Ovidiu C. Andronesi

Detection of 2-Hydroxyglutarate in IDH-Mutated Glioma Patients by In Vivo Spectral-Editing and 2D Correlation Magnetic Resonance Spectroscopy

2-Hydroxyglutarate, a metabolite overproduced in IDH-mutated gliomas, can be detected noninvasively in patients with brain tumors by optimized magnetic resonance spectroscopy. Spectroscopy Gets Inside Your Head Gliomas are diffuse brain tumors that are difficult to diagnose, with outcomes that are nearly impossible to predict—unless you can sample the diseased tissue itself via biopsy. This invasive procedure is typically performed at the time of surgery, with results available only after several weeks. Normally, it is a good thing that people can’t “see” inside your head; but, for gliomas, Andronesi and coauthors have found it to be beneficial by noninvasively imaging the brain to identify a glioma gene mutation that is correlated with patient survival. Mutations in the enzyme isocitrate dehydrogenase (IDH) lead to the accumulation of the metabolite 2-hydroxyglutarate (2HG). This mutation has been found in up to 86% of grade II to IV gliomas. Patients with IDH1 gene mutations have a greater 5-year survival rate than do patients with wild-type IDH1 gliomas, suggesting that such mutations could be used for prognosis. Andronesi et al. developed a strategy to detect IDH1 mutations in patients with glioma using magnetic resonance spectroscopy (MRS) imaging of 2HG. The similarity of 2HG to other metabolites, such as glutamate and glutamine, precludes detection with traditional one-dimensional spectroscopy; however, two-dimensional MRS allowed the authors to see the presence of 2HG in the brains of two glioma patients with IDH1 mutations, but not in healthy volunteers with wild-type IDH. The method was further validated ex vivo in tissue biopsies. With these results and those in the companion study by Elkhaled et al. (also in this issue), the authors show that in vivo brain imaging for genotyping cancer patients is a possibility—one that would avoid invasive clinical procedures and help doctors not only predict cancer outcomes but also effectively treat tumors on the basis of grade and genetic makeup. 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.

Glioblastoma Recurrence after Cediranib Therapy in Patients: Lack of “Rebound” Revascularization as Mode of Escape

Recurrent glioblastomas (rGBM) invariably relapse after initial response to anti-VEGF therapy. There are 2 prevailing hypotheses on how these tumors escape antiangiogenic therapy: switch to VEGF-independent angiogenic pathways and vessel co-option. However, direct evidence in rGBM patients is lacking. Thus, we compared molecular, cellular, and vascular parameters in autopsy tissues from 5 rGBM patients who had been treated with the pan-VEGF receptor tyrosine kinase inhibitor cediranib versus 7 patients who received no therapy or chemoradiation but no antiangiogenic agents. After cediranib treatment, endothelial proliferation and glomeruloid vessels were decreased, and vessel diameters and perimeters were reduced to levels comparable to the unaffected contralateral brain hemisphere. In addition, tumor endothelial cells expressed molecular markers specific to the blood-brain barrier, indicative of a lack of revascularization despite the discontinuation of therapy. Surprisingly, in cediranib-treated GBM, cellular density in the central area of the tumor was lower than in control cases and gradually decreased toward the infiltrating edge, indicative of a change in growth pattern of rGBMs after cediranib treatment, unlike that after chemoradiation. Finally, cediranib-treated GBMs showed high levels of PDGF-C (platelet-derived growth factor C) and c-Met expression and infiltration by myeloid cells, which may potentially contribute to resistance to anti-VEGF therapy. In summary, we show that rGBMs switch their growth pattern after anti-VEGF therapy--characterized by lower tumor cellularity in the central area, decreased pseudopalisading necrosis, and blood vessels with normal molecular expression and morphology--without a second wave of angiogenesis.

Characterization of Alzheimer's-like Paired Helical Filaments from the Core Domain of Tau Protein Using Solid-State NMR Spectroscopy

The polymerization of the microtubule-associated protein tau into paired helical filaments (PHFs) represents one of the hallmarks of Alzheimers disease. We employed solid-state nuclear magnetic resonance (NMR) to investigate the structure and dynamics of PHFs formed in vitro by the three-repeat-domain (K19) of protein tau, representing the core of Alzheimer PHFs. While N and C termini of tau monomers in PHFs are highly dynamic and solvent-exposed, the rigid segment consists of three major beta-strands. Combination of through-bond and through-space ssNMR transfer methods with water-edited ((15)N, (13)C) and ((13)C, (13)C) correlation experiments suggests the existence of a fibril core that is largely built by repeat unit R3, flanked by surface-exposed units R1 and R4. Solid-state NMR, circular dichroism, and the fibrillization behavior of a K19 mutant furthermore indicate that electrostatic interactions play a central role in stabilizing the K19 PHFs.

