Biochemical characterization of breast tumors by in vivo and in vitro magnetic resonance spectroscopy (MRS)

Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) have evolved as sensitive tools for anatomic and metabolic evaluation of breast cancer. In vivo MRS studies have documented the presence of choline containing compounds (tCho) as a reliable biochemical marker of malignancy and also useful for monitoring the tumor response to therapy. Recent studies on the absolute quantification of tCho are expected to provide cut-off values for discrimination of various breast pathologies. Addition of MRS investigation was also reported to increase the specificity of MRI. Further, ex vivo and in vitro MRS studies of intact tissues and tissue extracts provided several metabolites that were not be detected in vivo and provided insight into underlying biochemistry of the disease processes. In this review, we present briefly the role of various ¹H MRS methods used in breast cancer research and their potential in relation to diagnosis, monitoring of therapeutic response and metabolism.     

Feasibility of MR Metabolomics for Immediate Analysis of Resection Margins during Breast Cancer Surgery

In this study, the feasibility of high resolution magic angle spinning (HR MAS) magnetic resonance spectroscopy (MRS) of small tissue biopsies to distinguish between tumor and non-involved adjacent tissue was investigated. With the current methods, delineation of the tumor borders during breast cancer surgery is a challenging task for the surgeon, and a significant number of re-surgeries occur. We analyzed 328 tissue samples from 228 breast cancer patients using HR MAS MRS. Partial least squares discriminant analysis (PLS-DA) was applied to discriminate between tumor and non-involved adjacent tissue. Using proper double cross validation, high sensitivity and specificity of 91% and 93%, respectively was achieved. Analysis of the loading profiles from both principal component analysis (PCA) and PLS-DA showed the choline-containing metabolites as main biomarkers for tumor content, with phosphocholine being especially high in tumor tissue. Other indicative metabolites include glycine, taurine and glucose. We conclude that metabolic profiling by HR MAS MRS may be a potential method for on-line analysis of resection margins during breast cancer surgery to reduce the number of re-surgeries and risk of local recurrence.     

In vivo magnetic resonance spectroscopy in chronic fatigue syndrome

The pathogenic mechanisms of chronic fatigue syndrome (CFS) are not clearly known. Fatigue, poor short-term memory and muscle pain are the most disabling symptoms in CFS. Research data on magnetic resonance spectroscopy (MRS) of muscles and brain in CFS patients suggest a cellular metabolic abnormality in some cases. 31P MRS of skeletal muscles in a subset of patients indicate early intracellular acidosis in the exercising muscles. 1H MRS of the regional brain areas in CFS have shown increased peaks of choline derived from the cell membrane phospholipids. Cell membrane oxidative stress may offer a common explanation for the observed MRS changes in the muscles and brain of CFS patients and this may have important therapeutic implications. As a research tool, MRS may be used as an objective outcome measure in the intervention studies. In addition, regional brain 1H MRS has the potential for wider use to substantiate a clinical diagnosis of CFS from other disorders of unexplained chronic fatigue.     

In vitro expression of N-acetyl aspartate by oligodendrocytes: implications for proton magnetic resonance spectroscopy signal in vivo

Magnetic resonance spectroscopy (MRS) provides a noninvasive means of assessing in vivo tissue biochemistry. N-Acetyl aspartate (NAA) is a major brain metabolite, and its presence is used increasingly in clinical and experimental MRS studies as a putative neuronal marker. A reduction in NAA levels as assessed by in vivo 1H MRS has been suggested to be indicative of neuronal viability. However, temporal observations of brain pathologies such as multiple sclerosis, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), and hypothyroidism have shown reversibility in NAA levels, possibly reflecting recovery of neuronal function. A knowledge of the cellular localisation of NAA is critical in interpreting these findings. The assumption that NAA is specific to neurones is based on previous immunohistochemical studies on whole brain using NAA-specific antibodies. The neuronal localisation was further substantiated by cell culture experiments in which its presence in the oligodendrocyte-type 2 astrocyte progenitors and immature oligodendrocytes, but not in the mature oligodendrocytes, was observed. More recently, studies on oligodendrocyte biology have revealed the requirement for trophic factors to promote the generation, maturation, and survival of oligodendrocytes in vitro. Here, we have used this new information to implement a more pertinent cell cultivation procedure and demonstrate that mature oligodendrocytes can express NAA in vitro. This observation brings into question whether the NAA changes observed in clinical in vivo 1H MRS studies reflect neuronal function alone. The data presented here support the hypothesis that oligodendrocytes may express NAA in vivo and contribute to the NAA signal observed by 1H MRS.     

