Detection efficacy of [89Zr]Zr-PSMA-617 PET/CT in [68Ga]Ga-PSMA-11 PET/CT-negative biochemical recurrence of prostate cancer

Rationale In patients with biochemical recurrence of prostate cancer (BCR), preliminary data suggest that prostate-specific membrane antigen (PSMA) ligand radiotracers labeled with zirconium-89 (89Zr; half-life ~ 78.41 h), which allow imaging ≥ 24 h post-injection, detect suspicious lesions that are missed when using tracers incorporating short-lived radionuclides. Materials and methods To confirm [89Zr]Zr-PSMA-617 positron emission tomography/computed tomography (PET/CT) detection efficacy regarding such lesions, and compare quality of 1-h, 24-h, and 48-h [89Zr]Zr-PSMA-617 scans, we retrospectively analyzed visual findings and PET variables reflecting lesional [89Zr]Zr-PSMA-617 uptake and lesion-to-background ratio. The cohort comprised 23 men with BCR post-prostatectomy, median (minimum–maximum) prostate-specific antigen (PSA) 0.54 (0.11–2.50) ng/mL, and negative [68Ga]Ga-PSMA-11 scans 40 ± 28 d earlier. Primary endpoints were percentages of patients with, and classifications of, suspicious lesions. Results Altogether, 18/23 patients (78%) had 36 suspicious lesions (minimum–maximum per patient: 1–4) on both 24-h and 48-h scans (n = 33 lesions) or only 48-h scans (n = 3 lesions). Only one lesion appeared on a 1-h scan. Lesions putatively represented local recurrence in 11 cases, and nodal or bone metastasis in 21 or 4 cases, respectively; 1/1 lesion was histologically confirmed as a nodal metastasis. In all 15 patients given radiotherapy based on [89Zr]Zr-PSMA-617 PET/CT, PSA values decreased after this treatment. Comparison of PET variables in 24-h vs 48-h scans suggested no clear superiority of either regarding radiotracer uptake, but improved lesion-to-background ratio at 48 h. Conclusions In men with BCR and low PSA, [89Zr]Zr-PSMA-617 PET/CT seems effective in finding prostate malignancy not seen on [68Ga]Ga-PSMA-11 PET/CT. The higher detection rates and lesion-to-background ratios of 48-h scans versus 24-h scans suggest that imaging at the later time may be preferable. Prospective study of [89Zr]Zr-PSMA-617 PET/CT is warranted.

To some extent, this diagnostic dilemma may be mitigated when image acquisition using 68 Ga or 18 F tracers is performed at additional, delayed time points, e.g., 3 h post-[ 68 Ga]Ga-PSMA-11 administration [16]. Nonetheless, certain cases of missed lesions may relate to a key limitation of the shortlived tracers: inability to provide interpretable images beyond several hours post-injection. PET/CT with such tracers thus might not visualize lesions requiring longer times to internalize PSMA-targeted radiopharmaceuticals, e.g., lesions with low PSMA expression or low perfusion [17]. Alternatively, residual urinary tract activity associated with hours-long renal clearance of a short-lived radiopharmaceutical may obscure lesions in sites such as the ureter or urinary bladder [17,18].
Interest therefore has increased in imaging patients with BCR with tracers that combine PSMA ligands with the radionuclide zirconium-89 ( 89 Zr), with its much longer half-life, ~ 78.41 h [13,[17][18][19]. Building on earlier work by others [20,21], our group and our collaborators from Radboud University Medical Center/ the University of Nijmegen stably conjugated 89 Zr to PSMA-617 [17]; we then reported on this radiotracer's biodistribution, organ and whole-body dosimetry, and use in a small number of men with BCR (N = 8). We observed that [ 89 Zr]Zr-PSMA-617 PET/CT at ≥ 24 h post-injection frequently revealed lesions suspicious for prostate cancer that had been missed on ~ 1-h [ 68 Ga] Ga-PSMA-11 PET/CT images [17,19,22]. This favorable preliminary clinical experience, and experience reported with additional 89 Zr-conjugated PSMA-targeted tracers [13,17,18], led us to perform the present retrospective analysis of our use of [ 89 Zr]Zr-PSMA-617 PET/CT in a larger group of patients with BCR and recent prior negative [ 68 Ga] Ga-PSMA-11 PET/CT. Our goal was to confirm the detection efficacy of [ 89 Zr]Zr-PSMA-617 PET/CT, and to gather additional data regarding appropriate timing of image acquisition when using this novel modality.

