Patients with histologically proven EOC with radiologically FIGO (Fédération Internationale de Gynécologie et d’Obstétrique) stage IIIB/IIIC [12], who were scheduled to undergo cytoreductive surgery, were asked to participate in the study. Exclusion criteria for the study can be found in the supplemental material. Informed consent was obtained from all participants prior to inclusion and participants were instructed not to use vitamin B9/B11 (‘folate’ or ‘folic acid’) supplements or any other supplements containing vitamin B9/B11 at least 6–7 days before the start of the study.

After a low-dose CT scan (120 kVp) for attenuation correction and anatomical reference, a 90-min dynamic PET acquisition of the pelvis (16-cm field of view) was initiated concurrent with intravenous (cephalic or cubital vein) bolus administration of 185 MBq [18F]fluoro-PEG6-folate. Participants were scanned in supine position using a digital PET/CT scanner (Vereos, Philips Healthcare, Best, The Netherlands).

During this scan, arterial (contralateral radial artery) blood samples were collected manually at nineteen time points (7 × 15s, 2 × 30s, 2 × 60s,1 × 195s, 2 × 300s, 1 × 450s, 2 × 600s, 1 × 1200s, 1 × 1500s cumulative p.i., 2 mL samples) to determine the plasma input function. Plasma supernatant was separated from blood cells (2500 rpm for 10 min) and plasma activity concentrations were determined in an automatic gamma counter (Wizard 2480, PerkinElmer, MA, USA). Three of the samples (i.e. 12.5, 35 and 65 min p.i., 9 mL samples) were used to conjointly determine the plasma to whole blood ratio, the tracer fraction, and the metabolite fraction. Radioactivity concentrations of plasma and whole blood were determined in an automatic gamma counter. In addition, 1 mL of plasma was diluted with 2 mL of water and loaded onto an activated tC2 Sep-Pak cartridge (Waters, MA, USA). The metabolite fraction was obtained by washing the cartridge with 5 mL of water. The PEG-folate fraction was then eluted with 1.5 mL of methanol followed by 1.5 mL of water.

After acquisition, the dynamic scan was reconstructed in 30 time frames (3 × 10 s, 4 × 5 s, 2 × 10 s, 2 × 20 s, 4 × 30 s, 4 × 60 s, 1 × 150 s, 4 × 300 s, 6 × 600 s) using an EARL1-compliant (18F standards 1, EANM Forschungs GmbH, Vienna, Austria) iterative blob-based OSEM reconstruction with 3 iteration, 15 subsets and a 5.5 mm Gaussian filter. Isotropic 4 mm voxels were used.

Motion correction software (FALCON V.2.0, Medical University of Vienna, Austria) [13] was utilized to attempt correction of physiological motion.

Tumor lesions to perform pharmacokinetic modelling on were selected if they met all the following criteria: a) the lesion must show visual uptake of [18F]fluoro-PEG6-folate; b) lesion size (major axis) ≥ 20 mm; c) the lesion must not be directly adjacent to tissues with high uptake, such as bone, intestines, the urinary bladder or arteries. Adjacent, high-uptake tissues may render unreliable pharmacokinetic modelling due to spill-over effects; d) the lesion should not undergo movement more than the tumor's diameter during the scan due to physiological movement, such as bladder filling or bowel peristalsis. Substantial movement results in difficulties with delineation and in inaccurate quantification due to nonmatching scatter and attenuation correction.

For included lesions, a static (identical for all time frames) volume of interest (VOI) was drawn manually on the PET images. If the static VOI resulted in inaccurate delineation for some of the time frames, a VOI was drawn for all time frames individually. From the VOI, a time-activity curve was extracted.

Pharmacokinetic modelling was performed using PMOD (version 4.2, PMOD Technologies LLC, Fällanden, Switzerland). Incorporation of the blood sampling was accomplished by importing the plasma input function, the plasma/whole blood ratio and the tracer fraction into the software. Three different models without blood volume (Vb) correction were fit on the time activity curves: a 1-tissue compartment model (1T2k) and two 2-tissue compartment models, irreversible (2T3k) and reversible (2T4k). For all models, the goodness of fit was determined by extracting the Akaike information criterion (AIC) from PMOD. Due to the sample size being smaller than 40, a second order correction is used to calculate the AIC. [14]

Cytoreductive surgery was performed within 3 weeks after the [.18F]fluoro-PEG-folate PET/CT in all participants (range 3 – 21 days). None of the patients received NACT or any other interventions or treatments between the PET/CT and surgery. In each tumor lesion analyzed in this study the presence of folate receptor-alpha (FRα) expression was investigated by immunohistochemical analysis (assay kit BioCare Medical, Pacheco,USA). [4]