Standardization of radiological evaluation of dynamic contrast enhanced MRI: application in breast cancer diagnosis.

Dynamic contrast enhanced MRI is applied as an adjuvant tool for breast cancer detection, diagnosis, and follow-up of therapy. Despite improvements through the years in achieving higher spatial and temporal resolution, it still suffers from lack of scanning and processing standardization, and consequently, high variability in the radiological evaluation, particularly differentiating malignant from benign lesions. We describe here a hybrid method for achieving standardization of the radiological evaluation of breast dynamic contrast enhanced (DCE)-magnetic resonance imaging (MRI) protocols, based on integrating the model based three time point (3TP) method with principal component analysis (PCA). The scanning and image processing procedures consisted of three main steps: 1. 3TP standardization of the MRI acquisition parameters according to a kinetic model, 2. Applying PCA to test cases and constructing an eigenvectors' base related to the contrast-enhancement kinetics and 3. Projecting all new cases on the eigenvectors' base and evaluating the clinical outcome. Datasets of overall 96 malignant and 26 benign breast lesions were recorded on 1.5T and 3T scanners, using three different MRI acquisition parameters optimized by the 3TP method. The final radiological evaluation showed similar detection and diagnostic ability for the three different MRI acquisition parameters. The area under the curve of receiver operating characteristic analysis yielded a value of 0.88 ± 0.034 for differentiating malignant from benign lesions. This 3TP+PCA hybrid method is fast and can be readily applied as a computer aided diagnostic tool of breast cancer. The underlying principles of this method can be extended to standardize the evaluation of malignancies in other organs.

Hyperpolarized 13C NMR studies of glucose metabolism in living breast cancer cell cultures.

The recent development of dissolution dynamic nuclear polarization (DNP) gives NMR the sensitivity to follow metabolic processes in living systems with high temporal resolution. In this article, we apply dissolution DNP to study the metabolism of hyperpolarized U-(13)C,(2)H7-glucose in living, perfused human breast cancer cells. Spectrally selective pulses were used to maximize the signal of the main product, lactate, whilst preserving the glucose polarization; in this way, both C1-lactate and C3-lactate could be observed with high temporal resolution. The production of lactate by T47D breast cancer cells can be characterized by Michaelis-Menten-like kinetics, with K(m) = 3.5 ± 1.5 mM and V(max) = 34 ± 4 fmol/cell/min. The high sensitivity of this method also allowed us to observe and quantify the glycolytic intermediates dihydroxyacetone phosphate and 3-phosphoglycerate. Even with the enhanced DNP signal, many other glycolytic intermediates could not be detected directly. Nevertheless, by applying saturation transfer methods, the glycolytic intermediates glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, phosphoenolpyruvate and pyruvate could be observed indirectly. This method shows great promise for the elucidation of the distinctive metabolism and metabolic control of cancer cells, suggesting multiple ways whereby hyperpolarized U-(13)C,(2)H7-glucose NMR could aid in the diagnosis and characterization of cancer in vivo.

A compound herbal preparation (CHP) in the treatment of children with ADHD: a randomized controlled trial.

OBJECTIVE: Evaluation of the efficacy of a patented, compound herbal preparation (CHP) in improving attention, cognition, and impulse control in children with ADHD. DESIGN: A randomized, double-blind, placebo-controlled trial. SETTING: University-affiliated tertiary medical center. PARTICIPANTS: 120 children newly diagnosed with ADHD, meeting DSM-IV criteria. INTERVENTION: Random assignment to the herbal treatment group (n = 80) or control group (placebo; n = 40); 73 patients in the treatment group (91%) and 19 in the control group (48%) completed the 4-month trial. OUTCOME MEASURE: Test of Variables of Attention (TOVA) administered before and after the treatment period; overall score and 4 subscales. RESULTS: The treatment group showed substantial, statistically significant improvement in the 4 subscales and overall TOVA scores, compared with no improvement in the control group, which persisted in an intention-to-treat analysis. CONCLUSIONS: The well-tolerated CHP demonstrated improved attention, cognition, and impulse control in the intervention group, indicating promise for ADHD treatment in children.

