Supplementary Data s7

Supplementary Data

Table 1S. Baseline metabolite levels relative to creatine. Table 2S. Demographic, genomic, molecular, histological, radiological and clinical data. Table 3S. Fractional changes for all imaging biomarkers. Table 4S. General Linear Model with change of KPS as dependent variable and grade, age and fractional volume of decreased 2HG/Cre as covariates. Figure 1S. 3D MRSI pulse sequence for 2HG imaging, detailed diagram. Figure 2S. Quantum mechanics simulations of 2HG spectral editing for MEGA- LASER, MEGA-PRESS and PRESS-97 sequences. Figure 3S. Simulations of 2HG editing for MEGA-LASER and PRESS-97 sequences in the presence of B1 field inhomogeneity. Figure 4S. Mitigation of subtraction artifacts caused by motion and scanner instability in J-difference MR spectroscopy using real-time prospective motion correction, shim update and reacquisition. Figure 5S. Metabolic maps before image interpolation. Figure 6S. Examples of OFF and difference (DIFF) MEGA-LASER spectra from 3D MRSI voxels located in the tumor and contralateral normal appearing white matter, pre- and post-treatment respectively. Figure 7S. Distributions of 2HG/Cre in the test-retest and post-pre longitudinal data sets. Figure 8S. Comparison of functional spectroscopic maps (fSM) and functional diffusion maps (fDM). Figure 9S. Relation between residual tumor measured by FLAIR hyperintensity volume and the SNR and CRLB of 2HG. Notes. Details related to data analysis in Figures 7S and 9S.

1 Table 1S. Pre-treatment metabolite levels in all 25 patients. Patients had: (1-9) pre/post-treatment scans, (10-21) only pre-treatment scan, (22-25) gross total resection of tumor and 2HG was not detectable.

IDH1 2HG/ Glx/ Lac/ GPC/ NAA/ Patient # Grade Type mutation Cre Cre Cre Cre Cre Pt_01 2 A R132G 0.31 1.21 0.23 0.32 1.02 Pt_02 2 OA R132H 0.35 1.35 0.35 0.18 0.93 Pt_03 3 AA R132H 0.39 1.06 0.62 0.45 0.73 Pt_04 3 AA R132S 0.70 0.93 0.86 0.40 0.69 Pt_05 3 AA R132H 0.31 0.58 0.24 0.55 0.76 Pt_06 3 AOD R132H 0.40 0.77 0.17 0.32 0.65 Pt_07 3 AOA R132H 0.22 1.29 0.30 0.19 0.86 Pt_08 4 GBM R132H 0.31 0.99 0.29 0.44 0.71 Pt_09 4 GBMO R132H 0.23 1.38 0.09 0.17 1.10 Pt_10 2 OD R132H 0.17 0.84 0.13 0.44 0.95 Pt_11 3 AOD R132H 0.37 0.89 0.23 0.32 0.90 Pt_12 2 A R132H 0.30 1.76 0.30 0.28 0.95 Pt_13 4 GBM R132H 0.57 0.96 0.25 0.27 0.99 Pt_14 3 AA R132H 0.16 1.46 0.23 0.14 0.60 Pt_15 4 GBM R132H 0.27 1.20 0.37 0.40 0.90 Pt_16 3 AOD R132H 0.86 0.87 0.91 0.34 1.18 Pt_17 3 AOA R132H 0.35 1.65 0.36 0.35 0.90 Pt_18 3 AA R132H 0.18 1.35 0.24 0.19 1.11 Pt_19 3 AA R132H 0.19 1.76 0.93 0.27 1.13 Pt_20 2 A R132H 0.54 1.40 0.37 0.22 1.45 Pt_21 3 AA R132H 0.47 1.94 1.59 0.29 0.84 Pt_22 3 AOA R132H - 1.86 0.18 0.57 1.57 Pt_23 3 AA R132C - 1.39 0.17 0.25 0.87 Pt_24 2 A R132H - 1.23 - 0.24 1.13 Pt_25 2 OA R132H - 1.18 0.29 0.13 1.22

2 Table 2S. Demographic, genomic, molecular, histological, radiological and clinical data in patients with follow up scans and positive 2HG. A=Astrocytoma, AA/OA=Anaplastic/Oligo astrocytoma, AOD/A=Anaplastic oligo- dendroglioma/astrocytoma, GBM(O)=Glioblastoma (with oligodendroglial component).

Patient Age Grade Glioma IDH Molecular Tumor location Treatment Steroids KPS RANO # type mutation markers Pre/Post Left posterior 1 33 WHO-II A R132G - RT 0 90/100 PR cingulum/callosum 2 38 WHO-II OA R132H 19q only Right parietal RT 0 100/100 SD Left 3 33 WHO-III AA R132H - RT 0 90/90 SD frontal/temporal 4 42 WHO-III AA R132S - Left frontal RT 0 50/80 MR 5 38 WHO-III AA R132H 19q only Right parietal RT 0 90/80 SD 1p/19q, 6 45 WHO-III AOD R132H Right frontal RT 0 90/100 SD MGMT 7 46 WHO-III AOA R132H 1p/19q Left temporal CT 0 90/80 MR 4mg qD 8 63 WHO-IV GBM R132H - Left frontal RT 100/60 PD (stable) 9 55 WHO-IV GBMO R132H 1p/19q Right frontal CT 0 90/90 SD