In vivo magnetic resonance spectroscopy in the evaluation of mitochondrial disorders

Magnetic resonance spectroscopy (MRS) has been utilized to study several metabolic pathways in vivo and in live tissues in vitro non-invasively. Despite its inherited lack of sensitivity, its application has extended all the way to in situ human tissues and organs since proper technical advancements were devised. Examples of its application described here demonstrate the value of in vivo MRS as a technique that determines parameters of mitochondrial dysfunction directly and indirectly which could be of value for the diagnosis, prognosis, and follow-up of mitochondrial disorders.     

In Vivo NMR Studies of Neurodegenerative Diseases in Transgenic and Rodent Models

In vivo magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) provide unique quality to attain neurochemical, physiological, anatomical, and functional information noninvasively. These techniques have been increasingly applied to biomedical research and clinical usage in diagnosis and prognosis of diseases. The ability of MRS to detect early yet subtle changes of neurochemicals in vivo permits the use of this technology for the study of cerebral metabolism in physiological and pathological conditions. Recent advances in MR technology have further extended its use to assess the etiology and progression of neurodegeneration. This review focuses on the current technical advances and the applications of MRS and MRI in the study of neurodegenerative disease animal models including amyotrophic lateral sclerosis, Alzheimer's, Huntington's, and Parkinson's diseases. Enhanced MR measurable neurochemical parameters in vivo are described in regard to their importance in neurodegenerative disorders and their investigation into the metabolic alterations accompanying the pathogenesis of neurodegeneration.     

Magnetic Resonance Metabolic Profiling of Breast Cancer Tissue Obtained with Core Needle Biopsy for Predicting Pathologic Response to Neoadjuvant Chemotherapy

The purpose of this study was to determine whether metabolic profiling of core needle biopsy (CNB) samples using high-resolution magic angle spinning (HR-MAS) magnetic resonance spectroscopy (MRS) could be used for predicting pathologic response to neoadjuvant chemotherapy (NAC) in patients with locally advanced breast cancer. After institutional review board approval and informed consent were obtained, CNB tissue samples were collected from 37 malignant lesions in 37 patients before NAC treatment. The metabolic profiling of CNB samples were performed by HR-MAS MRS. Metabolic profiles were compared according to pathologic response to NAC using the Mann-Whitney test. Multivariate analysis was performed with orthogonal projections to latent structure-discriminant analysis (OPLS-DA). Various metabolites including choline-containing compounds were identified and quantified by HR-MAS MRS in all 37 breast cancer tissue samples obtained by CNB. In univariate analysis, the metabolite concentrations and metabolic ratios of CNB samples obtained with HR-MAS MRS were not significantly different between different pathologic response groups. However, there was a trend of lower levels of phosphocholine/creatine ratio and choline-containing metabolite concentrations in the pathologic complete response group compared to the non-pathologic complete response group. In multivariate analysis, the OPLS-DA models built with HR-MAS MR metabolic profiles showed visible discrimination between the pathologic response groups. This study showed OPLS-DA multivariate analysis using metabolic profiles of pretreatment CNB samples assessed by HR- MAS MRS may be used to predict pathologic response before NAC, although we did not identify the metabolite showing statistical significance in univariate analysis. Therefore, our preliminary results raise the necessity of further study on HR-MAS MR metabolic profiling of CNB samples for a large number of cancers.     