Detection of evolving acute tubular necrosis with renal 23Na MRI: studies in rats.

The clinical detection of evolving acute tubular necrosis (ATN) and differentiating it from other causes of renal failure are currently limited. The maintenance of the corticomedullary sodium gradient, an indicator of normal kidney function, is presumably lost early in the course of ATN. Herein, sodium magnetic resonance imaging (23Na MRI) was applied to study the early alteration in renal sodium distribution in rat kidneys 6 h after the induction of ATN. Three-dimensional gradient echo sodium images were recorded at 4.7 T with high spatial resolution. ATN was produced by the administration of radiologic contrast medium, combined with inhibition of nitric oxide and prostaglandin synthesis. The sodium images revealed that the sham-controlled kidney exhibited a linear increase in sodium concentration along the corticomedullary axis of 30+/-2 mmol/l/mm, resulting in an inner medulla to cortex sodium ratio of 4.3+/-0.3 (n=5). In the ATN kidney, however, the cortico-outer medullary sodium gradient was reduced by 21% (P<0.01, n=7) and the inner medulla to cortex sodium ratio was decreased by 40% (P<0.001, n=7). Small, though significant, increments in plasma creatinine at this time inversely correlated with the decline in the corticomedullary sodium gradient. Histological findings demonstrated outer medullary ATN involving 4% of medullary thick ascending limbs. Hence, 23Na MRI non-invasively quantified changes in the corticomedullary sodium gradient in the ATN kidney when morphologic tubular injury was still focal and very limited. MRI detection of corticomedullary sodium gradient abnormalities may serve to identify evolving ATN at its early phases.

Magnetic resonance imaging of breast cancer angiogenesis: a review.

The development and growth of solid tumors rely on a process known as perfusion which allows for the delivery and clearance of nutrients through their vasculature. The classical approach of studying tumor vasculature by histologic staining of endothelial cells provides a measure of microvessel density in areas of high vascularization (so-called "hot spots"). More advanced high-resolution, parametric, contrast-enhanced MRI techniques enable quantitative assessment of the vascular distribution and function over the entire tumor. Hence, MRI overcomes the intratumoral variation of the histological method. Clinical testing of the MRI approach, that evaluates the permeability and surface area of the tumor vasculature, demonstrates improved accuracy of breast cancer diagnosis.

Differential routing of choline in implanted breast cancer and normal organs.

Choline is an essential nutrient participating as the initial substrate in major metabolic pathways. The differential metabolic routing of choline was investigated in MCF7 human breast cancer implanted in nude mice and in the kidney, liver, and brain of these mice. The distribution of metabolites following infusion of [1,2-(13)C]-choline was monitored by (13)C magnetic resonance spectroscopy. This infusion led to an 18-fold increase in plasma choline and to concomitant changes in the content and distribution of choline metabolites. In vivo kinetic studies of the tumor during the infusion demonstrated accumulation of choline in the interstitium and intracellular synthesis of phosphocholine. The amount of unlabeled choline metabolites was 7.1, 4.1, 3.5, and 1.4 micromol/g in the kidney, liver, tumor, and brain, respectively. The variations in the labeled metabolites were more pronounced with high amounts in the kidney and liver (8.0 and 4.3 micromol/g, respectively) and very low amounts in the tumor and brain (0.33 and 0.12 micromol/g, respectively). In the kidney and liver, betaine (unlabeled and labeled) was the predominant choline metabolite. The dominant unlabeled metabolite in breast cancer was phosphocholine and in the brain glycerophosphocholine. Magn Reson Med 46:31-38, 2001.

Critical role of spatial resolution in dynamic contrast-enhanced breast MRI.

The spatial resolution of three-dimensional (3D) gradient-echo T1-weighted images, from 40 women with 25 malignant and 23 benign lesions, was purposely degraded to determine the role of spatial resolution in recording, analysis, and diagnosis of dynamic contrast-enhanced breast MRI. Images were recorded and analyzed at pixel resolution according to the 3TP method (Degani et al., Nat Med 1997;3:780-782). Reduction in spatial resolution degraded the appearance of foci with fast wash-in and fast washout dynamics. This resulted in an increase in false-negative diagnoses. The sensitivity for differentiating between malignant and benign lesions, using threshold criteria defined by the 3TP analysis, of 76% decreased to 60% and 24% for a 2- and 4-fold reduction in spatial resolution, respectively, without affecting significantly the high specificity (96-100%). In order to minimize false-negative diagnoses of contrast-enhanced breast MRI and maintain high specificity, it is essential to record and analyze the dynamic behavior at high spatial resolution. J. Magn. Reson. Imaging 2001;13:862-867.

