*2.1. Patients*

Thirty patients with biochemical recurrence of prostate cancer scheduled for routine clinical Ga-68PSMA PET/CT were prospectively enrolled in this pilot study (NL52809.100. 16/R16.058/Ga-68 PSMA test-retest study) between January 2018 and July 2019. Their scans were screened for evaluable PSMA-positive tumor lesions by two board certified nuclear physicians (MJR and CB). Seven patients had no evaluable tumor lesions and were excluded from the study. The remaining twenty-three had their second (retest) scan within on average 7.9 days (range 6 to 23) of their initial (test) scan and were evaluated for measurements in assigned tumor lesions. Only one patient had a relatively long interval of 23 days between test and retest due to logistical issues, where the next highest value was 10 days. Tumor lesions were classified as suspected local recurrence, lymph node metastases or bone metastases. Both baseline scans were performed before any treatment had begun. The study was approved by the local institutional ethics review board, and all patients had

given written approval before any scanning was done (NL52809.100.16/R16.058/Ga-68 PSMA test-retest study, date of approval 22 March 2017).

PET images were acquired on a PET/CT scanner (GE Healthcare Discovery 710), with a 2.5 min acquisition per bed position, on average 58 (range 55 to 69) min after injection of on average 1.4 (range 0.9 to 1.7) MBq/kg dose of Ga-68 PSMA. Two datasets were reconstructed: one standard ordered subset expectation maximization (OSEM) reconstruction including both time-of-flight (TOF) and point spread function (PSF) modelling and a second one using the Bayesian penalized likelihood (BPL) algorithm, called Q.Clear [17]. For the OSEM reconstruction, 2 iterations and 24 subsets were used. BPL reconstruction also included TOF and PSF. BPL (including TOF and PSF) is subsequently referred to as BPL, same as for OSEM. A matrix size of 256 × 256 was used, resulting in voxels of 2.73 × 2.73 × 3.27 mm3. With respect to filtering, a 6.4 mm Gaussian filter and 1:4:1 filter in axial direction were applied. Both used low dose CT for attenuation correction. For the BPL algorithm, a beta value of 600 was used. This value was found optimal in a Ga-68 PSMA phantom study using spheres of 5–37 mm diameter (this special issue).

#### *2.2. PET and CT Analysis*

For both the test and the retest datasets, the PET and low-dose CT images were processed independently. Imaging reading was performed using dedicated software for PET/CT imaging (Philips IntelliSpace Portal 9.0, Eindhoven, The Netherlands).

In PET, tumor lesion size was measured in the axial plane using a fixed PET windowing upper level (UL) of 10 used for stretching of the greyscale. Both long and short axis were measured; lesion area was calculated according to the simple formula for round and oval lesions: A = π × half long axis × half short axis. Within this area, the pixel with the highest standardized uptake value is designated the SUVmax (injected dose/kg body weight). Thus SUVmax was measured in all tumor lesions. The small size of most of the lesions did not allow for measurement of other meaningful SUVs such as SUVpeak that need lesions of at least 1 cm3. Low dose CT was used to check for appropriateness of the lesion area measured in PET if possible (i.e., with the exception of some bone metastases not visible on CT).
