumulation in the urinary bladder [34]. Our early dynamic PET/CT acquisition did not detect more PCa lesions, which indicated that early dynamic imaging may be of little use due to the shortcomings of technically demanding and time-consuming [35]. Of course, further analyses are needed to confirm this result. The European Association of Nuclear Medicine (EANM) and the Society of Nuclear Medicine and Molecular Imaging (SNMMI) procedure guideline suggested a 60-min interval (range 50-100 min) was appropriate for uptake time [36]. In our study, the images at 20 min on [ 68 Ga]Ga-P16-093 PET/ CT exhibited roughly equivalent tracer uptakes of tumor lesions and higher TBR to that at 60 min on [ 68 Ga]Ga-PSMA-617 PET/CT. We propose that [ 68 Ga]Ga-P16-093 PET/CT allows a more flexible imaging time, which greatly increases the practicability and alleviate concerns with regard to necessary coordination of patient activities. So, we suggest that a 20-60-min interval for uptake time for [ 68 Ga]Ga-P16-093 PET/CT scan may be appropriate.
There were some limitations in our study. Firstly, the studied cohort was relatively small. Nevertheless, it was surprising that there were statistically significant differences in many aspects, such as tumor uptake and detection capability, in this small sample. The second limitation was the lack of histological confirmation of both the [ 68 Ga]Ga-P16-093 and [ 68 Ga]Ga-PSMA-617 avid focal lesions in cases of recurrent PCa. Whereas, quite a few studies had confirmed the high correlation between immunohistochemical findings and PSMA PET/CT imaging in PCa [29,37]. Therefore, the results of PET/CT are reliable and consistent with pathological examination.
In the past 10 years, precision medicine based on PSMAtargeting theranostic agents has created a dramatic shift in the practice of nuclear medicine. Significant impact of PSMA PET imaging in diagnosis and management of prostate cancer at different stages is well recognized [21,38]. Recently, [ 68 Ga]Ga-PSMA-11 and [ 18 F]PYL (Pylarify; piflufolastat F 18) have received approval by Food and Drug Administration (FDA) [39,40], and PSMA PET imaging is now becoming an important part of standard clinical practice for diagnosis and treatment of prostate cancers all over the world. [ 68 Ga]Ga-PSMA-11 and [ 68 Ga]Ga-PSMA-617 are two of the most widely used 68 Ga-labeled imaging agents for the diagnosis of prostate cancer. Based on numerous advantages of [ 68 Ga]Ga-P16-093, described herein, it could be an excellent alternative agent, which can be prepared readily in high yield and purity at local hospitals. In the past 3 years, [ 68 Ga]Ga-P16-093 was under clinical studies in the Indiana University and University of Pennsylvania (IND #133,222). We hope that [ 68 Ga]Ga-P16-093, if it is successfully tested and approved by the local authority, could serve the unmet clinical need worldwide.
In addition, with the improvement of positron emission tomography and hybrid imaging technologies, PET/MRI combines the molecular imaging of PET and the anatomical imaging benefits of MRI over CT, without radiation or CT beam-hardening imaging artifacts, which plays a more and more important role in the diagnosis and management of common genitourinary cancers. We also hope to apply PSMA-targeting radiotracers, such as [ 68 Ga]Ga-P16-093, to PET/MRI, which enable us to identify malignant lesions more successfully than ever and result in better patient care and outcomes [41,42].