**4. Discussion**

In this prospective test–retest study, we formally report the repeatability of Ga-68 PSMA in both relapsing and metastatic tumor. The main finding of this study is the relatively high day-to-day variability of tumor SUVmax with repeatability levels of agreemen<sup>t</sup> varying between +43% and −46% for all lesions taken together. For local recurrences, values vary between +23% and −33%; for the smaller lymph node and bone metastases, values are +40% to −49% and +56% to −47%, respectively. In addition to this, we show a significant correlation between lesion size and SUVmax repeatability levels of agreement.

With respect to our second aim, no significant differences in repeatability between standard OSEM reconstruction and BPL reconstruction could be shown in this pilot study. There was a small but not significant difference in favor of the standard OSEM reconstruction. However, BPL reconstructions resulted in significantly higher SUVmax of tumor lesions as compared to standard OSEM reconstructions, with significantly higher relative increases in smaller lesions.

Pollard et al. reported on the repeatability of Ga-68 PSMA-HBED-CC (PSMA-11) SUVmax in relapsing prostate cancer [20]. Repeatability levels of agreemen<sup>t</sup> were given for lymph node and bone metastases only and were lower than in our study: 30–40% versus 40–60%. Moreover, for other radiotracers like F-18 DCFPyL, F-18 FDG and F-18 FLT, values in the 30–40% range were reported for SUVmax [21,22]. A possible explanation for the higher day-to-day variability in our study is the relative high number of small lesions. Our study hints at a negative correlation between lesion SUVmax variability and lesion size (Figure 2A). We believe the patient cohort of this pilot study to be representative for patients with relapsing prostate cancer showing relatively small tumor lesions in lymph nodes and bones. Pollard et al. did not find any relationship between lesion size and SUVmax variability [20]. A possible explanation for this is that they only reported on relationships within classes, i.e., within typically smaller lymph node and bone metastases, where we report on all lesions including the larger relapses of the primary tumor in the prostate bed. Olde Heuvel et al. also reported on larger tumors in the setting of primary staging of prostate cancer and found smaller day-to-day variability [23].

A larger day-to-day variability may have implications for lesion-specific response in treatment monitoring. With respect to treatment response monitoring using F-18 FDG, Wahl et al. proposed a minimum of 30% SUVmax decrease for a true response in their PERCIST response criteria [24]. Although the PERCIST criteria have not been validated for Ga-68 PSMA PET yet, we think that a minimum of 30% response is probably not appropriate for the majority of the relatively small lesions that are typically found in (early) relapsing prostate cancer. Our study shows that a minimum response of 50% might be more appropriate in these cases, when using Ga-68 PSMA. Being deferred from a single center single vendor study, an even more conservative approach might be warranted when multiple centers and/or vendors are involved.

When confronted with the relatively small tumor lesions in patients with (early) relapsing prostate cancer, partial volume effects have to be anticipated. This will be particularly the case when quantifying tracer expression in small lesions using tracers with high tumor-to-background ratios like Ga-68 PSMA but is not always appreciated [25,26]. For example, in a report on the effects of androgen deprivation therapy on Ga-68 PSMA SUVmax in primary prostate cancer, lymph node and bone metastases as recent as April 2020, possible impact of partial volume effects was not discussed at all [27].

The possible importance of partial volume effects can be illustrated by the BPL versus standard reconstructions, as applied in our study. Two factors that contribute to the partial volume effect (spill-in and spill-out) are the finite spatial resolution of the imaging system (involving scanner hardware, acquisition parameters and reconstruction method) and image sampling on a discrete voxel grid imperfectly matching the actual contours of tracer distribution [12]. Impact of partial volume effects is strongly dependent on lesion size and is coming into play when lesion size falls below 2 to 3 times the resolution of the system, i.e., below 10 to 15 mm for an average PET/CT scanner system [10]. With respect to tracer SUVmax measurements, partial volume effect will generally result in lower SUVmax values. As SUVmean is usually defined as the average SUV in a 3D isocontour at a certain threshold set as a percentage of the SUVmax [28]. In standard OSEM reconstruction, the number of iterations is very limited, to prevent the emergence of excessive noise [13]. The Bayesian penalized likelihood (BPL) reconstruction algorithm called Q.Clear allows for improved spatial resolution because of its convergence, applying a noise penalty at the voxel level during image reconstruction [14]. The resulting higher uptake/expression values in small lesions (compared to standard reconstruction) have been reported for several radiotracers, including F-18 PSMA-1007 in prostate cancer patients [29]. In the latter study, SUVmax with BPL reconstruction has been compared to standard OSEM reconstruction, stratified for lesion size. A significant reported increase in SUVmax with BPL reconstruction for lesions smaller than 10 mm diameter only was reported. In our study, we confirm these findings for the 68Ga-PSMA tracer, including the relationship with lesion size (Table 2), thus highlighting the importance of partial volume effects and its correction.

We hypothesized that the BPL reconstruction algorithm Q.Clear has the ability to lower signal variability in the small lesions typically encountered in early relapse or early metastatic disease of prostate cancer. Our study did not confirm our hypothesis. On the contrary, signal variability tended to be higher with BPL reconstruction compared to standard reconstruction (although not significant). A possible explanation for this could be the 'overshoot' reported with BPL reconstruction for small spheres at high sphere-tobackground ratios in a phantom study using F-18 FDG [30]. The high sphere-to-background ratios in this study may correspond to the high tumor-to-background ratios typically encountered in Ga-68 PSMA avid prostate cancer lesions.

Limitations. This single site study has several limitations. For a formal test–retest study, there is a relatively wide range in administered activity (0.9–1.7 MBq/kg) and time from injection to acquisition (55–69 min). However, in most patients, the activity was administered within reasonable limits with respect to dose and equilibration time, thus reflecting daily practice. Especially, with regard to relatively short-lived radiopharmaceuticals like Ga-68, exact dosing can be cumbersome. For example, timely administration of the radiopharmaceutical was hampered by the pending results of the quality control just prior to injection in some patients. The assignment of tumor lesions was only done by two experienced image readers, and inter-observer variability was not assessed. This was considered acceptable because the exact nature of the lesions was less important in light of the general aim of the study, i.e., assessment of signal variability. The same holds for the fact that for the majority of the lesions there has been no confirmation by histopathology. Moreover, potential detrimental effects of patient motion cannot be ruled out. This may be especially relevant because most lesions were small.

With respect to the comparison of the BPL and standard OSEM reconstructions, the image reading was not blinded. This was deemed acceptable because the visual appearance of both reconstructions is different, precluding true blinded image reading.

No significant difference was found between BPL and standard OSEM reconstruction signal variability, probably because the study was underpowered. Being a pilot study, the results can be used for power calculations for a possible future study.