Development, standardization, and testing of a lexicon for reporting contrast-enhanced breast magnetic resonance imaging studies.

The purpose of this study was to develop, standardize, and test reproducibility of a lexicon for reporting contrast-enhanced breast magnetic resonance imaging (MRI) examinations. To standardize breast MRI lesion description and reporting, seven radiologists with extensive breast MRI experience developed consensus on technical detail, clinical history, and terminology reporting to describe kinetic and architectural features of lesions detected on contrast-enhanced breast MR images. This lexicon adapted American College of Radiology Breast Imaging and Data Reporting System terminology for breast MRI reporting, including recommendations for reporting clinical history, technical parameters for breast MRI, descriptions for general breast composition, morphologic and kinetic characteristics of mass lesions or regions of abnormal enhancement, and overall impression and management recommendations. To test morphology reproducibility, seven radiologists assessed morphology characteristics of 85 contrast-enhanced breast MRI studies. Data from each independent reader were used to compute weighted and unweighted kappa (kappa) statistics for interobserver agreement among readers. The MR lexicon differentiates two lesion types, mass and non-mass-like enhancement based on morphology and geographical distribution, with descriptors of shape, margin, and internal enhancement. Lexicon testing showed substantial agreement for breast density (kappa = 0.63) and moderate agreement for lesion type (kappa = 0.57), mass margins (kappa = 0.55), and mass shape (kappa = 0.42). Agreement was fair for internal enhancement characteristics. Unweighted kappa statistics showed highest agreement for the terms dense in the breast composition category, mass in lesion type, spiculated and smooth in mass margins, irregular in mass shape, and both dark septations and rim enhancement for internal enhancement characteristics within a mass. The newly developed breast MR lexicon demonstrated moderate interobserver agreement. While breast density and lesion type appear reproducible, other terms require further refinement and testing to lead to a uniform standard language and reporting system for breast MRI. J. Magn. Reson. Imaging 2001;13:889-895.

Glucose transporters and transport kinetics in retinoic acid-differentiated T47D human breast cancer cells.

The rates of glucose transport and of glycolysis and the expression of the glucose transporters GLUT-1 through GLUT-4 were measured in T47D human breast cancer cells that underwent differentiation by retinoic acid. Glucose transport was found to be the rate-limiting step of glycolysis in control and differentiated cells. The transporters GLUT-1, GLUT-3, and GLUT-4 were present in the cell membrane and in the cytoplasm, and GLUT-2 was present solely in the cytoplasm. Differentiation led to a reduction in GLUT-1 and to an increase in cytoplasmic GLUT-2 and GLUT-3 with no change in GLUT-4. Differentiation also caused a reduction in the maximal velocity of glucose transport by approximately 40% without affecting the Michaelis-Menten constant of glucose transport. These changes did not alter the steady-state concentration of the phosphate metabolites regulating cell energetics but increased the content of phospholipid breakdown phosphodiesters. In conclusion, differentiation of human breast cancer cells appears to be associated with decreased glycolysis by a mechanism that involves a reduction in GLUT-1 and a slowdown of glucose transport.

Kinetics of cyclocreatine and Na(+) cotransport in human breast cancer cells: mechanism of activity.

The growth-inhibitory effect of cyclocreatine (CCr) and the kinetics of CCr and Na(+) cotransport were investigated in MCF7 human breast cancer cells and its adriamycin-resistant subline with use of (31)P- and (23)Na-NMR spectroscopy. The growth-inhibitory effect in the resistant line occurred at a lower CCr concentration and was more pronounced than in the wild-type line. This correlated with an approximately 10-fold higher affinity of CCr to the transporter in the resistant line. The passive diffusion coefficient of CCr was also higher in the resistant line by three- to fourfold. The transport of CCr was accompanied by a rapid increase in intracellular Na(+). This increase was found to depend on the rate of CCr transport and varied differently with CCr concentration in the two cell lines. It is proposed that the cotransport of CCr and Na(+) followed by increased Na(+) concentration, together with the accumulation of the highly charged phosphocyclocreatine, are responsible for cell swelling and death.