Noninvasive quantitative in vivo mapping and metabolism of boronophenylalanine (BPA) by nuclear magnetic resonance (NMR) spectroscopy and imaging

10B-enriched L-p-boronophenylalanine (BPA) is one of the compounds used in boron neutron capture therapy (BNCT). In this study, several variations of nuclear magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) were applied to investigate the uptake, clearance and metabolism of the BPA-fructose complex (BPA-F) in normal mouse kidneys, rat oligodendroglioma xenografts, and rat blood. Localized 1H MRS was capable of following the uptake and clearance of BPA-F in mouse kidneys with temporal resolution of a few minutes, while 1H MRSI was used to image the BPA distribution in the kidney with a spatial resolution of 9 mm3. The results also revealed significant dissociation of the BPA-F complex to free BPA. This finding was corroborated by 1H and 11B NMR spectroscopy of rat blood samples as well as of tumor samples excised from mice after i.v. injection of BPA-F. This investigation demonstrates the feasibility of using 1H MRS and MRSI to follow the distribution of BPA in vivo, using NMR techniques specifically designed to optimize BPA detection. The implementation of such procedures could significantly improve the clinical efficacy of BNCT.     

In vivo imaging of apoptosis in oncology: an update

In this review, data on noninvasive imaging of apoptosis in oncology are reviewed. Imaging data available are presented in order of occurrence in time of enzymatic and morphologic events occurring during apoptosis. Available studies suggest that various radiopharmaceutical probes bear great potential for apoptosis imaging by means of positron emission tomography and single-photon emission computed tomography (SPECT). However, for several of these probes, thorough toxicologic studies are required before they can be applied in clinical studies. Both preclinical and clinical studies support the notion that 99mTc-hydrazinonicotinamide-annexin A5 and SPECT allow for noninvasive, repetitive, quantitative apoptosis imaging and for assessing tumor response as early as 24 hours following treatment instigation. Bioluminescence imaging and near-infrared fluorescence imaging have shown great potential in small-animal imaging, but their usefulness for in vivo imaging in humans is limited to structures superficially located in the human body. Although preclinical tumor-based data using high-frequency-ultrasonography (US) are promising, whether or not US will become a routinely clinically useful tool in the assessment of therapy response in oncology remains to be proven. The potential of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) for imaging late apoptotic processes is currently unclear. Neither 31P MRS nor 1H MRS signals seems to be a unique identifier for apoptosis. Although MRI-measured apparent diffusion coefficients are altered in response to therapies that induce apoptosis, they are also altered by nonapoptotic cell death, including necrosis and mitotic catastrophe. In the future, rapid progress in the field of apoptosis imaging in oncology is expected.     

Reproducibility and Absolute Quantification of Muscle Glycogen in Patients with Glycogen Storage Disease by 13C NMR Spectroscopy at 7 Tesla

Carbon-13 magnetic resonance spectroscopy (¹³C MRS) offers a noninvasive method to assess glycogen levels in skeletal muscle and to identify excess glycogen accumulation in patients with glycogen storage disease (GSD). Despite the clinical potential of the method, it is currently not widely used for diagnosis or for follow-up of treatment. While it is possible to perform acceptable ¹³C MRS at lower fields, the low natural abundance of ¹³C and the inherently low signal-to-noise ratio of ¹³C MRS makes it desirable to utilize the advantage of increased signal strength offered by ultra-high fields for more accurate measurements. Concomitant with this advantage, however, ultra-high fields present unique technical challenges that need to be addressed when studying glycogen. In particular, the question of measurement reproducibility needs to be answered so as to give investigators insight into meaningful inter-subject glycogen differences. We measured muscle glycogen levels in vivo in the calf muscle in three patients with McArdle disease (MD), one patient with phosphofructokinase deficiency (PFKD) and four healthy controls by performing ¹³C MRS at 7T. Absolute quantification of the MRS signal was achieved by using a reference phantom with known concentration of metabolites. Muscle glycogen concentration was increased in GSD patients (31.5±2.9 g/kg w. w.) compared with controls (12.4±2.2 g/kg w. w.). In three GSD patients glycogen was also determined biochemically in muscle homogenates from needle biopsies and showed a similar 2.5-fold increase in muscle glycogen concentration in GSD patients compared with controls. Repeated inter-subject glycogen measurements yield a coefficient of variability of 5.18%, while repeated phantom measurements yield a lower 3.2% system variability. We conclude that noninvasive ultra-high field ¹³C MRS provides a valuable, highly reproducible tool for quantitative assessment of glycogen levels in health and disease.     

