Kirstie S. Opstad

Metabolic profiles of human brain tumors using quantitative in vivo 1H magnetic resonance spectroscopy

Proton spectroscopy can noninvasively provide useful information on brain tumor type and grade. Short‐ (30 ms) and long‐ (136 ms) echo time (TE) 1H spectra were acquired from normal white matter (NWM), meningiomas, grade II astrocytomas, anaplastic astrocytomas, glioblastomas, and metastases. Very low myo‐Inositol ([mI]) and creatine ([Cr]) were characteristic of meningiomas, and high [mI] characteristic of grade II astrocytomas. Tumor choline ([Cho]) was greater than NWM and increased with grade for grade II and anaplastic astrocytomas, but was highly variable for glioblastomas. Higher [Cho] and [Cr] correlated with low lipid and lactate (P < 0.05), indicating a dilution of metabolite concentrations due to necrosis in high‐grade tumors. Metabolite peak area ratios showed no correlation with lipids and mI/Cho (at TE = 30 ms), and Cr/Cho (at TE = 136 ms) best correlated with tumor grade. The quantified lipid, macromolecule, and lactate levels increased with grade of tumor, consistent with progression from hypoxia to necrosis. Quantification of lipids and macromolecules at short TE provided a good marker for tumor grade, and a scatter plot of the sum of alanine, lactate, and δ1.3 lipid signals vs. mI/Cho provided a simple way to separate most tumors by type and grade. Magn Reson Med 49:223–232, 2003.

Differentiation of metastases from high-grade gliomas using short echo time 1H spectroscopy†

To determine if short echo time (TE) 1H magnetic resonance spectroscopy (MRS) can distinguish between intracranial metastases and glioblastomas.

Detection of elevated glutathione in meningiomas by quantitative in vivo 1H MRS

Glutathione has major roles in removing free radicals and toxins from normal tissues, but its presence in tumor cells hinders the effectiveness of many anticancer therapies. Analysis of short echo time brain tumor 1H spectra at 1.5 T using a linear combination of metabolite spectra (LCModel) suggested a significant contribution of glutathione to meningioma spectra. By in vivo MRS (TE = 30 ms, TR = 2020 ms), reduced glutathione was found to be significantly elevated in meningiomas (3.3 ± 1.5 mM, Mann Whitney, P < 0.005) compared to normal white matter (1.2 ± 0.15 mM) and low‐grade gliomas (1.0 ± 0.26 mM), in agreement with published histofluorescence studies of tumor biopsies. Glx concentrations were also found to be elevated in meningiomas compared to astrocytomas or normal white matter, indicative of metabolic differences. The ability to noninvasively quantify reduced glutathione in vivo may aid selection of treatment therapies and also provide an indication of tumor aggressiveness. Magn Reson Med 49:632–637, 2003.

An investigation of human brain tumour lipids by high‐resolution magic angle spinning 1H MRS and histological analysis

NMR‐visible lipid signals detected in vivo by 1H MRS are associated with tumour aggression and believed to arise from cytoplasmic lipid droplets. High‐resolution magic angle spinning (HRMAS) 1H MRS and Nile Red staining were performed on human brain tumour biopsy specimens to investigate how NMR‐visible lipid signals relate to viable cells and levels of necrosis across different grades of glioma. Presaturation spectra were acquired from 24 adult human astrocytoma biopsy samples of grades II (8), III (2) and IV (14) using HRMAS 1H MRS and quantified using LCModel to determine lipid concentrations. Each biopsy sample was then refrozen, cryostat sectioned, and stained with Nile Red, to determine the number of lipid droplets and droplet size distribution, and with Haematoxylin and Eosin, to determine cell density and percentage necrosis. A strong correlation (R = 0.92, P < 0.0001) was found between the number of Nile Red‐stained droplets and the ∼1.3 ppm lipid proton concentration by 1H MRS. Droplet sizes ranged from 1 to 10 µm in diameter, and the size distribution was constant independent of tumour grade. In the non‐necrotic biopsy samples, the number of lipid droplets correlated with cell density, whereas in the necrotic samples, there were greater numbers of droplets that showed a positive correlation with percentage necrosis. The correlation between 1H MRS lipid signals and number of Nile Red‐stained droplets, and the presence of lipid droplets in the non‐necrotic biopsy specimens provide good evidence that the in vivo NMR‐visible lipid signals are cytoplasmic in origin and that formation of lipid droplets precedes necrosis. Copyright