Breast fibroadenoma: mapping of pathophysiologic features with three-time-point, contrast-enhanced MR imaging--pilot study.

The capability of three-time-point, gadolinium-enhanced magnetic resonance imaging to depict vascular permeability and extracellular volume fraction of breast fibroadenoma was evaluated with histopathologic correlation. This method demonstrated an even distribution of high extracellular volume fraction and low to moderate microvascular permeability in these common breast lesions, providing a nonsurgical means of improving the accuracy of diagnosis of fibroadenoma.

Stimulation of fructose 1,6-bisphosphate production in melanoma cells by alpha-melanocyte-stimulating hormone-- 31P/13C-NMR and 32P-labeling studies.

In a 31P-NMR spectroscopic study of cultured M2R mouse melanoma cells, we previously demonstrated the acute stimulation of three peaks in the phosphomonoester region of the spectrum by [Ahx4, DPhe7]alpha-melanotropin (concomitant with an increase in cellular adenosine 3',5'-phosphate (cAMP) and a decrease in ATP [Degani, H., DeJordy, J. O. & Salomon, Y. (1991) Proc. Natl Acad. Sci. USA 88, 1506-1510]. Chemical identification of these metabolites was performed in this study using 32P metabolic labeling and polyethyleneimine-cellulose thin layer chromatography in combination with 31P-NMR and 13C-NMR spectroscopic methods. Two of the stimulated signals were identified as P1 and P6 of fructose 1,6-bisphosphate (FruP2) and their mode of regulation by alpha-melanotropin was examined. The FruP2 response to alpha-melanotropin coincided in time and dose with a rise in cAMP and a decrease in levels of ATP, while elevation of cAMP by forskolin alone did not increase FruP2. The stimulatory effect of alpha-melanotropin was not associated with a change in the overall rate of glycolysis, suggesting that FruP2 levels were not rate limiting in this process. The data suggest the presence of a previously unknown response of M2R melanoma cells to alpha-melanotropin, which coincides in time with enhanced cAMP accumulation but is not mediated by cAMP and may relate to the control of FruP2 in a non glycolytic context.

Altered brain glucose metabolism in transgenic-PFKL mice with elevated L-phosphofructokinase: in vivo NMR studies.

The gene for the liver-type subunit of phosphofructokinase (PFKL) resides on chromosome 21 and is overexpressed in Down syndrome (DS) patients. Transgenic PFKL (Tg-PFKL) mice with elevated levels of PFKL were used to determine whether, as in DS, overexpression of PFKL was also associated with altered sugar metabolism. We found that Tg-PFKL mice had an abnormal glucose metabolism with reduced clearance rate from blood and enhanced metabolic rate in brain. Transgenic-PFKL mice exhibited elevated activity of phosphofructokinase in both blood and brain, as compared to control non-transgenic (ntg) mice. Following glucose infusion, the rate of glucose clearance from the blood of Tg-PFKL mice was significantly slower than that of control ntg mice, although the basal blood glucose levels were similar. However, unlike the slower rate of glucose metabolism in blood, the initial rate of glucose utilization in the brain of the transgenic mice, was 58% faster than in control ntg mice. This was determined by infusion of [1-13C]-glucose followed by in vivo nuclear magnetic resonance (NMR) measurements of brain glucose metabolism. The faster utilization of glucose in Tg-PFKL brain is similar to the increased rate of cerebral glucose metabolism found in the brain of young adult DS patients, which may play a role in the etiology of their cognitive disabilities.

Reversible induction of ATP synthesis by DNA damage and repair in Escherichia coli. In vivo NMR studies.