Endpoints
The primary endpoints of this analysis were the percentage of the study sample with clear visual evidence of lesions suspicious for prostate cancer, and the classification of those lesions (local recurrence, lymph node metastasis, bone metastasis) on scans performed 1 h, 24  scans were performed to assess image acquisition at times that presumably could be used effectively with the longlived 89 Zr, but not the short-lived 68 Ga.
Secondary endpoints were the values on each scan for each suspicious lesion of four key [ 89 Zr]Zr-PSMA-617 PET variables reflecting lesional radiotracer uptake or lesion-to-background ratio, and comparison of these variables between scans at different time points. The variables reflecting lesional radiotracer uptake were the maximum and peak standardized uptake values (SUV max and SUV peak , respectively) of the lesions; the variables reflecting lesion-to-background ratio were the tumor-to-background ratios of SUV max or SUV peak (TBR max or TBR peak , respectively), i.e., the SUV max or SUV peak of the lesion/mean standardized uptake value (SUV mean ) of the tissue used as background, healthy gluteal muscle.
An additional secondary endpoint of the analysis was short-term safety, i.e., side effects or vital signs abnormalities that we believed to be associated with [ 89 Zr] Zr-PSMA-617 PET/CT. Also, we compiled data regarding prostate cancer-related interventions post-[ 89 Zr]Zr-PSMA-617 PET/CT and these interventions' results.

Patients and ethics
The cohort comprised 23 consecutive men with BCR, defined as increasing PSA following radical prostatectomy. These patients underwent imaging for that indication at our center between 27   Zr]Zr-PSMA-617 PET/CT after receiving comprehensive information regarding the risks of radiation exposure from the procedure, and regarding the potential for side effects of the novel PET agent. The latter information included a summary of adverse events associated to date with current PSMA-targeted radiotracers, and those associated with PSMA radioligand therapy (RLT). The consent also covered use of the resulting data, in de-identified form, for scientific publications. Data regarding 5/23 cases (22%) were reported previously [17,19,22].
All imaging was performed in 3D mode on a Biograph mCT 40 scanner (Siemens Medical Solutions, Knoxville, TN, USA). PET acquisition time was 3 min/bed position for the 1-h scan, 4 min/bed position for the 24-h scan, and 5 min/bed position for the 48-h scan, with an extended fieldof-view of 21.4 cm.
For attenuation correction and anatomical localization, low-dose CT was performed employing a 120-keV x-ray tube voltage and tube current modulation using CARE Dose4D software (Siemens Healthineers, Erlangen, Germany), with 30 mAs as the reference. Data were reconstructed with a soft tissue kernel (B31f/Be32) to a slice thickness of 5 mm (increment: 2-4 mm).
Along with attenuation correction, PET emission data underwent decay correction, random correction, and scatter correction. PET images were reconstructed applying an iterative 3-dimensional ordered-subset expectation maximization algorithm (3 iterations; 24 subsets) with Gaussian filtering to a transaxial resolution of 5 mm at full width at half maximum. The matrix size was 200 × 200 and the pixel size, 3.0 mm.  Each lesion that appeared to be suspicious for prostate cancer was analyzed using SyngoVia software (Enterprise VB 60, Siemens, Erlangen, Germany) to measure SUV max and SUV peak , and calculate TBR max and TBR peak. The latter two variables were respectively defined as SUV max or SUV peak of the lesion divided by SUV mean of healthy gluteal muscle, the tissue used as background. SUV mean was calculated within a volume of interest applying a threshold of 20% of SUV max .

Monitoring for potential adverse events related to [ 89 Zr]Zr-PSMA-617 PET/CT
We recorded adverse events and clinically-relevant abnormalities in vital signs that we believed to be related to [ 89 Zr] Zr-PSMA-617 PET/CT and that were observed by health care professionals, reported by the patient, or both during imaging and up to 4 weeks thereafter. Questions about specific potential side effects as well as open-ended queries about the occurrence of side effects in general were posed to patients in telephone calls made shortly after scanning and/or after the first follow-up visit.

Statistics
Data are presented as descriptive statistics, including mean ± SD, median (minimum-maximum), and number (percentage) or vice versa, as appropriate. The Wilcoxon matched-pairs signed rank test was used for intra-individual comparison of values for PET variables between scans acquired at different time points.
Prism version 8 (GraphPad Software, San Diego, USA) was used for the statistical analyses. p < 0.05 was considered to be statistically significant.

Visual findings
Eighteen of the 23 patients (78%) included in this analysis had clear visual evidence of lesions suspicious for prostate cancer on one or more [ 89 Zr]Zr-PSMA-617 PET/CT scans, while 5 (22%) had no such findings ( Table 2). PSA ranged from 0.19 ng/mL to 2.5 ng/mL in the patients with positive scans and from 0.11 ng/ mL to 1.55 ng/mL in the patients with negative scans. Altogether 36 lesions (minimum-maximum 1-4 per patient) were detected in the 18 patients with positive [ 89 Zr] Zr-PSMA-617 PET/CT scans. Of these lesions, 11 were suspected to be local (prostate bed) recurrence, 21, lymph node metastases, and 4, bone metastases. Figures 2, 3 and 4 display representative images of lesions that were classified as either a local recurrence, a lymph node metastasis, or a bone metastasis.
Thirty-three lesions (92%) were visible on the 24-h scan, all of which also were visible on the 48-h scan. Three additional lesions (8%), all presumed to be local (prostate bed) recurrences, were seen only on the 48-h scan. Of these 36 lesions, only one, which was classified as a bone metastasis, was visible on the 1-h scan; this lesion was also noted on both the 24-h and 48-h scans.