Serum α2-HS Glycoprotein Predicts Survival in Patients with Glioblastoma

BACKGROUND Glioblastoma, the most common primary brain tumor, has variable prognosis. We aimed to identify serum biomarkers that predict survival of patients with glioblastoma. METHODS In phase 1 (biomarker discovery), SELDI-TOF mass spectra were studied in 200 serum samples from 58 control subjects and 36 patients with grade II astrocytoma, 15 with anaplastic astrocytoma, and 91 with glioblastoma. To identify potential biomarkers, we searched for peptide peaks that changed progressively in size with increasing malignancy. One peak, identified as the B-chain of alpha 2-Heremans-Schmid glycoprotein (AHSG), was less prominent with increasing tumor grade. We therefore investigated AHSG as a survival predictor in glioblastoma. We measured serum AHSG by turbidimetry and determined indices of malignancy, including tumor proliferation (Ki67 immunolabel) and necrosis (tumor lipids on magnetic resonance spectroscopy). In phase 2 (biomarker validation), the prognostic power of AHSG was validated in an independent group of 72 glioblastoma patients. RESULTS Median survival was longer (51 vs 29 weeks) in glioblastoma patients with normal vs low serum AHSG concentrations (hazard ratio 2.7, 95% CI 1.5-5.0, P <0.001), independent of age and Karnofsky score. Serum AHSG inversely correlated with Ki-67 immunolabeling and tumor lipids. A prognostic index combining serum AHSG with patient age and Karnofsky score separated glioblastoma patients with short (<3 months) and long (>2 years) median survival. The prognostic value of serum AHSG was validated in a different cohort of glioblastoma patients. CONCLUSIONS We conclude that serum AHSG concentration, measured before starting treatment, predicts survival in patients with glioblastoma.

Correlations between in vivo (1)H MRS and ex vivo (1)H HRMAS metabolite measurements in adult human gliomas.

To assess how accurately ex vivo high‐resolution magic angle spinning (HRMAS) proton magnetic resonance spectroscopy (1H MRS) from small biopsy tissues relate to in vivo 1H MRS (from larger tumor volumes) in human astrocytomas.

An assessment of the effects of sample ischaemia and spinning time on the metabolic profile of brain tumour biopsy specimens as determined by high-resolution magic angle spinning 1H NMR

High‐resolution magic angle spinning (HRMAS) 1H NMR of biopsy tissue provides a biochemical profile that has potential diagnostic and prognostic value, and can aid interpretation of the lower‐resolution 1H‐NMR spectra obtained in vivo. However, the biochemical profile obtained may be affected by experimental factors such as a period of ischaemia before snap‐freezing of the biopsy tissue for subsequent analysis and the mechanical stress of the spinning procedure of HRMAS itself. We have used normal rat brain cortex as a ‘gold standard’, either funnel‐frozen or deliberately allowed to become ischaemic for set periods of time before snap‐freezing, to quantitatively investigate these two effects. In addition, we have compared biochemical changes that occur in normal rat brain during HRMAS (spun continuously at 5 kHz for 4 h at 4°C as could be required for a two‐dimensional acquisition) with those that occur in biopsy samples from low‐grade and high‐grade adult human astrocytomas, during the same HRMAS procedure. Significant changes due to delayed initial sample freezing were noted in metabolites associated with glycolysis (alanine, glucose and lactate), as expected. However, for the funnel‐frozen rat tissue at 4°C, there were even more significant changes, which appear to be the result of extended spinning at 5 kHz. In particular, the 18% total creatine increase observed is unlikely to be de novo synthesis of creatine. More likely, the asymptotic exponential increase in creatine suggests an exponential release of an NMR‐invisible bound creatine store as a result of tissue damage from mechanical stress of sample spinning. Overall, it appears that tissue ischaemia during biopsy excision and delays in snap‐freezing may have less significant effects on metabolite profile than the prolonged spinning times required for two‐dimensional HRMAS, and this must be accounted for when results are being interpreted. Copyright

Pattern recognition of MRSI data shows regions of glioma growth that agree with DTI markers of brain tumor infiltration.

Gliomas are the most common primary brain tumors and the majority are highly malignant, with one of the worst prognoses for patients. Gliomas are characterized by invasive growth into normal brain tissue that makes complete surgical resection and accurate radiotherapy planning extremely difficult. We have performed independent component analysis of magnetic resonance spectroscopy imaging data from human gliomas to segment brain tissue into tumor core, tumor infiltration, and normal brain, with confirmation by diffusion tensor imaging analysis. Our data are consistent with previous studies that compared anomalies in isotropic and anisotropic diffusion images to determine regions of potential glioma infiltration. We show that coefficients of independent components can be used to create colored images for easy visual identification of regions of infiltrative tumor growth. Magn Reson Med, 2009.

Toward accurate quantification of metabolites, lipids, and macromolecules in HRMAS spectra of human brain tumor biopsies using LCModel

High‐resolution magic angle spinning (HRMAS) 1H MR spectroscopy of biopsy samples provides detailed biochemical profiles that can be related to the lower‐resolution spectra obtained in vivo. Nevertheless, there is still significant overlap of many resonance peaks and contributions from broad lipid and macromolecule resonances that impede accurate quantification. We determined a minimum set of in vitro metabolite and simulated lipid and macromolecule resonances needed for LCModel analysis and quantification of brain tumor biopsy HRMAS spectra. We also demonstrate the quality of the LCModel fit for the four main brain tumor types (astrocytoma grade II, glioblastoma, metastasis, and meningioma). Our data suggest that when fitting resonances of coupled spins systems in high‐resolution spectra, interactions between metabolites and the macromolecular environment of the biopsy may cause small peak shifts not found in the solution spectra. However, LCModel is shown to provide a user‐independent method of analyzing HRMAS brain tumor spectra. Magn Reson Med, 2008.

Apparent T2 relaxation times of lipid and macromolecules: A study of high‐grade tumor spectra

To determine T2 relaxation times of lipid and macromolecules (Lip/MMs) observed by 1H magnetic resonance spectroscopy (1H MRS) of metastases (MET) and glioblastomas (GBM), so that they may be better characterized.