a) Population and treatment
This prospective study included 21 patients between July 2016 and November 2019 in Rouen’s Henri Becquerel Cancer Centre presenting with biopsy-proven lymphoma. To avoid partial volume effect on RGD PET, only patients with a target lesion of at least 3 cm on CT were included. All patients were treated according to standard routine practice and national guidelines and to their histological subtype. Patients with indolent lymphomas and no treatment criteria may not receive treatment. All patients were staged using FDG PET/CT (FDG). Additional RGD-K5 PET/CT (RGD) was performed before or after initial and intermediate FDG (median +7 days, range -4; +22 for initial FDG and median –0.5 days, range -5; +1 for intermediate FDG). Intermediate imaging using FDG and RGD was performed after two cycles of chemotherapy for patients receiving treatment. No additional treatment was performed between FDG and RGD imaging. All patients had final FDG for final Deauville score staging at end of treatment. Patients with final Deauville score of 4 or 5 were considered non-responders and final Deauville score of 1, 2 or 3 as responders (9). All participants were followed at least 12 months after end of treatment. The study was conducted in accordance with the precepts of the Helsinki declaration and received approval by the Ethical Committee. All patients gave written consent for the study. Favourable opinion from the North-west Committee for the protection of persons was given (ref CPP 02/008/2014). EudraCT number was 2015-000757-20 and the study National Clinical Trial identifier was NCT02490891 first posted on the 7th of july 2015, https://www.clinicaltrials.gov/ct2/show/NCT02490891.
b) PET-CT imaging
Patients had baseline (C0), and intermediate (C2) FDG and RGD PET. Regarding protocol, intermediate RGD was not performed at C2 if RGD was negative at C0. FDG was performed according to EANM procedure guidelines (10). An activity of 3.5 MBq/kg of [18F] FDG was injected after 20 minutes of rest. Sixty minutes later (±10 min), the acquisition began with non-injected CT in the cephalocaudal direction on a General Electric 710 PET/CT (Buc, FRANCE). The images were acquired with the patients’ arms positioned over the head while breathing freely. The PET data were then acquired in the caudocephalic direction using a whole-body protocol (2 min per bed position). The delay between injection and acquisition was standardized to 60 minutes in order to obtain a normalized counting rate for all patients.
RGD was performed using Siemens® PETnet’s RGD-K5. Patients were not at fast. Standard dose of 4 MBq/kg (Maximum of 450 MBq) was administrated after 60 minutes at rest. Images were acquired on same PET machine as for FDG, using the same bed protocol, but with 100kV and 80 mAs CT parameters to avoid unnecessary irradiation. Estimated additional estimated dose delivered for RGD was 10 mSv. The tracer uptake was quantified using the standardized uptake value (SUV) calculated as tissue concentration (Bq/g)/[injected dose (Bq)/body weight (g)].
c) PET analysis
All FDG and RGD PET were analysed by one junior and one senior nuclear physicians. On FDG, all lesions (primary tumor and involved lymph nodes) with significant uptake were considered allowing Maximal Standardized Uptake Value (SUVmax), mean SUV (SUVmean), and Metabolic Tumor Volume (MTV) using a 41% of SUVmax threshold.
On RGD, on addition to SUVmax and SUVmean on all lesions, SUVmax and SUVmean were also considered in 13 organs: occipital cortex, thyroid, ascending aorta, inside left ventricular, lung, liver, spleen, gall bladder, kidney cortex, iliopsoas muscle, femoral head, T12 vertebra and inferior vena cava. Organs were excluded of normal uptake analysis if pathological on FDG PET or CT. For RGD’s MTV, threshold was determined by adaptive method and if necessary, by expert’s visual sampling.
d) Treatment and follow-up
All patients were treated according to national guidelines according to their histological subtype. Chemotherapy was performed using standard protocols. The patients were followed up by physical examination and FDG imaging. Biopsy was performed for any suspicious residual/recurrent tumors whenever possible.
e) Histopathological analysis
All patients had a biopsy proven lymphoma. All tumor tissues were routinely fixed in 4% buffered formaldehyde and processed by standard methods into paraffin blocks. Four micrometer slides were prepared and stained with hematoxylin and eosin. A senior pathologist noted the number of vascular sections and the presence of capillary vascularization via ERGmarker by field of view. For the ERG antibody (EP111, 1/100, pH9, Epitomics, Burlingame, USA), the immunochemistry was performed with VECTASTAIN ABC Rabbit IgG Kit (Vector, California). Samples were examined under an Olympus DX51 microscope (Olympus, Paris, France). All pathological samples were reviewed by one senior pathologist.
f) Statistical analysis
Mean and standard deviation were used for descriptive data. Student test (or Mann-Whiney if population sample beneath five) were performed for patients or PET comparison. All the significance thresholds were set at 0.05 (2-tailed test). Statistics were performed using MedCalc® software. (MedCalc Statistical Software version 13.1.2; MedCalc Software bvba, Ostend, Belgium)