Semi-parametric time-domain quantification of HR-MAS data from prostate tissue.

High Resolution--Magic Angle Spinning (HR-MAS) spectroscopy provides rich biochemical profiles that require accurate quantification to permit biomarker identification and to understand the underlying pathological mechanisms. Meanwhile, quantification of HR-MAS data from prostate tissue samples is challenging due to significant overlap between the resonant peaks, the presence of short T2* metabolites such as citrate or polyamines (T2 from 25 to 100 msec) and macromolecules, and variations in chemical shifts and T2*s within a metabolite's spin systems. Since existing methods do not address these challenges completely, a new quantification method was developed and optimized for HR-MAS data acquired with an ultra short T(E) and over 30,000 data points. The proposed method, named HR-QUEST (High Resolution--QUEST), iteratively employs the QUEST time-domain semi-parametric strategy with a new model function that incorporates prior knowledge from whole and subdivided metabolite signals. With these features, HR-QUEST is able to independently fit the chemical shifts and T2*s of a metabolite's spin systems, a necessity for HR-MAS data. By using the iterative fitting approach, it is able to account for significant contributions from macromolecules and to handle shorter T2 metabolites, such as citrate and polyamines. After subdividing the necessary metabolite basis signals, the root mean square (RMS) of the residual was reduced by 52% for measured HR-MAS data from prostate tissue. Monte Carlo studies on simulated spectra with varied macromolecular contributions showed that the iterative fitting approach (6 iterations) coupled with inclusion of long T2 macromolecule components in the basis set improve the quality of the fit, as assessed by the reduction of the RMS of the residual and of the RMS error of the metabolite signal estimate, by 27% and 71% respectively. With this optimized configuration, HR-QUEST was applied to measured HR-MAS prostate data and reliably quantified 16 metabolites and reference signals with estimated Cramér Rao Bounds ≤5%.

Kinetic modeling of hyperpolarized 13C1-pyruvate metabolism in normal rats and TRAMP mice.

PURPOSE: To investigate metabolic exchange between (13)C(1)-pyruvate, (13)C(1)-lactate, and (13)C(1)-alanine in pre-clinical model systems using kinetic modeling of dynamic hyperpolarized (13)C spectroscopic data and to examine the relationship between fitted parameters and dose-response. MATERIALS AND METHODS: Dynamic (13)C spectroscopy data were acquired in normal rats, wild type mice, and mice with transgenic prostate tumors (TRAMP) either within a single slice or using a one-dimensional echo-planar spectroscopic imaging (1D-EPSI) encoding technique. Rate constants were estimated by fitting a set of exponential equations to the dynamic data. Variations in fitted parameters were used to determine model robustness in 15 mm slices centered on normal rat kidneys. Parameter values were used to investigate differences in metabolism between and within TRAMP and wild type mice. RESULTS: The kinetic model was shown here to be robust when fitting data from a rat given similar doses. In normal rats, Michaelis-Menten kinetics were able to describe the dose-response of the fitted exchange rate constants with a 13.65% and 16.75% scaled fitting error (SFE) for k(pyr-->lac) and k(pyr-->ala), respectively. In TRAMP mice, k(pyr-->lac) increased an average of 94% after up to 23 days of disease progression, whether the mice were untreated or treated with casodex. Parameters estimated from dynamic (13)C 1D-EPSI data were able to differentiate anatomical structures within both wild type and TRAMP mice. CONCLUSIONS: The metabolic parameters estimated using this approach may be useful for in vivo monitoring of tumor progression and treatment efficacy, as well as to distinguish between various tissues based on metabolic activity.

Methods for metabolic evaluation of prostate cancer cells using proton and (13)C HR-MAS spectroscopy and [3-(13)C] pyruvate as a metabolic substrate.

Prostate cancer has been shown to undergo unique metabolic changes associated with neoplastic transformation, with associated changes in citrate, alanine, and lactate concentrations. (13)C high resolution-magic angle spinning (HR-MAS) spectroscopy provides an opportunity to simultaneously investigate the metabolic pathways implicated in these changes by using (13)C-labeled substrates as metabolic probes. In this work, a method to reproducibly interrogate metabolism in prostate cancer cells in primary culture was developed using HR-MAS spectroscopy. Optimization of cell culture protocols, labeling parameters, harvesting, storage, and transfer was performed. Using [3-(13)C] pyruvate as a metabolic probe, (1)H and (13)C HR-MAS spectroscopy was used to quantify the net amount and fractional enrichment of several labeled metabolites that evolved in multiple cell samples from each of five different prostate cancers. Average enrichment across all cancers was 32.4 +/- 5.4% for [3-(13)C] alanine, 24.5 +/- 5.4% for [4-(13)C] glutamate, 9.1 +/- 2.5% for [3-(13)C] glutamate, 25.2 +/- 5.7% for [3-(13)C] aspartate, and 4.2 +/- 1.0% for [3-(13)C] lactate. Cell samples from the same parent population demonstrated reproducible fractional enrichments of alanine, glutamate, and aspartate to within 12%, 10%, and 10%, respectively. Furthermore, the cells produced a significant amount of [4-(13)C] glutamate, which supports the bioenergetic theory for prostate cancer. These methods will allow further characterization of metabolic properties of prostate cancer cells in the future. Magn Reson Med, 2009. (c) 2009 Wiley-Liss, Inc.