Early metabolic events in Escherichia coli exposed to nalidixic acid, a topoisomerase II inhibitor and an inducer of the SOS system, were investigated by in vivo NMR spectroscopy, a technique that permits monitoring of bacteria under controlled physiological conditions. The energetics of AB1157 (wild type) and of its isogenic, SOS-defective mutants, recBC, lexA, and DeltarecA, were studied by 31P and 19F NMR before, during, and after exposure to nalidixic acid. The content of the NTP in E. coli embedded in agarose beads and perfused at 36 degreesC was found to be 4.3 +/- 1.1 x 10(-18) mol/cell, yielding a concentration of approximately 2.7 +/- 0.7 mM. Nalidixic acid induced in the wild type and mutants a rapid 2-fold increase in the content of the NTP, predominantly ATP. This induction did not involve synthesis of uracil derivatives or breakdown of RNA and caused cell proliferation to stop. Removal of nalidixic acid after 40 min of treatment rescued the cells and resulted in a decrease of ATP to control levels and resumption of proliferation. However, in DeltarecA cells, which were more sensitive to the activity of the drug, ATP elevation could not be reversed, and ATP content continued to increase faster than in control cells. The results ruled out association between the elevation of ATP and the induction of the SOS system and suggested involvement of a process reminiscent of apoptosis in the stimulation of ATP synthesis. Thus, the presence of the RecA protein was found to be essential for reversing the ATP increase and cell rescue, possibly by its function in repair of DNA damage.

Choline metabolism in breast cancer; 2H-, 13C- and 31P-NMR studies of cells and tumors.

Choline metabolism in breast cancer cells and tumors has been investigated by multinuclear NMR in order to provide the biochemical basis for the presence of high phosphocholine in breast carcinoma relative to benign breast tumors and normal breast tissue. Choline was found to be transported into MCF7 human breast cancer cells and rapidly phosphorylated to phosphocholine which was then accumulated in the cells to high concentrations. The increased level of phosphocholine did not affect the rate of synthesis of phosphatidylcholine, indicating tight regulation of this pathway. The incorporation of [1,2-13C]choline (100 microM) into phosphocholine and phosphatidylcholine after 24 h was 69.5 and 36% of the total respective pools. Incorporation of 2H9-choline to tumors implanted in nude mice was achieved by infusing the deuterated choline to the blood circulation. The metabolism of deuterated choline was then monitored by 2H localized MRS. The blood level of choline before the infusion was 58.6 +/- 10.3 microM (measured by 1H-NMR of plasma samples) and increased approximately 5-fold during the infusion (measured by 2H-NMR). This increase in the blood level resulted in a gradual increase of a signal at 3.2 ppm due to deuterated choline metabolites. It appears that the increased availability of choline in the blood circulation leads to accumulation of phosphocholine in the tumors by the same mechanism as in the cells.

Response of MCF7 human breast cancer to tamoxifen: evaluation by the three-time-point, contrast-enhanced magnetic resonance imaging method.

Variations in the cellular volume fraction and in the microvascular permeability of MCF7 human breast tumors were used to assess response to tamoxifen. These pathophysiological features were mapped by applying the three-time-point, contrast-enhanced, high resolution magnetic resonance imaging method (H. Degani et al, Nat. Med., 3: 780-782, 1997). Short-term treatment with tamoxifen caused a highly significant increase in the fraction of pixels displaying intermediate contrast agent clearance pattern and a significant increase in the fraction of pixels displaying high rate of contrast agent entrance. These changes resulted from a marked rise in the extracellular volume fraction, indicating increased necrosis, and from an augmentation in the microvascular permeability, predominantly in the vicinity of the high extracellular volume fraction areas, as a result of stress-induced angiogenesis.

High resolution MRI of MCF7 human breast tumors: complemented use of iron oxide microspheres and Gd-DTPA.