Mitochondrial function in vivo: spectroscopy provides window on cellular energetics

Mitochondria integrate the key metabolic fluxes in the cell. This role places this organelle at the center of cellular energetics and, hence, mitochondrial dysfunction underlies a growing number of human disorders and age-related degenerative diseases. Here we present novel analytical and technical methods for evaluating mitochondrial metabolism and (dys)function in human muscle in vivo. Three innovations involving advances in optical spectroscopy (OS) and magnetic resonance spectroscopy (MRS) permit quantifying key compounds in energy metabolism to yield mitochondrial oxidation and phosphorylation fluxes. The first of these uses analytical methods applied to optical spectra to measure hemoglobin (Hb) and myoglobin (Mb) oxygenation states and relative contents ([Hb]/[Mb]) to determine mitochondrial respiration (O₂ uptake) in vivo. The second uses MRS methods to quantify key high-energy compounds (creatine phosphate, PCr, and adenosine triphosphate, ATP) to determine mitochondrial phosphorylation (ATP flux) in vivo. The third involves a functional test that combines these spectroscopic approaches to determine mitochondrial energy coupling (ATP/O₂), phosphorylation capacity (ATP_max) and oxidative capacity (O_2max) of muscle. These new developments in optical and MR tools allow us to determine the function and capacity of mitochondria noninvasively in order to identify specific defects in vivo that are associated with disease in human and animal muscle. The clinical implication of this unique diagnostic probe is the insight into the nature and extent of dysfunction in metabolic and degenerative disorders, as well as the ability to follow the impact of interventions designed to reverse these disorders.     

磁共振功能成像在监测乳腺癌新辅助化疗中的应用和进展

近年来,新辅助化疗在原发乳腺癌治疗中运用越来越广泛。影像学手段在评价新辅助化疗疗效、指导临床治疗方案的制定中发挥重要作用。磁共振功能成像的引入,可以加深对恶性乳腺肿瘤的病理生理活动及分子生物学特性的了解,监测化疗疗效,提高早期预测的准确性。本文总结功能学MRI(磁共振成像)探测乳腺癌患者生物标志物的研究现状及发展情况,描述各种生物学标志物特性,评价其潜在的临床应用价值和局限性。以下将对动态增强磁共振成像、弥散加权成像、血氧水平依赖成像以及波谱成像等几种磁共振功能学成像方法的原理进行描述,重点对其在监测乳腺癌新辅助化疗中的应用进行综述。     

A metabolomics perspective of human brain tumours

During the past decade or so, a wealth of information about metabolites in various human brain tumour preparations (cultured cells, tissue specimens, tumours in vivo) has been accumulated by global profiling tools. Such holistic approaches to cellular biochemistry have been termed metabolomics. Inherent and specific metabolic profiles of major brain tumour cell types, as determined by proton nuclear magnetic resonance spectroscopy ((1)H MRS), have also been used to define metabolite phenotypes in tumours in vivo. This minireview examines the recent advances in the field of human brain tumour metabolomics research, including advances in MRS and mass spectrometry technologies, and data analysis.     

The INTERPRET Decision-Support System version 3.0 for evaluation of Magnetic Resonance Spectroscopy data from human brain tumours and other abnormal brain masses

Background  

Proton Magnetic Resonance (MR) Spectroscopy (MRS) is a widely available technique for those clinical centres equipped with MR scanners. Unlike the rest of MR-based techniques, MRS yields not images but spectra of metabolites in the tissues. In pathological situations, the MRS profile changes and this has been particularly described for brain tumours. However, radiologists are frequently not familiar to the interpretation of MRS data and for this reason, the usefulness of decision-support systems (DSS) in MRS data analysis has been explored.     

