Detection of 2-hydroxyglutarate in IDH-mutated glioma patients by in vivo spectral-editing and 2D correlation magnetic resonance spectroscopy

Abstract

Mutations in the gene isocitrate dehydrogenase 1 (IDH1) are present in up to 86% of grade II and III gliomas and secondary glioblastoma. Arginine 132 (R132) mutations in the enzyme IDH1 result in excess production of the metabolite 2-hydroxyglutarate (2HG), which could be used as a biomarker for this subset of gliomas. Here, we use optimized in vivo spectral-editing and two-dimensional (2D) correlation magnetic resonance spectroscopy (MRS) methods to unambiguously detect 2HG noninvasively in glioma patients with IDH1 mutations. By comparison, fitting of conventional 1D MR spectra can provide false-positive readouts owing to spectral overlap of 2HG and chemically similar brain metabolites, such as glutamate and glutamine. 2HG was also detected using 2D high-resolution magic angle spinning MRS performed ex vivo on a separate set of glioma biopsy samples. 2HG detection by in vivo or ex vivo MRS enabled detailed molecular characterization of a clinically important subset of human gliomas. This has implications for diagnosis as well as monitoring of treatments targeting mutated IDH1.

Fig. 1

2D LASER-COSY and 1D MEGA-LASER spectra from brain phantoms at 3 T, with 3×3×3 cm³ voxels used in all measurements. (A) Overlay of 2D LASER-COSY spectra from a phantom containing a mixture of normal brain metabolites (red contours) and a phantom where 2HG was added to the mixture of normal brain metabolites (blue contours). The H_α-H_β crosspeak of 2HG is at 4.02/1.91 (δ₂/δ₁) ppm. (B) Overlay of 1D MEGA-LASER from the same phantoms. The position of Hα peak of 2HG at 4.02 ppm lines with the crosspeak in the 2D spectrum above (dashed line). (C) Intensity of 2HG signal in 2D LASER-COSY and 1D MEGA-LASER at different 2HG concentrations (error bars represent one standard deviation of two independent measures, signal intensity is normalized (I_norm) to the maximum intensity). Other metabolites shown: choline (Cho), glutamate (Glu), lactate (Lac), myo-inositol (Myo), N-acetyl-aspartate (NAA).

Fig. 2

HR-MAS spectra recorded at 14 T ex vivo on biopsy tissue from patients with and without IDH1 mutations. ¹H-¹H 2D TOBSY spectra are shown for all biopsies (the minimum contour levels were set five times the noise level). (A) For anaplastic astrocytoma biopsy tissue with IDH1_R132 mutation (n=1), the spectra are shown in green-blue contours. The phantom is shown in red-yellow. Projections along δ₁ and δ₂ show the 2HG crosspeaks, outlined by a red rectangle. (B and C) Spectra for wt-IDH1 patients: primary glioblastoma (B; n=1) and non-tumor (C; n=1). The region where 2HG crosspeaks would be expected is outlined by a red rectangle. For all 2D TOBSY brain spectra, several other metabolites can be identified. Amino acids: alanine (Ala), aspartate (Asp), glutamate (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), proline (Pro), serine (Ser), threonine (Thr). Membrane phospholipid-related compounds: choline (Cho), ethanolamine (Etn), glycerol (Glr), glycerophosphocholine (GPC), glycerophosphoethanolamine (GPE), phosphocholine (PC), phosphoethanolamine (PE). Sugars: l-fucose (lFuc), β-glucose (bGlc), myo-inositol (Myo). Miscellaneous: glutathione (GSH), lactate (Lac), lipids (Lip), N-acetyl-aspartate (NAA), and taurine (Tau).

Fig. 3

2D LASER-COSY spectra in human subjects at 3 T. (A) An anaplastic astrocytoma patient with IDH1_R132C. The 2D LASER-COSY shows at 4.02/1.91 ppm the H_α-H_β crosspeak of 2HG. Projections along both spectral dimensions through 2HG crosspeak indicate the SNR and spectral quality. The single voxel (3×3×3 cm³, red rectangle) was placed on the FLAIR images to include most of the tumor abnormality. (B) A primary glioblastoma patient (wt-IDH1). The 2D LASER-COSY does not contain any 2HG crosspeak in the H_α-H_β region outlined by the green rectangle. Projections through Glu+Gln crosspeak indicate spectral quality. The single voxel (3.5×3.5×3.5 cm³, red rectangle) was placed on the FLAIR images to include most of the tumor abnormality. (C) A healthy volunteer (wt-IDH1). 2HG is not found in the H_α-H_β region of 2D LASER-COSY outlined by the green rectangle. Projections through Glu+Gln indicate spectral quality. The single voxel (3×3×3 cm³, red rectangle) was placed on the MEMPRAGE images in the white matter of the occipital lobe, in a region similar to tumor locations from patients in (A) and (B).

Fig. 4

1D MEGA-LASER spectra in human subjects at 3 T. In all subjects two voxels (3×3×3 cm³ each) were placed in both brain hemispheres, symmetrically from the middle line. (A) A secondary glioblastoma patient with IDH1_R132H mutation. (B and C) The spectra from subjects with wtIDH1—primary glioblastoma (B) and healthy volunteer (C). 2HG is present only in the tumor voxel of IDH1_R132H patient. (MM denotes contamination of GABA signal with macromolecule signal).

Fig. 5

Signal intensity ratios of 2HG to the sum of glutamate and glutamine (Glu+Gln). (A and B) Ratios are shown for all phantom and in vivo human spectra: 2D correlation MRS (LASER-COSY) (A), 1D spectral-edited MRS (MEGA-LASER) (B), and 1D conventional MRS (LASER) (A and B). Ratios are given as averages +/− one standard deviation. n = 2 for phantoms and IDH1_R132 patients; n = 4 for wt-IDH1 subjects.