417 [18F]FDG uptake and expression of immunohistochemical markers 8 the ultrasonographic size and location of the index nodule, to ensure its correct identification. For the visual assessment, any focal [18F]FDG uptake within the thyroid that was visually higher than the physiological background [18F]FDG uptake of the surrounding normal thyroid tissue and that corresponded to the index nodule in size and location, was considered positive (Figure 1). Quantitative image analyses were performed using OsiriX Lite DICOM-viewer (Pixmeo SARL, Bernex, Switzerland). SUV-computation was validated after each mandatory software version update. The SUVmax and peak SUV (SUVpeak, defined as the maximum average SUV within a 1 cm3 spherical volume) of the index nodule were semi-automatically measured. Body weight corrected values were used. The SUVmax-ratio was calculated by dividing the SUVmax of the nodule by the background SUVmax of normal thyroid tissue in the contralateral lobe. [18F]FDG-positive foci in the thyroid that did not correspond to the index nodule in size and location (i.e., thyroid incidentalomas) were not analysed in the current study. Histopathology During the EfFECTS trial, all postoperative patient management was based on the local histopathological diagnosis. For scientific purposes and to limit the effect of any interobserver variability, all (hemi)thyroidectomy specimens were centrally reviewed by a dedicated thyroid pathologist (AE) in accordance with the WHO classification (4th edition) [20]. In case of discordance with the local diagnosis, an additional dedicated thyroid pathologist (BK) was consulted to reach consensus. Local and central histopathologists were blinded to the [18F]FDG-PET/CT result; the two central pathologists were also blinded to the local histopathological diagnosis. Incidentally detected (micro)carcinomas located outside the index nodule were not considered for the main outcome measure. Immunohistochemistry Indirect chromogenic immunohistochemical staining was performed on 5 mm thick paraffinembedded tissue sections on coated slide glasses. We used primary antibodies against GLUT1 (RB-9052-P, Thermo Fisher Scientific, Waltham, MA, USA; dilution 1:500), GLUT3 (RB-9096-P, Immunologic, WellMed BV, Duiven, the Netherlands; 1:300), GLUT4 (ab654, Abcam, Cambridge, United Kingdom: 1:1000), HK1 (MA5-15680, Invitrogen™, Thermo Fisher Scientific; 1:7500), HK2 (MA5-15679, Invitrogen™, Thermo Fisher Scientific; 1:200), HIF1a (ab2185, Abcam; 1:250), MCT4 (anti SLC16A4, HPA046986, Sigma-Aldrich®, Saint Louis, MO, USA; 1:200), CA-IX (NB100-417, Novus Biologicals™, Bio-techne, Centennial, CO, USA; 1:250), VEGF (555036, Pharmingen™, Becton Dickinson Biosciences, San Diego, CA, USA; 1:100), NIS (ABC1453, Merck Millipore, Burlington, MA, USA; 1:2000), and Ki-67 (M724001, DAKO Agilent, Santa Clara CA, USA; 1:25).
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