Spectroscopy Voxel-wise IDH-1 Mutation and Non-Enhancing Component of Glioblastoma Daniel M Fountain1, Timothy J Larkin1,2, Natalie R Boonzaier1,2, Jiun-Lin Yan1, and Stephen J Price1,2 1 The Brain Tumour Imaging Laboratory, Division of Neurosurgery and 2 Wolfson Brain Imaging Centre, University of Cambridge, United Kingdom Introduction Isocitrate dehydrogenase 1 gene (IDH-1) mutations have been shown to be present in over 80% of secondary glioblastoma (GBM), but only in 5-12% of primary GBM.1 IDH-1 mutated tumors show increased survival benefit compared to wild-type tumors following further resection of non-enhancing disease.2 We investigated the non-enhancing lesion to identify intrinsic differences between IDH-1 mutated and wild-type tumors using multimodality imaging data. Hypothesis – Non-enhancing components of IDH-1 mutated and wild-type GBM differed significantly. Methods 54 patients were imaged on a Siemens 3T Magnetom Trio with conventional anatomical, diffusion tensor (DTI), dynamic susceptibility contrast perfusion (DSC) and 1H chemical shift imaging (CSI) performed. Of the 54 patients, 8 were diagnosed with IDH-1 mutated GBM. All images were coregistered to the T2-weighted image using the FLIRT toolbox in FSL. The fluid attenuated inversion recovery (FLAIR) region of interest (ROI) was determined excluding the contrast-enhancing tumor and necrosis, thus forming the non-enhancing component for analysis (Figure 1). Analysis was performed at three levels of data (Figure 2): The first and second levels applied a mixed model design to DTI voxelwise and spectroscopy voxelwise data (Figure 2a and 2b respectively). The third level of analysis approached the FLAIR VOI as a whole by averaging all variables across the total volume and modelling using a generalised linear model (Figure 2c). Significance was assessed at the 95% level with Bonferroni correction for multiple testing. Area under the receiver operator characteristics curve (AUC) was computed following multiple logistic regression. All statistical analysis was performed in 64-bit R version 3.0.2.  Figure 1 – Multi-modality data for Patient 1 (IDH-1 wild-type). Data available across all axial slices are shown for each variable of interest. FLAIR ROI – Fluid attenuated inversion recovery peritumoral region of interest, p – isotropic component, q – anisotropic component, rCBV – relative cerebral blood volume, Cho – choline, NAA – N-acetyl-aspartate, mI – myo-inositol, Cr – creatine, Glu – glutamate and glutamine, GSH – glutathione. Figure 2 – Three levels of resolution of imaging data for statistical analysis. A) Voxel-wise DTI data with 2 × 2 × 5mm voxels. B) Voxel-wise spectroscopy data with 10 × 10 × 15-20mm. C) Imaging data averaged over the whole region of interest to provide a single value per variable per patient for statistical analysis. Variable DTI Voxel-wise Spectroscopy Voxel-wise FLAIR ROI Estimate SE t p rCBV 0.096 0.364 0.265 0.775 -0.285 0.348 -0.818 0.384 0.153 0.347 0.440 0.662 0.093 0.103 0.908 0.328 -0.007 0.122 -0.056 0.960 0.101 0.110 0.920 0.363 q -0.087 0.070 -1.248 0.182 -0.148 0.091 -1.637 0.081 -0.105 0.060 -1.747 0.088 Cho:NAA 0.557 0.241 2.311 0.016* 0.801 0.317 2.524 0.008** 0.510 0.236 2.157 0.037* mI:Cr 0.112 0.279 0.400 0.665 0.193 0.277 0.698 0.442 0.179 0.268 0.668 0.508 Cho:Cr 0.190 0.068 2.781 0.004** 0.205 2.525 0.197 2.909 0.006** NAA:Cr 0.003 0.126 0.027 0.977 -0.064 0.131 -0.490 0.571 -0.034 0.127 -0.270 0.789 Glu:Cr -0.603 0.255 -2.359 0.014** -0.878 0.343 -2.557 0.007** 0.211 0.115 1.838 0.073 GSH:Cr -0.293 0.177 -1.656 0.078 -0.369 0.185 -1.992 0.029* -0.195 -1.544 0.130 Results Across all patients, a total of 1,300,632 DTI voxels with data for individual p, q, and rCBV were included with 520 spectroscopy voxels. All statistical results are shown in Table 1. No significant difference in rCBV between IDH-1 wild-type and mutated patients (Estimate -0.285 – 0.153, p = 0.384 – 0.775), or Isotropic (p) and Anisotropic (q) Components (p estimate -0.007 – 0.101, p = 0.328 – 0.960 and q estimate -0.087 – -0.148 p = 0.081 – 0.182) Non-enhancing component showed significant increases in choline relative to creatine in IDH-mutated tumors (Estimate 0.190 – 0.205, p = 0.004-0.008), and associated choline relative to N-acetyl-aspartate (Estimate 0.510–0.801, p = 0.008–0.037) Significantly lower levels of glutamate and glutamine were observed relative to creatine in patients with an IDH-1 mutation (Estimate -0.603 – -0.878, p = 0.007–0.014) Combining these findings with age provided an area under the curve (AUROC) of 0.943. Table 1 – Results of Voxel-wise and ROI multivariable statistical analysis of R132H mutation status. Discussion We propose two hypotheses to explain these findings and the improved survival in patients with IDH-1 mutated tumors: Invasive margins of IDH-mutated tumors are more sensitive to treatment. This correlates with in vitro findings,3 and in vivo IDH-1 mutated tumours with a higher Cho/NAA in the peritumoral margin may be more sensitive to cytotoxic therapies.4 Reduced concentration of glutamate and glutamine identified in IDH-1 mutated tumors is neuroprotective. Glutamate is known to be released in excitotoxic concentrations in glioma cells and in GBM, and is involved in the mechanism of glioma invasion.5 1 Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2 Mutations in Gliomas. N Engl J Med. 2009;360:765–773. 2 Beiko J, Suki D, Hess KR, et al. IDH1 mutant malignant astrocytomas are more amenable to surgical resection and have a survival benefit associated with maximal surgical resection. Neuro-Oncol. 2014;16:81–91 3 Mohrenz IV, Antonietti P, Pusch S, et al. Isocitrate dehydrogenase 1 mutant R132H sensitizes glioma cells to BCNU-induced oxidative stress and cell death. Apoptosis Int J Program Cell Death.2013;18(11):1416-1425 4 Price SJ, Young AHM, Scotton WJ, et al. Metabolic activity of the invasive microenvironment of glioblastomas determines time to progression: a multimodal MR study. Proc Intl Soc Mag Reson Med. 2015;23:2252 5 Sontheimer H. A role for glutamate in growth and invasion of primary brain tumors. J Neurochem. 2008;105(2):287-295