The differential capacity of iron oxide microspheres and of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) to serve as contrast agents that can map the microcirculation of MCF7 human breast cancer implanted in nude mice has been examined by high resolution MRI. Modulation of signal intensity in T2*-weighted, gradient-echo images after iron oxide administration and the temporal signal enhancement after Gd-DTPA administration were monitored and analyzed at a spatial resolution of 98 x 98 x 500 microm and 195 x 390 x 1,000 microm, respectively. The pathophysiologic features revealed in the contrast-enhanced images were analyzed in reference to those obtained from the corresponding high resolution T2-weighted, spin-echo images and from histologic sections stained with hematoxylin and eosin and with an endothelial cell marker. The results showed that iron oxide microspheres can aid in the characterization of gross histopathologic features and in the assessment of the distribution of the microvasculature, whereas Gd-DTPA estimates the permeability of the microvessels to this agent and determines the cellularity (cell volume fraction) in the vicinity of the vessels.

TNF-induced modulations of phospholipid metabolism in human breast cancer cells.

Tumor necrosis factor alpha (TNF) is a cytokine that is cytocidal for certain tumor cells and induces necrotic and apoptotic forms of cell death. Flow cytometry and transmission electron microscopy analysis demonstrated that in human breast cancer cells (MCF7) TNF induces cell cycle arrest in G0+G1/S, accompanied by apoptosis. 31P and 13C NMR spectroscopy was applied to study cellular metabolism of MCF7 cells during TNF-induced signal to apoptosis. Deuterated choline and 2H NMR spectroscopy were utilized to monitor the kinetics of the rate limiting reactions in phosphocholine metabolism. The NMR measurements revealed that immediately after administration of TNF, choline transport was inhibited by 52+/-6%. Later (approximately 15 h), the activity of phosphocholine:cytidine triphosphate cytidylyltransferase, a key enzyme in the biosynthesis of phosphatidylcholine, was enhanced two-fold. These two opposing changes led to a decrease in the level of phosphocholine. Throughout these changes the energetic state of the cells, determined by the level of nucleoside triphosphates and the rate of glucose metabolism via glycolysis, remained constant. The results indicate that TNF specifically modulates the kinetics of membrane-bound enzymes of the rate determining steps in phosphatidylcholine biosynthesis, possibly as part of early events involved in apoptosis.

Enhancement of ATP levels and glucose metabolism during an infection by Chlamydia. NMR studies of living cells.

The Chlamydia species are obligate intracellular bacteria that proliferate only within the infected cell. Since the extracellular bacteria are metabolically inert and there are no cell-free systems for characterizing Chlamydia metabolism, we studied metabolic changes related to ATP synthesis and glycolysis in HeLa cells infected with Chlamydia psittaci during the course of the 2-day infection cycle using noninvasive 31P and 13C NMR methods. We find that the infection stimulates ATP synthesis in the infected cell, with a peak of ATP levels occurring midway through the infection cycle, when most of the metabolically active bacteria are proliferating. The infection also stimulates synthesis of glutamate with a similar time course as for ATP. The stimulation is apparently due to an enhancement in glucose consumption by the infected cell, which also results in an increased rate of lactate production and glutamate synthesis as well as higher glycogen accumulation during the infection. Concurrently, infection leads to an increase in the expression of the glucose transporter, GLUT-1, on HeLa cells, which may account for the enhanced glucose consumption. The chlamydiae are thus able to stimulate glucose transport in the host cell sufficiently to compensate for the extra energy load on the cell represented by the infection.

Dynamic contrast-enhanced imaging and analysis at high spatial resolution of MCF7 human breast tumors.

High resolution, dynamic GdDTPA-enhanced images of MCF7 human breast tumors in immunodeficient mice were analyzed at pixel resolution. The analysis, based on a physiological model, was performed by applying a nonlinear least-square algorithm using a color coded scale. The final output mapped at pixel resolution capillary permeability times surface area and fraction of extracellular volume, for each tumor slice. In addition, the output included assessment of the fit to the model by determining the proportion of variability (R2) for each pixel. The spatial variation in the R2 values served to identify regions where the predominant mechanism of enhancement was leakage from the intravascular volume to the extracellular volume (R2 close to 1). In regions with low R2 other mechanisms of enhancement appear to be dominating presumably diffusion within the extracellular space. As expected, in necrotic regions lacking microcapillaries and identified by analyzing T2-weighted images of the same tumors, the model failed to fit the dynamic contrast enhanced data. The heterogeneous distribution of the determined pathophysiological features demonstrates the importance of recording and analyzing breast tumor images at high spatial resolution.