The purpose of this study was to develop and apply strategies for monitoring serial changes in parameters derived from H-1 MRSI for patients with newly diagnosed GBM being treated with combination therapy. This is particularly important for situations where anti-angiogenic agents such as bevacizumab are included because they cause a reduction in the size of the lesion observed with post-gadolinium T1-weighted images and mean that the assessment of response depends upon the identification and evaluation of non-enhancing tumor¹. Thirty-one patients being treated with radiation (RT), temozolomide, erlotinib and bevacizumab as part of a single institution Phase II clinical trial, received serial 3T scans. Anatomic imaging sequences comprised FLAIR T2-weighted, pre- and post-gadolinium T1-weighted scans. Lactate edited 3D H-1 MRSI data were obtained with CHESS water suppression, VSS outer volume suppression and PRESS volume selection (TE=144ms, TR= 1.3s, acquisition matrix of 16x16x16 or 18x18x16 and nominal spatial resolution =1cm3). Fly-back trajectories were applied in the S/I dimension as described previously². The contrast enhancing (CEL) and T2 lesion were defined from anatomic images. The spectral data were processed³ to provide levels of choline (Cho), creatine (Cre), N-acetylaspartate (NAA), lactate (Lac) and lipid (Lip), as well as indices such as the CNI that describes the increase in Cho and decrease in NAA relative to values from normal appearing brain tissue⁴. Serial changes in imaging parameters were evaluated using Wilcoxon rank sign tests and association with progression free survival (PFS) and overall survival (OS) was assessed using Cox proportional hazards analysis, taking into account patient age, extent of resection and KPS.As reported in other clinical studies⁵, the PFS for these patients was longer (median 404 days, 27 events, 4 censored) but the OS was similar to patients receiving standard of care (median 603 days, 23 events, 8 censored). The mean T2 lesion volume decreased from 36.2cm3 at pre-RT to 15.3cm3 (post-RT, p<0.0001) and to 14.7cm3 (Fup1, p=0.0002). The mean CEL volume decreased from 5.2 cm3 (pre-RT) to 3.0 cm3 (mid-RT, p=0.018), 2.1 cm3 (post-RT,p=0.0002) and 0.7cm3 (Fup1, p <0.0001). Figure 1 shows changes in metabolite parameters from these first 4 exams for sub-populations with PFS either greater or less than the median value. Overall these metrics of abnormal metabolism showed a continued reduction with time, and values for the sub-group with longer PFS were lower than those with shorter PFS. Figures 2 and 3 show examples of serial anatomic images and overlays of CNI maps from baseline to the time of progression for 2 subjects with similar PFS but different OS. Note that there was minimal residual enhancement for both subjects and the clinical assessment of treatment response relied upon changes in the T2 lesion. The metabolic (CNI) lesion at the time of recurrence was relatively small for patient A (OS=706 days), but much larger and increasing in size for patient B (OS=434 days). When considering the population as a whole, a number of imaging parameters were associated with PFS and OS. These included the volume of the CEL at mid-RT (p=0.025 for PFS and p=0.039 for OS) but not other time points. For metabolic parameters such as the volume with CNI>2, the sum(CNI), sum(Lac/nNAA) and sum(Lip/nNAA) values from the Fup1 examination showed strong associations with PFS and OS (see Table 1). There was no association of the T2 lesion volume with outcome detected these time points. The RANO criteria for assessing response to therapy in patients with GBM depend upon changes in the CEL, with secondary evaluation of the T2 lesion⁶. This is problematic for treatments including anti-angiogenic agents because the enhancing portion of the lesion may disappear, leaving behind a mixture of non-enhancing tumor and non-specific treatment effects. An alternative approach to assessing tumor burden is to use 3-D H-1 MRSI to monitor changes in regions with abnormal metabolism. The results of this study indicate that maps of the CNI were able to visualize temporal changes in the spatial extent of tumor and that metrics based upon CNI values and levels of Lac and Lip within the region with abnormal CNI are able to predict outcome. Integrating 3D H-1 MRSI and the metabolic parameters that they provide into serial MR scans of patients with GBM would be helpful for resolving ambiguities cause by non-specific treatment effects and for determining at an early stage whether alternative therapies should be considered.