Detection of oncogenic IDH1 mutations using magnetic resonance spectroscopy of 2-hydroxyglutarate

The investigation of metabolic pathways disturbed in isocitrate dehydrogenase (IDH) mutant tumors revealed that the hallmark metabolic alteration is the production of D-2-hydroxyglutarate (D-2HG). The biological impact of D-2HG strongly suggests that high levels of this metabolite may play a central role in propagating downstream the effects of mutant IDH, leading to malignant transformation of cells. Hence, D-2HG may be an ideal biomarker for both diagnosing and monitoring treatment response targeting IDH mutations. Magnetic resonance spectroscopy (MRS) is well suited to the task of noninvasive D-2HG detection, and there has been much interest in developing such methods. Here, we review recent efforts to translate methodology using MRS to reliably measure in vivo D-2HG into clinical research.

Real-time motion and B0 corrected single voxel spectroscopy using volumetric navigators.

In population groups where head pose cannot be assumed to be constant during a magnetic resonance spectroscopy examination or in difficult‐to‐shim regions of the brain, real‐time volume of interest, frequency, and shim optimization may be necessary. We investigate the effect of pose change on the B0 homogeneity of a (2 cm)3 volume and observe typical first‐order shim changes of 1 μT/m per 1° rotation (chin down to up) in four different volumes of interest in a single volunteer. An echo planar imaging volume navigator was constructed to measure and apply in real‐time within each pulse repetition time: volume of interest positioning, frequency adjustment, and first‐order shim adjustment. This volume navigator is demonstrated in six healthy volunteers and achieved a mean linewidth of 4.4 Hz, similar to that obtained by manual shim adjustment of 4.9 Hz. Furthermore, this linewidth is maintained by the volume navigator at 4.9 Hz in the presence of pose change. By comparison, a mean linewidth of 7.5 Hz was observed, when no correction was applied. Magn Reson Med, 2011.

Structural characterization of Ca2+-ATPase-bound phospholamban in lipid bilayers by solid-state nuclear magnetic resonance (NMR) spectroscopy.

Phospholamban (PLN) regulates cardiac contractility by modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. While PLN and SERCA1a, an isoform from skeletal muscle, have been structurally characterized in great detail, direct information about the conformation of PLN in complex with SERCA has been limited. We used solid-state NMR (ssNMR) spectroscopy to deduce structural properties of both the A 36F 41A 46 mutant (AFA-PLN) and wild-type PLN (WT-PLN) when bound to SERCA1a after reconstitution in a functional lipid bilayer environment. Chemical-shift assignments in all domains of AFA-PLN provide direct evidence for the presence of two terminal alpha helices connected by a linker region of reduced structural order that differs from previous findings on free PLN. ssNMR experiments on WT-PLN show no significant difference in binding compared to AFA-PLN and do not support the coexistence of a significantly populated dynamic state of PLN after formation of the PLN/SERCA complex. A combination of our spectroscopic data with biophysical and biochemical data using flexible protein-protein docking simulations provides a structural basis for understanding the interaction between PLN and SERCA1a.

Spectroscopic imaging with improved gradient modulated constant adiabaticity pulses on high-field clinical scanners.

The purpose of this work was to design and implement constant adiabaticity gradient modulated pulses that have improved slice profiles and reduced artifacts for spectroscopic imaging on 3T clinical scanners equipped with standard hardware. The newly proposed pulses were designed using the gradient offset independent adiabaticity (GOIA, Tannus and Garwood[13]) method using WURST modulation for RF and gradient waveforms. The GOIA-WURST pulses were compared with GOIA-HSn (GOIA based on nth-order hyperbolic secant) and FOCI (frequency offset corrected inversion) pulses of the same bandwidth and duration. Numerical simulations and experimental measurements in phantoms and healthy volunteers are presented. GOIA-WURST pulses provide improved slice profile that have less slice smearing for off-resonance frequencies compared to GOIA-HSn pulses. The peak RF amplitude of GOIA-WURST is much lower (40% less) than FOCI but slightly higher (14.9% more) to GOIA-HSn. The quality of spectra as shown by the analysis of lineshapes, eddy currents artifacts, subcutaneous lipid contamination and SNR is improved for GOIA-WURST. GOIA-WURST pulse tested in this work shows that reliable spectroscopic imaging could be obtained in routine clinical setup and might facilitate the use of clinical spectroscopy.

Treatment Response Assessment in IDH-Mutant Glioma Patients by Noninvasive 3D Functional Spectroscopic Mapping of 2-Hydroxyglutarate.

