PurposeThe definition of tumor boundaries in newly diagnosed glioma is crucial for optimal local therapies and/or non-invasive tumor characterization. The widespread use of [18F]FDOPA PET in this setting is hampered by the lack of a histologically validated standardized method. This study aimed to determine the best way to delineate the overall tumor volume in untreated gliomas regarding diagnostic performance and reliability.MethodsWe retrospectively analyzed 33 biopsy sites and 33 biopsy sites and 33 contralateral virtual biopsies, from fourteen patients. Six sets of 10 minutes images were reconstructed starting at three different time points (10, 20, and 30 minutes post-injection) using two sets of features for the iterative algorithm. Four reference regions (“hemispheric,” “banana-shaped,” “striatum” and “white matter”) were drawn independently by four readers. A step-by-step selection was applied based on areas under the ROC curve (AUC) for the identification of tumor tissue, intraclass correlation coefficients (ICC) across reconstruction features, and inter-reader agreement. The best cut-off value was calculated as maximizing Youden's index.ResultsTumor infiltration was confirmed in all biopsy samples. TargetSUVmax-to-StriatumSUVmean (TSRmean) and TargetSUVmax-to-StriatumSUVmax (TSRmax) on images performed at 20 minutes provided the best overall performances with AUCs = 0.91, inter-algorithm ICCs = 0.99 and inter-reader Fleiss’ kappas = 0.99 for both. Optimal threshold values of TSRmean20–30 = 0.90 and TSRmax20–30 = 0.68 provided sensitivities of 85% and 83%, respectively, and specificities of 86% for both.ConclusionThis study completes the current EANM guidelines and is a step forward in standardizing the delineation methods in untreated glioma patients. TSRmean20–30 or TSRmax20–30 provided the best combination of accuracy and reliability.