Clinicians and scientists interested in brain tumor patients and bevacizumab treatment The current standard of care for recurrent glioblastoma (GBM) involves treatment with the anti-vascular endothelial growth factor (VEGF) drug bevacizumab. Curiously, a subset of patients develops focal regions of diffusion restriction following the onset of therapy. Conflicting studies have shown these regions to be either diffusion restricted necrosis (DRN)¹ or exclusively hypercellular tumor². One recent manuscript reports regions of both, where the DRN was shown to have significantly lower apparent diffusion coefficient (ADC) values³. This study explores regions of bevacizumab induced diffusion restriction in human whole brain samples and determines appropriate ADC cutoffs for differentiating DRN from viable hypercellular tumor. An additional population analysis shows overall survival consequences dependent on whether these lesions appear and whether they progress.Part 1: (histological validation): Patient Population Five patients with recurrent GBM and regions of focal diffusion restriction (while on bevacizumab therapy) were recruited for brain donation and enrolled into this study. Imaging Prior to death patients were clinically imaged using diffusion weighted imaging (DWI), T1 pre and post gadolinium, and T2 weighted images. Apparent diffusion coefficient (ADC) maps were calculated from DWI. Histology Processing Post-mortem pathological slicing of the brains was done using customized slicing jigs 3d-printed for each patient. Areas of diffusion restriction were defined using radiological criteria and histological samples were taken from regions co-localized with diffusion restriction. Samples were paraffin embedded, H&E stained, and digitized for interpretation. Each sample was graded to differentiate necrotic regions from viable tumor. Pathologist-confirmed coagulative necrosis and hypercellularity were then compared to MR imaging. Figure 1 shows one patient’s scans compared to matching brain slice with targeted section sampled and stained. Figure 1 also shows a comparison of regions within the diffusion restriction zone and directly neighboring regions. ADC values from regions of diffusion restricted necrosis were compared to those classified as hypercellular. An ROC analysis was performed iteratively adjusting ADC cut-off values until a maximal area under the curve (AUC) was obtained for each patient. Part 2 (population study): Patient Population 64 patients undergoing bevacizumab treatment for recurrent GBM were retrospectively analyzed to determine if diffusion restricted lesions developed following treatment onset. Patients were separated into three groups, no diffusion restriction developed (NDR), diffusion restriction that progressively grew within 3months (PrDR), and patients that developed diffusion restriction that was stable for at least 3 months (StDR). The overall survival (OS) was then compared using Kaplan-Meier curves and a log-rank test.Part 1: Regions of diffusion-restricted necrosis had significantly lower ADC values than nearby hypercellularity. The average ADC threshold (shown in table 1) that best differentiated these tissue types was 0.6996x10-3mm2/s corresponding to an average AUC of 0.7364. In four of the five patients, the diffusion-restricted necrosis was progressively growing at the time of the patient’s final scan. The necrotic regions were surrounded by viable hypercellular non-enhancing tumor. The patient without progressively growing diffusion restriction had recurrent tumor elsewhere, not directly adjacent to the diffusion restriction. Part 2: Figure 3 shows the survival curves comparing NDR, PrDR and StDR, where StDR patients had a significantly greater OS than the NDR group, while the PrDR group significantly lower OS than the NDR group (p<0.05). It should be noted that at the time of death, all diffusion-restricted lesions were expanding for both the StDR and PrDR groups.We pathologically confirmed that progressively expanding diffusion restriction in patients undergoing bevacizumab treatment indicated coagulative necrosis surrounded by viable hypercellular tumor. We also determined an optimal ADC cutoff for differentiating diffusion-restricted necrosis from hypercellular tumor. Population-wise, patients with progressively growing regions of diffusion restriction have decreased overall survival suggesting the lesions themselves are necrosis surrounded by viable tumor when expanding. Patients with stable lesions however showed increased OS over the NDR group. Further research is necessary to establish the biological basis for bevacizumab causing these lesions.