2-Hydroxyglutarate (2HG) is elevated in gliomas harboring IDH1/2-mutations, suggesting the potential of 2HG as a biomarker for the diagnosis of IDH-mutant gliomas^1-2. A recent development of 1H MRS for in-vivo detection of 2HG has provided a major breakthrough in cancer research^3-4. Development of therapeutics to tackle 2HG production would require a preclinical model system. Thus development of in-vivo MRS methodologies for precise measurement of 2HG in IDH-mutant mouse model of gliomas, and further application of 2HG optimized MRS method to evaluate the efficacy of 2HG or IDH-inhibiting drugs would provide a clinical advancement for therapeutic strategies. In-vivo measurement of 2HG is of high significance, but accurate evaluation in-vivo remains challenging, especially when 2HG elevation is moderate. Hence the present study was undertaken to develop PRESS MRS Method for 2HG detection with enhanced precision in Patient-Derived Xenograft Orthotropic Mouse model (PDX). Methodology was further extended to detect 2HG and metabolic profile with tumor-progression in PDX-mice.Volume-localized density-matrix simulations were performed to optimize echo time (TE1 and TE2) of PRESS for 2HG detection, and to create the model spectra of metabolites for LCModel spectral fitting. Several PDX-mouse lines were generated from the inoculum of tumor cells from different IDH-mutant patients. In-vivo MRS data was obtained from PDX mice (n=7), and wild-type (n=3) at 9.4T horizontal-bore animal MR-scanner (Agilent) using a home-built 15mm-diameter single-loop transmit/receive surface-coil. The voxel localization was achieved with a 550μs 90°-RF pulse (bandwidth=10.7 kHz) and two 3.0ms 180°-RF pulses (bandwidth=5.4 kHz), at an RF-field intensity of 60μT. In-vivo PRESS MRS was acquired at short-TE (TE1=8ms, TE2=6.7ms), and long-TE (TE1=19ms, TE2=77ms) with signal-averages=128 and 512 respectively (TR=3s). 2HG MRS with tumor-progression was conducted at intervals of ~10-12 days over a period of 35-days after tumor establishment. Spectral-fitting was performed with LCModel software⁵, using in-house basis spectra calculated upon incorporating the volume-localizing RF and gradient pulses of PRESS. Following the normalization of LCModel estimates to water signal, metabolite concentration was estimated with reference to total creatine at 8 mM.Volume-localized numerical simulations indicated that strongly-coupled 2HG signal at 2.25-ppm depends on PRESS sub-echo times, TE1 and TE2 (Fig.1A(1)). The signal had positive polarity at short-TE (=TE1+TE2< 40ms) but inverted at TE>60ms and <110ms, for which TE1 is shorter than TE2. Maximum amplitude was obtained for TE=96ms (TE1=19ms and TE2=77ms). Numerical calculation at TE1=8ms, TE2=6.7ms (Fig.1A(2)) for signal strength and pattern for 2HG, glutamate (Glu), GABA, and glycine showed that 2HG signal is masked at short-TE while Fig.1A(3) depicts that at TE=96ms, the 2HG signal is clearly discernible as it shows negative-polarity, while neighboring signals have positive-polarity. In-vivo spectra obtained with 2HG-optimized PRESS TE=96ms from IDH-mutant mice glioma showed a large inverted peak at 2.25-ppm, which was well reproduced by the LCModel fit generated using basis set with 2HG, and was estimated to be 26.1mM (CRLB=1%) (Fig.1B(1)). 2HG was detected in all of the 7 IDH-mutant gliomas using optimized PRESS, the level ranging from 1.6±0.25 to 26.1±0.26 mM. IDH-mutation was confirmed with IDH-immunohistochemistry (Fig.1B(2)). tCho which was found to be 2.8±0.25 mM in wild-type mouse brain was increased to 11±4 mM in the IDH-mutant gliomas. In-vivo PRESS spectrum obtained from a low-2HG PDX tumor line at long-TE resulted in a residual at 2.25-ppm when fitted to basis set without-2HG (Fig.2A(2), marked with arrow), indicating that inverted-signal at 2.25-ppm was primarily attributed to 2HG H4-resonances. However, when short-TE in-vivo spectrum was fitted with and without-2HG, the residuals obtained were about the same in both cases, indicating that the 2HG signal detection at short-TE is not very selective. Moreover, absence of 2HG in the basis set altered the estimation of neighboring metabolites (Fig.2A-B). Application of 2HG optimized long-TE PRESS-method at different stages of glioma growth showed that amount of 2HG became precisely detectable (2.4mM, CRLB=7%) in smaller tumor volume after 20-days of its establishment, and rose to 4mM (CRLB=4%) after 35-days (Fig.3), indicating the potential of the method to pinpoint the exact stage for application of 2HG-inhibiting drugs based upon 2HG kinetics. Mean 2HG and tCho level in low-2HG cohort of mice rose from 1.7±1 to 3.3±0.6 mM, and 2.1±0.4 to 8.3±2.0 mM respectively with tumor growth, while NAA (11.7±0.5 to 5.2±1.6 mM, p=0.01), and Glu (7.3±0.5 to 4.5±0.6 mM, p=0.007) showed significant reduction suggestive of altered metabolic-homeostasis. At 9.4T, PRESS TE=96ms gives an inverted 2HG signal at 2.25-ppm with minimal interference from neighboring resonances, thus provides selective and improved detection of 2HG compared to short-TE MRS, and can be effectively used for in-vivo evaluation of drugs targeted to treat IDH-mutant gliomas.