Purpose: Measurements of objective response rates are critical to evaluate new glioma therapies. The hallmark metabolic alteration in gliomas with mutant isocitrate dehydrogenase (IDH) is the overproduction of oncometabolite 2-hydroxyglutarate (2HG), which plays a key role in malignant transformation. 2HG represents an ideal biomarker to probe treatment response in IDH-mutant glioma patients, and we hypothesized a decrease in 2HG levels would be measureable by in vivo magnetic resonance spectroscopy (MRS) as a result of antitumor therapy. Experimental Design: We report a prospective longitudinal imaging study performed in 25 IDH-mutant glioma patients receiving adjuvant radiation and chemotherapy. A newly developed 3D MRS imaging was used to noninvasively image 2HG. Paired Student t test was used to compare pre- and posttreatment tumor 2HG values. Test–retest measurements were performed to determine the threshold for 2HG functional spectroscopic maps (fSM). Univariate and multivariate regression were performed to correlate 2HG changes with Karnofsky performance score (KPS). Results: We found that mean 2HG (2HG/Cre) levels decreased significantly (median = 48.1%; 95% confidence interval = 27.3%–56.5%; P = 0.007) in the posttreatment scan. The volume of decreased 2HG correlates (R2 = 0.88, P = 0.002) with clinical status evaluated by KPS. Conclusions: We demonstrate that dynamic measurements of 2HG are feasible by 3D fSM, and the decrease of 2HG levels can monitor treatment response in patients with IDH-mutant gliomas. Our results indicate that quantitative in vivo 2HG imaging may be used for precision medicine and early response assessment in clinical trials of therapies targeting IDH-mutant gliomas. Clin Cancer Res; 22(7); 1632–41. ©2015 AACR.

High-resolution magic angle spinning magnetic resonance spectroscopy detects glycine as a biomarker in brain tumors

The non-essential amino acid neurotransmitter glycine (Gly) may serve as a biomarker for brain tumors. Using 36 biopsies from patients with brain tumors [12 glioblastoma multiforme (GBM); 10 low-grade (LG), including 7 schwannoma and 3 pylocytic astrocytoma; 7 meningioma (MN); 7 brain metastases (MT), including 3 adenocarcinoma and 4 breast cancer] and 9 control biopsies from patients undergoing surgery for epilepsy, we tested the hypothesis that the presence of glycine may distinguish among these brain tumor types. Using high-resolution magic angle spinning (HRMAS) 1H magnetic resonance spectroscopy (MRS), we determined a theoretically optimum echo time (TE) of 50 ms for distinguishing Gly signals from overlapping myo-inositol (Myo) signals and tested our methodology in phantom and biopsy specimens. Quantitative analysis revealed higher levels of Gly in tumor biopsies (all combined) relative to controls; Gly levels were significantly elevated in LG, MT and GBM biopsies (P≤0.05). Residual Myo levels were elevated in LG and MT and reduced in MN and GBM (P<0.05 vs. control levels). We observed higher levels of Gly in GBM as compared to LG tumors (P=0.05). Meanwhile, although Gly levels in GBM and MT did not differ significantly from each other, the Gly:Myo ratio did distinguish GBM from MT (P<0.003) and from all other groups, a distinction that has not been adequately made previously. We conclude from these findings that Gly can serve as a biomarker for brain tumors and that the Gly:Myo ratio may be a useful index for brain tumor classification.

3D GABA imaging with real-time motion correction, shim update and reacquisition of adiabatic spiral MRSI

Gamma-aminobutyric acid (GABA) and glutamate (Glu) are the major neurotransmitters in the brain. They are crucial for the functioning of healthy brain and their alteration is a major mechanism in the pathophysiology of many neuro-psychiatric disorders. Magnetic resonance spectroscopy (MRS) is the only way to measure GABA and Glu non-invasively in vivo. GABA detection is particularly challenging and requires special MRS techniques. The most popular is MEscher-GArwood (MEGA) difference editing with single-voxel Point RESolved Spectroscopy (PRESS) localization. This technique has three major limitations: a) MEGA editing is a subtraction technique, hence is very sensitive to scanner instabilities and motion artifacts. b) PRESS is prone to localization errors at high fields (≥3T) that compromise accurate quantification. c) Single-voxel spectroscopy can (similar to a biopsy) only probe steady GABA and Glu levels in a single location at a time. To mitigate these problems, we implemented a 3D MEGA-editing MRS imaging sequence with the following three features: a) Real-time motion correction, dynamic shim updates, and selective reacquisition to eliminate subtraction artifacts due to scanner instabilities and subject motion. b) Localization by Adiabatic SElective Refocusing (LASER) to improve the localization accuracy and signal-to-noise ratio. c) K-space encoding via a weighted stack of spirals provides 3D metabolic mapping with flexible scan times. Simulations, phantom and in vivo experiments prove that our MEGA-LASER sequence enables 3D mapping of GABA+ and Glx (Glutamate+Gluatmine), by providing 1.66 times larger signal for the 3.02ppm multiplet of GABA+ compared to MEGA-PRESS, leading to clinically feasible scan times for 3D brain imaging. Hence, our sequence allows accurate and robust 3D-mapping of brain GABA+ and Glx levels to be performed at clinical 3T MR scanners for use in neuroscience and clinical applications.