Evaluation of the ERETIC method as an improved quantitative reference for 1H HR-MAS spectroscopy of prostate tissue.

The Electronic REference To access In vivo Concentrations (ERETIC) method was applied to (1)H HR-MAS spectroscopy. The accuracy, precision, and stability of ERETIC as a quantitative reference were evaluated in solution and human prostate tissue samples. For comparison, the reliability of 3-(trimethylsilyl)propionic-2,2,3,3-d(4) acid (TSP) as a quantitation reference was also evaluated. The ERETIC and TSP peak areas were found to be stable in solution over the short-term and long-term, with long-term relative standard deviations (RSDs) of 4.10% and 2.60%, respectively. Quantification of TSP in solution using the ERETIC peak as a reference and a calibrated, rotor-dependent conversion factor yielded results with a precision < or =2.9% and an accuracy error < or =4.2% when compared with the expected values. The ERETIC peak area reproducibility was superior to TSP's reproducibility, corrected for mass, in both prostate surgical and biopsy samples (4.53% vs. 21.2% and 3.34% vs. 31.8%, respectively). Furthermore, the tissue TSP peaks exhibited only 27.5% of the expected area, which would cause an overestimation of metabolite concentrations if used as a reference. The improved quantification accuracy and precision provided by ERETIC may enable the detection of smaller metabolic differences that may exist between individual tissue samples and disease states.

Hyperpolarized 13C lactate, pyruvate, and alanine: noninvasive biomarkers for prostate cancer detection and grading.

An extraordinary new technique using hyperpolarized (13)C-labeled pyruvate and taking advantage of increased glycolysis in cancer has the potential to improve the way magnetic resonance imaging is used for detection and characterization of prostate cancer. The aim of this study was to quantify, for the first time, differences in hyperpolarized [1-(13)C] pyruvate and its metabolic products between the various histologic grades of prostate cancer using the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Fast spectroscopic imaging techniques were used to image lactate, alanine, and total hyperpolarized carbon (THC = lactate + pyruvate + alanine) from the entire abdomen of normal mice and TRAMP mice with low- and high-grade prostate tumors in 14 s. Within 1 week, the mice were dissected and the tumors were histologically analyzed. Hyperpolarized lactate SNR levels significantly increased (P < 0.05) with cancer development and progression (41 +/- 11, 74 +/- 17, and 154 +/- 24 in normal prostates, low-grade primary tumors, and high-grade primary tumors, respectively) and had a correlation coefficient of 0.95 with the histologic grade. In addition, there was minimal overlap in the lactate levels between the three groups with only one of the seven normal prostates overlapping with the low-grade primary tumors. The amount of THC, a possible measure of substrate uptake, and hyperpolarized alanine also increased with tumor grade but showed more overlap between the groups. In summary, elevated hyperpolarized lactate and potentially THC and alanine are noninvasive biomarkers of prostate cancer presence and histologic grade that could be used in future three-dimensional (13)C spectroscopic imaging studies of prostate cancer patients.

Evaluation of lactate and alanine as metabolic biomarkers of prostate cancer using 1H HR-MAS spectroscopy of biopsy tissues.

The goal of this study was to investigate the use of lactate and alanine as metabolic biomarkers of prostate cancer using (1)H high-resolution magic angle spinning (HR-MAS) spectroscopy of snap-frozen transrectal ultrasound (TRUS)-guided prostate biopsy tissues. A long-echo-time rotor-synchronized Carr-Purcell-Meiboom-Gill (CPMG) sequence including an electronic reference to access in vivo concentrations (ERETIC) standard was used to determine the concentrations of lactate and alanine in 82 benign and 16 malignant biopsies (mean 26.5% +/- 17.2% of core). Low concentrations of lactate (0.61 +/- 0.28 mmol/kg) and alanine (0.14 +/- 0.06 mmol/kg) were observed in benign prostate biopsies, and there was no significant difference between benign predominantly glandular (N = 54) and stromal (N = 28) biopsies between patients with (N = 38) and without (N = 44) a positive clinical biopsy. In biopsies containing prostate cancer there was a highly significant (P < 0.0001) increase in lactate (1.59 +/- 0.61 mmol/kg) and alanine (0.26 +/- 0.07 mmol/kg), and minimal overlap with lactate concentrations in benign biopsies. This study demonstrates for the first time very low concentrations of lactate and alanine in benign prostate biopsy tissues. The significant increase in the concentration of both lactate and alanine in biopsy tissue containing as little as 5% cancer could be exploited in hyperpolarized (13)C spectroscopic imaging (SI) studies of prostate cancer patients.