In vivo MRS measurement of liver lipid levels in mice

A magnetic resonance spectroscopy (MRS) procedure for in vivo measurement of lipid levels in mouse liver is described and validated. The method uses respiratory-gated, localized spectroscopy to collect proton spectra from voxels within the mouse liver. Bayesian probability theory analysis of these spectra allows the relative intensities of the lipid and water resonances within the liver to be accurately measured. All spectral data were corrected for measured spin-spin relaxation. A total of 48 mice were used in this study, including wild-type mice and two different transgenic mouse strains. Different groups of these mice were fed high-fat or low-fat diets or liquid diets with and without the addition of alcohol. Proton spectra were collected at baseline and, subsequently, every 4 weeks for up to 16 weeks. Immediately after the last MRS measurement, mice were killed and their livers analyzed for triglyceride level by conventional wet-chemistry methods. The excellent correlation between in vivo MRS and ex vivo wet-chemistry determinations of liver lipids validates the MRS method. These results clearly demonstrate that in vivo MRS will be an extremely valuable technique for longitudinal studies aimed at providing important insights into the genetic, environmental, and dietary factors affecting fat deposition and accumulation within the mouse liver.     

2-Hydroxyglutarate as a Magnetic Resonance Biomarker for Glioma Subtyping

Mutations in the isocitrate dehydrogenase (IDH) genes are frequently found in gliomas and in a fraction of acute myeloid leukemia patients. This results in the production of an oncometabolite, 2-hydroxyglutarate (2-HG). Glioma patients harboring IDH mutations have a longer survival than their wild-type counterparts. 2-HG has been detected noninvasively in gliomas with IDH mutations using magnetic resonance spectroscopy (MRS), suggesting its potential clinical relevance for identifying glioma subtypes with better prognosis. In this paper, the recent developments in the MRS detection of the 2-HG in gliomas are reviewed, including the therapeutic potentials and translational values.     

Magnetic resonance spectroscopy: an in vivo tool for monitoring cerebral injury in SIV-infected macaques

The purpose of our study was to demonstrate the feasibility of using in vivo proton Magnetic Resonance Spectroscopy (MRS) to monitor the brain manifestations of SIV infection in the macaque model of AIDS. Previous spectroscopy work on macaque brain tissue and in vivo work in humans is reviewed to provide the motivation and context for this study. We collected 34 MRS data sets on 14 uninfected rhesus macaques. From this data, we demonstrate that we are capable of detecting changes similar to those observed in human MRS studies for most metabolites using less than 10 animals. The juvenile macaques utilized in this study demonstrate age-related changes in the levels of N-acetyl aspartate (NAA), a neuronal marker. The quantity and distribution of neurochemicals in the macaque are found to be slightly, but significantly, different than in the human.     

Quantification of ethanol methyl 1H magnetic resonance signal intensity following intravenous ethanol administration in primate brain

In vivo ¹H magnetic resonance spectroscopy (MRS) can be used to directly monitor brain ethanol. Previously, studies of human subjects have lead to the suggestion that the ethanol methyl ¹H MRS signal intensity relates to tolerance to ethanol’s intoxicating effects. More recently, the ethanol ¹H MRS signal intensity has been recognized to vary between brain gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF) due to differences in T₂ within these environments. The methods presented here extend ethanol MRS techniques to non-human primate subjects. Twelve monkeys were administered ethanol while sedated and positioned within a 3T MRI system. Chemical shift imaging (CSI) measurements were performed following intravenous infusion of 1 g/kg ethanol. Magnetic resonance imaging (MRI) data were also recorded for each monkey to provide volume fractions of GM, WM, and CSF for each CSI spectrum. To estimate co-variance of ethanol MRS intensity with GM, WM, and CSF volume fractions, the relative contribution of each tissue subtype was determined following corrections for radiofrequency pulse profile non-uniformity, chemical shift artifacts, and differences between the point spread function in the CSI data and the imaging data. The ethanol MRS intensity per unit blood ethanol concentration was found to differ between GM, WM, and CSF. Individual differences in MRS intensity were larger in GM than WM. This methodology demonstrates the feasibility of ethanol MRS experiments and analysis in non-human primate subjects, and suggests GM may be a site of significant variation in ethanol MRS intensity between individuals.