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Simple Summary: For the treatment of high-grade gliomas, radiolabeled amino acid PET/CT could allow for a better tumor delineation for radiotherapy planning and to target aggressive tumor areas for radiotherapy dose escalation guiding. The aim of this ancillary study from the IMAGG prospective trial is to demonstrate a spatial similarity between the areas of high uptake on 18F-FET PET/CT before radio-chemotherapy (MTV), the residual tumor on post-therapy NADIR MRI (GTV 2), and the area of recurrence on MRI (GTV 3). These results on 23 patients showed modest similarity indices between MTV, GTV 2, and GTV 3. Nevertheless, their similarities improved in patients who underwent only biopsy or partial surgery. Delineation methods based on TBR ≥ 1.6 and 80–90% SUVmax showing a good agreement in the hotspot concept for targeting standard dose and radiation boost. The standard therapy strategy for high-grade glioma (HGG) is based on the maximal surgery followed by radio-chemotherapy (RT-CT) with insufficient control of the disease. Recurrences are mainly localized in the radiation field, suggesting an interest in radiotherapy dose escalation to better control the disease locally. We aimed to identify a similarity between the areas of high uptake on O-(2-[18F]-fluoroethyl)-L-tyrosine (FET) positron emission tomography/computed tomography (PET) before RT-CT, the residual tumor on post-therapy NADIR magnetic resonance imaging (MRI) and the area of recurrence on MRI. This is an ancillary study from the IMAGG prospective trial assessing the interest of FET PET imaging in RT target volume definition of HGG. We included patients with diagnoses of HGG obtained by biopsy or tumor resection. These patients underwent FET PET and brain MRIs, both after diagnosis and before RT-CT. The follow-up consisted of sequential brain MRIs performed every 3 months until recurrence. Tumor delineation on the initial MRI 1 (GTV 1), post-RT-CT NADIR MRI 2 (GTV 2), and progression MRI 3 (GTV 3) were performed semi-automatically and manually adjusted by a neuroradiologist specialist in neuro-oncology. GTV 2 and GTV 3 were then co-registered on FET PET data. Tumor volumes on FET PET (MTV) were delineated using a tumor to background ratio (TBR) ≥ 1.6 and different % SUVmax PET thresholds. Spatial similarity between different volumes was performed using the dice (DICE), Jaccard (JSC), and overlap fraction (OV) indices and compared together in the biopsy or partial surgery group (G1) and the total or subtotal surgery group (G2). Another overlap index (OV') was calculated to determine the threshold with the highest probability of being included in the residual volume after RT-CT on MRI 2 and in MRI 3 (called "hotspot"). A total of 23 patients were included, of whom 22% (n = 5) did not have a NADIR MRI 2 due to a disease progression diagnosed on the first post-RT-CT MRI evaluation. Among the 18 patients who underwent a NADIR MRI 2, the average residual tumor was approximately 71.6% of the GTV 1. A total of 22% of patients (5/23) showed an increase in GTV 2 without diagnosis of true progression by the multidisciplinary team (MDT). Spatial similarity between MTV and GTV 2 and between MTV and GTV 3 were higher using a TBR ≥ 1.6 threshold. These indices were significantly better in the G1 group than the G2 group. In the FET hotspot analysis, the best similarity (good agreement) with GTV 2 was found in the G1 group using a 90% SUVmax delineation method and showed a trend of statistical difference with those (poor agreement) in the G2 group (OV' = 0.67 vs. 0.38, respectively, p = 0.068); whereas the best similarity (good agreement) with GTV 3 was found in the G1 group using a 80% SUVmax delineation method and was significantly higher than those (poor agreement) in the G2 group (OV'= 0.72 vs. 0.35, respectively, p = 0.014). These results showed modest spatial similarity indices between MTV, GTV 2, and GTV 3 of HGG. Nevertheless, the results were significantly improved in patients who underwent only biopsy or partial surgery. TBR ≥ 1.6 and 80–90% SUVmax FET delineation methods showing a good agreement in the hotspot concept for targeting standard dose and radiation boost. These findings need to be tested in a larger randomized prospective study. [ABSTRACT FROM AUTHOR]