Synthesis of 18F-rhPSMA-7 and 18F-rhPSMA-7.3
The rhPSMA-7 and rhPSMA-7.3 peptide precursors were labelled with 18F and dissolved in phosphate buffered saline solution as previously described [11]. The 18F-labeling of rhPSMA-7 and rhPSMA-7.3 was performed in a fully automated, Good Manufacturing Practice-compliant procedure using a GRP™ synthesis module (Scintomics, Fürstenfeldbruck, Germany).
Preclinical biodistribution and dosimetry
Severe combined immunodeficiency (SCID) mice were supplied by Charles River Laboratories. (Freiburg, Germany). The preclinical evaluation study was performed in accordance with the German Animal Welfare Act (Deutsches Tierschutzgesetz, approval #55.2-1-54-2532-216-2015). Male mice (≥ 6 weeks old) were used for the study after reaching sexual maturation.
The biodistribution study was performed at multiple timepoints; 10, 20, 40, 60, 120 and 180 min post-injection of 18F-rhPSMA-7, and at 10, 60, 120, 180 and 300 min post-injection of 18F-rhPSMA-7.3. Based on initial experiments exhibiting prolonged renal uptake for 18F-rhPSMA-7.3, a late timepoint (300 min) was applied for the final experiments. At each timepoint, 3–6 mice were injected intravenously in the tail vein with a mean 25.6 ± 3.6 MBq of 18F-rhPSMA-7 and 28.5 ± 4.8 MBq of 18F-rhPSMA-7.3, respectively. For the biodistribution study, the mice were dissected and samples collected from urine, blood, heart, lung, spleen, pancreas, liver, stomach (emptied), small intestine (emptied), large intestine (emptied), kidneys, bladder, testis, fat, muscle (partial, femoral), femur, tail and brain. An automatic gamma counter (PerkinElmer-Wallac, Waltham, USA) was used to measure count rate and the percentages of the injected dose (%ID and %ID/g) were calculated.
Of note, to achieve a similar number of timepoints for both calculations, the 18F-rhPSMA-7 10 min and 20 min timepoints were interpolated to a 15 min timepoint. The time-integral of activity for the accumulation in the investigated source organs (AUCs) were generated both with numerical integration and physical decay [12].
To extrapolate from preclinical data to human dosimetry, linear scaling of %ID from the mice by the ratio of the organ weights and total body weights of phantoms compared to humans was necessary [13]. Normal-organ radiation doses were estimated for the 70 kg standard adult anatomic model using time-dependent organ activity concentrations in %ID/g and total-body activities measured in the biodistribution studies in mice. Tissue activity concentrations in mice were converted to tissue fractional activities in the 70 kg standard adult using the relative fractional organ masses in the standard adult and the standard 25 g mouse. Time-dependent total-body activity was fit to an exponential function and the difference between the injected activity and the total-body activity was assumed to be excreted via the urine because activity concentrations in the liver and gastrointestinal tract were low at all timepoints studied.
Organ residence time was calculated by numerical integration using the trapezoidal rule and the rest-of-body. 18F residence times were calculated as the difference between the total-body residence time and the sum of the organ and urine residence times. The bladder contents residence time was estimated using the dynamic voiding model in the OLINDA/EXM 1.0 dosimetry software (Vanderbilt University, Nashville, TN, USA). Finally, the standard adult mean absorbed dose to organs (in µGy/MBq) and total effective dose (in µSv/MBq) were calculated using OLINDA/EXM 1.0 [14].
Human biodistribution
Patients
Data from patients with histopathologically proven prostate cancer who underwent a clinically indicated PET/CT with 18F-rhPSMA-7 or 18F-rhPSMA-7.3 between October 2017 and November 2018 were retrospectively analyzed. 18F-rhPSMA-7 PET/CT was performed in 47 patients (mean [range] age: 69.8 [52–80] years) with a mean injected activity of 324 (range, 236–424) MBq and mean acquisition time of 84 (range, 42–166) min. 18F-rhPSMA-7.3 PET/CT was performed in 33 patients (mean [range] age: 70.8 [57–85] years) with a mean injected activity of 345 (range, 235–420) MBq and mean acquisition time of 76 (range, 59–122) min. The mean prostate-specific antigen level at the time of imaging was 42.9 ng/mL (median range, 0–1459 ng/mL) for 18F-rhPSMA-7 and 20.0 ng/mL (range, 0–202 ng/mL) for 18F-rhPSMA-7.3, respectively. Patients received an injection of 20 mg of furosemide at the time of tracer application. A comparison of the disease stages and primary Gleason Scores of both cohorts are presented in Additional file 1: Table S5.
All patients gave written informed consent for the original procedure. All reported investigations were conducted in accordance with the Helsinki Declaration and with national regulations. This retrospective analysis was approved by the Local Ethics Committee (permits 290/18S and 99/19S) and the need for patient consent was waived. The administration of 18F-rhPSMA-7 and 18F-rhPSMA-7.3 was in accordance with The German Medicinal Products Act (AMG §13 2b) and the responsible regulatory body (Government of Oberbayern).
PET/CT imaging
All the PET/CT scans were obtained from the skull base to mid-thigh using a Biograph mCT Flow scanner (Siemens Medical Solutions, Erlangen, Germany). PET scanning was operated in 3D mode with an acquisition time of 1.1 mm/s in continuous table movement, and a diagnostic CT scan (240 mAs, 120 kV, 5 mm slice thickness) was acquired in the portal venous phase 80 s after the intravenous injection of an iodinated contrast agent (Imeron 400, Bracco Imaging Deutschland GmbH, Konstanz, Germany). Reconstruction of the PET images was performed based on iterative algorithms with an ordered-subsets expectation maximization (4 iterations, 8 subsets) followed by a post-reconstruction smoothing Gaussian filter (5 mm full width at one-half maximum).
Image analysis
For both quantitative and qualitative analyses PET datasets (non-Time-of-Flight/non-True X) were used. The maximum standardized uptake value (SUVmax) and the mean standardized uptake value (SUVmean) with an isocontour of 50% of the SUVmax were determined applying circular volumes of interest (VOIs) using OsiriX MD® 11.0.2 (Pixmeo SARL, Geneva, Switzerland).
The circular VOIs were placed over normal organs; parotid gland, submandibular gland, mediastinal aortic arch (blood pool), lungs, liver, spleen, pancreas, duodenum, kidneys, bladder, sacral promontory, and background (gluteus maximus muscle). Up to 3 lesions per patient were analyzed in decreasing order of SUVmax. Organ/tumour to background ratios (ratio-SUVmean, ratio-SUVmax) were calculated.
To evaluate overall image quality, non-specific blood pool activity and background uptake in bone/marrow was analysed using 3- or 4-point scales as previously described [7]. All the analyses were performed by a board-certified nuclear medicine physician.
Statistical analysis
Prior to analysis, the Kolmogorov–Smirnov test was used to assess the normality of the data distribution. The independent Student t-test was performed to compare means between groups for the normal parameters. The Mann–Whitney U test was conducted for the non-normal parameters. The Chi-square test or the Fisher's exact test was adopted to compare differences among groups for the analyses of ordinal variables. Data were expressed as mean ± standard deviation (SD) for continuous variables and frequencies (percentages, %) for categorical variables, respectively. All statistical analyses were performed using the IBM SPSS Statistics version 25 (IBM Inc. Armonk, NY, USA) and R version 3.5.2 (http://www.r-project.org). P values less than 0.05 were considered statistically significant.