PET variables
In intra-lesion comparisons involving all 33 lesions visible on both scans, SUV max did not differ significantly between 24-h scans versus 48-h scans (p = 0.104), while SUV peak was slightly but significantly lower in 48-h scans (p = 0.01) (Fig. 5). Results of PET variables across scans and lesion types are compiled in Table 2.
In contrast, intra-lesion comparisons, also involving all 33 lesions visible on both scans, of variables reflecting lesionto-background ratio, i.e., TBR max and TBR peak , showed that values were significantly higher in the 48-h scan versus the 24-h scan (both p < 0.001) ( Table 2; Fig. 5). The pattern of improved lesion-to-background ratio from the 24-h scan to the 48-h scan also held true across lesion types (Table 2; Fig. 6).

Safety
No adverse events, including clinically-relevant vital signs abnormalities, were noted during the [ 89 Zr]Zr-PSMA-617 PET/CT or the 4 weeks thereafter.

Follow-up
All patients with positive [ 89 Zr]Zr-PSMA-617 PET/CT imaging (n = 18) received treatment due to scan findings. One patient underwent lymphadenectomy of a solitary nodal lesion. The surgical specimen was histopathologically confirmed to contain prostate cancer using immunohistochemistry. The other patients underwent radiotherapy with the dose and fields based on the [ 89 Zr] Zr-PSMA-617 scan observations (n = 15) or received systemic antiandrogen therapy (n = 2). In the patients who received radiotherapy, PSA decreased following that intervention in all cases, by an average of 72% ± 25%. Post-radiotherapy, the PSA value became undetectable in Post-treatment PSA became also undetectable in the 2 men given antiandrogen therapy. Zr]Zr-PSMA-617 PET/CT findings max., maximum; min., minimum; PET/CT, positron emission tomography/computed tomography; PSMA, prostate-specific membrane antigen; SD, standard deviation; SUV max , maximum standardized uptake value; SUV peak , peak standardized uptake value; TBR max , tumor-to-background ratio of SUV max : SUV max of presumed tumor lesion/SUV mean of healthy gluteal muscle; TBR peak , tumor-to-background ratio of SUV peak : SUV peak of presumed tumor lesion/SUV mean of healthy gluteal muscle a Calculation based on lesions seen in both 24-  Second, our analysis of PET variables of lesional tracer uptake and lesion-to-background ratio confirmed a pair of earlier observations regarding [ 89 Zr]Zr-PSMA-617 pharmacokinetics [19]. Namely, lesional accumulation of this tracer remains broadly stable in the 24-h to 48-h postadministration interval, reflected in the present analysis by the lack of significant differences in SUV max and by only slightly (albeit significantly) lower SUV peak at 48 h. On the other hand, decreased physiological accumulation of [ 89 Zr] Zr-PSMA-617 during that period resulted in an increased lesion-to-background ratio in the later scans, in the form of a statistically-significant, and roughly twofold higher, TBR max and TBR peak at 48 h vs 24 h. These pharmacokinetics likely explain our detection of 33 lesions on both the 24-h and 48-h scans, but 3 additional lesions only on 48-h images. Indeed, these pharmacokinetic findings, along with the persistent visibility on 48-h scans of all lesions seen on 24-h scans, and the greater detection efficacy of the later imaging, suggest that 48-h imaging may be preferable to 24-h imaging. However, it remains for future studies to more definitively determine the most appropriate timing for [ 89 Zr]Zr-PSMA-617 PET/CT scanning.
Third, we found evidence that patients with suspicious lesions on [ 89 Zr]Zr-PSMA-617 PET/CT appeared to have had true-positive scans. One patient underwent surgery based on scan findings, and true positivity was  48 h post-injection: a) SUV max , b) SUV peak , c) TBR max , and d) TBR peak . p values refer to intra-lesion comparisons of the values between the 24-h and 48-h post-injection scans for all lesions visible on both scans (n = 33). SD, standard deviation; SUV max , maximum standardized uptake value; SUV peak , peak standardized uptake; TBR max , tumor-to-background ratio of SUV max : SUV max of presumed tumor lesion/SUV mean of healthy gluteal muscle; TBR peak , tumor-to-background ratio of SUV peak : SUV peak of presumed tumor lesion/SUV mean of healthy gluteal muscle Zr-PSMA-617 PET/CT findings, PSA decreased afterwards. The average PSA reduction was 72% ± 25% and the minimum decrease, 30%, with the analyte becoming undetectable in 5 of these men. Fourth, our analysis supported previous published observations of the safety of [ 89 Zr]Zr-PSMA-617 PET/CT, at least over the short term [19]. No undesirable effects were noted by health care professionals or patients up to 4 weeks post-scanning.
Our . Aligned with our experience, Dietlein and colleagues also detected putative local recurrence, lymph node metastasis, and/or distant metastasis. Additionally, like us, these investigators reported no side effects of the novel imaging procedure.
Limitations of the present work should be kept in mind. This was a single-center, retrospective analysis of a relatively small number of patients, which merits a caveat regarding generalizability. Additionally, in the everyday practice setting reported here, malignancy of 35/36 suspected prostate cancer lesions was not assessed histopathologically. However, 1 patient who underwent excision of a solitary nodal lesion seen on [ 89 Zr]Zr-PSMA-617 PET/CT had this finding histologically confirmed as prostate cancer. Additionally, true scan positivity was suggested by marked to complete biochemical responses following salvage radiotherapy in all 15 additional patients who received that intervention. Nonetheless, presence of detectable PSA post-radiotherapy in some of these men means that the presence of lesions missed by [ 89 Zr]Zr-PSMA-617 PET/CT as well as by [ 68 Ga] Ga-PSMA-11 PET/CT cannot be excluded. Thus, to more definitively characterize the diagnostic performance of this novel radiotracer, it will be useful to more systematically analyze biochemistry, imaging, and clinical follow-up after [ 89 Zr]Zr-PSMA-617 PET/CT. Additionally, [ 68 Ga]Ga-PSMA-11 PET acquisition at delayed time points, e.g., 3 h post-injection was not performed in our cohort. Further, it cannot be excluded that not only the different radionuclides used in this study, but also the different PSMA ligands played a role in our results. Compared with PSMA-11, PSMA-617 has a longer persistence in the blood pool and is associated with higher background in PET images. However, no clinically relevant differences in tumor uptake of these ligands have been reported, thus an impact on our results seems to be unlikely [24]. Lastly, we did not evaluate longer-term safety of this imaging procedure, or comprehensively assess short-term safety. However, some reassurance is provided by safety having been demonstrated using zirconium-labeled radiopharmaceuticals in other settings [25][26][27][28].
[ 89 Zr]Zr-PSMA-617 PET/CT may ultimately be somewhat of a "niche modality", largely reserved for patients with negative or indeterminate conventional (early) PET/CT despite BCR. In such cases, [ 89 Zr]Zr-PSMA-617 may be particularly suitable for patients wishing to avoid systemic treatment in favor of metastasis-directed approaches [13,[29][30][31][32]. The potentially treatment-altering and outcome-altering information to be gained from the examination would seem to substantially outweigh its disadvantage of an approximately 2.5 times higher radiation exposure than that associated with conventional PSMA PET/CT [19], and to justify application of this novel procedure, especially in the above-mentioned cases. Given the identical PSMA ligands in this pair of radiopharmaceuticals, another potential application of [ 89 Zr]Zr-PSMA-617 PET/CT may be for pre-lutetium-177-PSMA-617 RLT dosimetry, to attempt to individualize, and thereby optimize, dosing to improve safety and efficacy.