3 Table 3S. Fractional changes for all imaging biomarkers.

Patient 2HG/Cre GLX/ tCho/Cre Lac/Cre NAA/Cre tCho/NAA A Volume Volume 2HG 2HG ADC ADC # Cre D FLAIR 2HG Vd/Vt Vi/Vt Vd/Vt Vi/Vt C

1 -37.37 -19.8 -11.5 -12.2 -4.7 -10.4 - 39.90 -50.09 60.01 0.00 - - 2 -69.32 -13.7 -51.6 -29.0 -0.7 -50.9 2 -15.31 -51.87 58.23 24.58 0.65 1.42 . 7 2 3 -54.01 17.2 -17.5 -11.0 6.1 -32.2 - 6.90 -61.55 76.74 0.86 - - 4 -65.39 -25.7 7.4 -20.0 -18.1 23.5 5 -16.45 -34.61 99.12 0.00 5.11 45.27 . 7 6 5 2.88 24.2 -0.3 -19.8 16.6 -30.3 - -14.37 -32.15 24.82 8.75 - - 6 -34.34 -26.6 13.8 8.8 3.2 11.99 - -9.35 -14.86 60.76 1.09 - - 7 -48.09 0.1 2.9 -12.5 -19.0 24.96 6 -8.80 -42.53 49.11 3.29 3.04 22.75 . 7 6 8 -22.30 -1.0 -29.8 0.8 -2.3 -27.6 - 69.80 -26.29 19.78 7.95 7.01 15.22 1 . 3 3 9 -48.89 -5.3 -13.2 25.9 -2.7 23.3 - -11.85 -37.70 58.01 0.00 0.31 4.63 2 . 6 6

4 Table 4S. General Linear Model of KPS with grade, age and fractional volume of decreased 2HG/Cre as covariates.

Patient # Grade Age 2HG/Cre dKPS Vd/Vt (<-0.11) (KPSpost – KPSpre) 1 2 33 60.01 10 2 2 38 58.23 0 3 3 33 76.74 0 4 3 42 99.12 30 5 3 38 24.82 -10 6 3 45 60.76 10 7 3 46 49.11 -10 8 4 63 19.78 -40 9 4 55 58.01 0 P-value 0.72317 0.74637 0.015248 R2 = 0.82, aR2 = 0.71 dKPS = 0.61Vd - 0.24Age - 3.48Grade - 14.82

5 Figure 1S. 3D MRSI pulse sequence for 2HG imaging. MEGA-LASER is used to edit 2HG signal at 4.02ppm. More details are given in Ref (1).

6 Figure 2S. GAMMA (2) quantum mechanics simulations of 2HG editing for MEGA-LASER, MEGA-PRESS (3) and PRESS-97 (4). Experimental pulses, 2HG spin system (5), and relaxation (T2 = 180ms) were considered (6).

7 Figure 3S. Simulations of 2HG editing for MEGA-LASER and PRESS-97 sequences in the presence of B1 field inhomogeneity (15% from the nominal value).

8 Figure 4S. Mitigation of subtraction artifacts caused by motion and scanner instability in J-difference MR spectroscopy using real-time prospective motion correction, shim update and reacquisition.

9 Figure 5S. Metabolic maps before image interpolation. The same intensity range is shown in both pre- and post-treatment maps.

10 Figure 6S. Examples of OFF and difference (DIFF) MEGA-LASER spectra from tumor and contralateral normal appearing white matter (fit red, experimental black, 3 Hz line broadening).

11 Figure 7S. Distributions of 2HG/Cre in test-retest (A) and longitudinal (B) data sets. Normality of distributions was verified by Kolmogorov-Smirnov test (red line, further details in Notes).

12 Figure 8S. Comparison of functional spectroscopic maps (fSM) and functional diffusion maps (fDM). Regions of decreased 2HG/Cre partially overlap regions of increased ADC.

13 Figure 9S. Relation between residual tumor measured by FLAIR volume and the SNR and CRLB of 2HG (further details in Notes).

14 Notes: 1) In Figure 7S the threshold of 0.115 for significant 2HG/Cre change was determined from the test-retest 95% CI, and corresponds to a change in 2HG level of approximately 1mM assuming total Cre (creatine + phosphocreatine) levels of 8-10 mM (7). 2) In Figure 9S there is a trend for positive correlation between the SNR and FLAIR volume, and a trend for negative correlation between relative CRLB and FLAIR volume. The correlations are not statistical significant (P > 0.05) and can be explained by a range of biological factors (efficiency of 2HG production, tumor cell density and metabolic activity do not scale with FLAIR hyperintensity) and methodological factors (linewidths and shim quality influence the signal maximum intensity while residual unfitted signal can amplify the noise estimation). 3) SNR is underestimated by LCModel that overestimates noise by considering the entire residual (experimental-fit), which often includes unfitted signal. 4) The upper threshold for relative CRLB was set to 25%. While the proper CRLB threshold and metric for quality of fit of MRS is still a matter of debate among experts, investigators mostly agree on excluding metabolites with CRLB larger than 50%. Metabolites with CRLB less than 20% are generally considered very well fitted, CRLB less than 30% acceptable, and CRLB between 30%-50% as poor fits but potentially acceptable in a given context (8-12). In particular, for multiplet lines of spin coupled metabolites the CRLB threshold may be higher than for singlet lines of uncoupled spins. 1)

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