Hyperpolarized C-13 spectroscopic imaging of the TRAMP mouse at 3T-initial experience.

The transgenic adenocarcinoma of mouse prostate (TRAMP) mouse is a well-studied murine model of prostate cancer with histopathology and disease progression that mimic the human disease. To investigate differences in cellular bioenergetics between normal prostate epithelial cells and prostate tumor cells, in vivo MR spectroscopic (MRS) studies with non-proton nuclei, such as (13)C, in the TRAMP model would be extremely useful. The recent development of a method for retaining dynamic nuclear polarization (DNP) in solution permits high signal-to-noise ratio (SNR) (13)C MRI or MRSI data to be obtained following injection of a hyperpolarized (13)C agent. In this transgenic mouse study, this method was applied using a double spin-echo (DSE) pulse sequence with a small-tip-angle excitation RF pulse, hyperbolic-secant refocusing pulses, and a flyback echo-planar readout trajectory for fast (10-14 s) MRSI of (13)C pyruvate (pyr) and its metabolic products at 0.135 cm(3) nominal spatial resolution. Elevated (13)C lactate (lac) was observed in both primary and metastatic tumors, demonstrating the feasibility of studying cellular bioenergetics in vivo with DNP hyperpolarized (13)C MRSI.

Double spin-echo sequence for rapid spectroscopic imaging of hyperpolarized 13C.

Dynamic nuclear polarization of metabolically active compounds labeled with (13)C has been introduced as a means for imaging metabolic processes in vivo. To differentiate between the injected compound and the various metabolic products, an imaging technique capable of separating the different chemical-shift species must be used. In this paper, the design and testing of a pulse sequence for rapid magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized (13)C is presented. The pulse sequence consists of a small-tip excitation followed by a double spin echo using adiabatic refocusing pulses and a "flyback" echo-planar readout gradient. Key elements of the sequence are insensitivity to calibration of the transmit gain, the formation of a spin echo giving high-quality spectral information, and a small effective tip angle that preserves the magnetization for a sufficient duration. Experiments in vivo showed three-dimensional coverage with excellent spectral quality and SNR.

Proton-decoupled 31P MRS in untreated pediatric brain tumors.

Proton-decoupled (31)P and (1)H MRS was used to quantify markers of membrane synthesis and breakdown in eight pediatric patients with untreated brain tumors and in six controls. Quantitation of these compounds in vivo in humans may provide important indicators for tumor growth and malignancy, tumor classification, and provide prognostic information. The ratios of phosphoethanolamine to glycerophosphoethanolamine (PE/GPE) and phosphocholine to glycerophosphocholine (PC/GPC) were significantly higher in primitive neuroectodermal tumors (PNET) (16.30 +/- 5.73 and 2.97 +/- 0.93) when compared with controls (3.42 +/- 1.62, P < 0.0001 and 0.45 +/- 0.13, P < 0.0001) and with other tumors (3.93 +/- 3.42, P < 0.001 and 0.65 +/- 0.30, P < 0.0001). Mean PC/PE was elevated in tumors relative to controls (0.48 +/- 0.11 versus 0.24 +/- 0.05, P < 0.001), but there was no difference between PNET and other tumors. Total choline concentration determined with quantitative (1)H MRS was significantly elevated (4.78 +/- 3.33 versus 1.73 +/- 0.56 mmol/kg, P < 0.05), whereas creatine was reduced in tumors (4.89 +/- 1.83 versus 8.28 +/- 1.50 mmol/kg, P < 0.05). A quantitative comparison of total phosphorylated cholines (PC+GPC)/ATP measured with (31)P MRS and total choline measured with (1)H MRS showed that in tumors a large fraction of the choline signal (>54 +/- 36%) was not accounted for by PC and GPC. The fraction of unaccounted choline was particularly large in PNET (>78 +/- 7%). The pH of tumor tissue was higher than the pH of normal brain tissue (7.06 +/- 0.03 versus. 6.98 +/- 0.03, P < 0.001).