Conclusions
In men with BCR, even when PSA levels are low, [ 89 Zr] Zr-PSMA-617 PET/CT seems to be effective (positivity rate 78% in our cohort) in detecting lesions suspicious for local recurrence and metastasis that eluded detection using conventional imaging such as [ 68 Ga]Ga-PSMA-11 PET/CT. Prospective study of this novel imaging modality in men with BCR is warranted. Prostate-specific membrane antigen; RLT: Radioligand therapy; SD: Standard deviation; SUV max : Maximum standardized uptake value; SUV mean : Mean standardized uptake value; SUV peak : Peak standardized uptake value; TBR max : Tumor-to-background ratio of SUV max : SUV max of presumed tumor lesion/SUV mean of healthy gluteal muscle; TBR peak : Tumorto-background ratio of SUV peak : SUV peak of presumed tumor lesion/ SUV mean of healthy gluteal muscle Funding Open Access funding enabled and organized by Projekt DEAL.

Data availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate All procedures performed in the patients described herein were in accordance with the ethical standards of the Institutional and/or National Research Ethics Committees and with the 1964 Helsinki Declaration and its later amendments, or with comparable ethical standards. This analysis was approved by the Institutional Review Board of the Ärztekammer des Saarlandes/ Saarbrücken (approval number: 170/22, approval date: 13 September 2022). This report does not include any animal studies. Written informed consent was obtained from all participants.
Consent for publication All patients have given written consent to publication.

Conflict of interest
The authors declare that they have no conflict of interest.
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