tCho Levels Research Articles

AB0589 Steroid-sparing effect of tocilizumab in the treatment of rheumatoid arthritis

BackgroundLong-term use of corticosteroid is often required in rheumatoid arthritis(RA), but it is suggested that dose dependent relationship between prednisone treatment and the risk of pneumonia in RA patients1. Therefore,...

Improved diagnostic accuracy in differentiating malignant and benign lesions using single-voxel proton MRS of the breast at 3 T MRI

To investigate the diagnostic accuracy of single-voxel proton magnetic resonance spectroscopy (SV (1)H MRS) by quantifying total choline-containing compounds (tCho) in differentiating malignant from benign lesions, and subsequently, to analyse the relationship of tCho levels in malignant breast lesions with their histopathological subtypes. A prospective study of SV 1H MRS was performed following dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in 61 women using a 3 T MR system. All lesions (n=57) were analysed for characteristics of morphology, contrast-enhancement kinetics, and tCho peak heights at SV (1)H MRS that were two-times above baseline. Subsequently, the tCho in selected lesions (n=32) was quantified by calculating the area under the curve, and a tCho concentration equal to or greater than the cut-off value was considered to represent malignancy. The relationship between tCho in invasive ductal carcinomas (IDCs) and their Bloom & Richardson grading of malignancy was assessed. Fifty-two patients (57 lesions; 42 malignant and 15 benign) were analysed. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), of predicting malignancy were 100, 73.3, 91.3, and 100%, respectively, using DCE-MRI and 95.2, 93.3, 97.6, and 87.5%, respectively, using SV (1)H MRS. The tCho cut-off for receiver operating characteristic (ROC) curve was 0.33mmol/l. The relationship between tCho levels in malignant breast lesions with their histopathological subtypes was not statistically significant (p=0.3). Good correlation between tCho peaks and malignancy, enables SV (1)H MRS to be used as a clinically applicable, simple, yet non-invasive tool for improved specificity and diagnostic accuracy in detecting breast cancer.

Abstract 1327: IGF-I and insulin signaling enhances levels of choline kinase in breast cancer cells: Implications for developing noninvasive magnetic resonance spectroscopy of choline containing compounds as a biomarker of response to IGF1R targeted therapy.

Abstract Insulin-like growth factor I (IGF-I) and insulin signal through the type I IGF receptor (IGF1R) and insulin receptor (IR) respectively. Multiple drugs targeting IGF1R and IGF1R/IR are being tested in clinical trials. Biomarkers that enable the selection of patients with IGF driven tumors or the monitoring of response to this therapy have not yet been identified. Previously, we found that intratumoral total choline (tCho) metabolites decreased in MCF-7 xenograft tumors in mice twenty-four hours following administration of an inhibitory antibody against IGF1R. The tCho was measured by 1H magnetic resonance spectroscopy (MRS) and included three species of choline containing compounds, choline, phosphocholine (pCho), and glycerophosphocholine. These data suggested that noninvasive measurement of tCho may be a useful surrogate marker of response to IGF1R targeted therapy. The objective of the current study is to investigate whether there is a mechanistic link between IGF-I and insulin signaling and choline phospholipid metabolism to understand if decreased tCho levels could be a specific marker of inhibition of IGF signaling in cancer cells. In this study, we examined the effect of IGF-I and insulin signaling on levels of choline kinase (CK). CK is the enzyme that generates pCho, one of the species measured by MRS in our prior in vivo study. Estrogen receptor positive (ER+) human breast cancer cell lines (MCF-7, T47-D, ZR-75-1) and triple negative cells (MDA-MB-231, MDA-231-LM2 and MDA-MB-435A/LCC6) were treated with IGF-I or insulin for 10 minutes, 4h or 24h and levels of CK were examined. IGF-I and insulin signaling upregulated CK levels in ER+ breast cancer cells (MCF-7, T47-D, and ZR-75-1) but not in triple negative breast cancer cells (MDA-MB-231, MDA-231-LM2 and MDA-MB-435A/LCC6). In ER+ breast cancer cells, IGF-I and insulin activate the mitogen-activated protein kinase (MAPK) pathway. However, in triple negative breast cancer cells phosphorylation of p44/p42 MAPK are unaffected by IGF-I and insulin signaling or IGF1R inhibition. Inhibition of the MAPK pathway with U0126 blocked IGF-I and insulin induced upregulation of CK. These data suggest that IGF-I and insulin induce upregulation of CK via a MAPK dependent mechanism. We next investigated the effect of EM164, an inhibitory antibody against IGF1R, on IGF-I and insulin induced upregulation of CK. EM164 blocked IGF-I induced upregulation of CK but did not affect insulin induced upregulation of CK. These results provide molecular evidence for the observed decrease in intratumoral tCho after IGF1R inhibition in xenograft tumors. Our study is clinically significant as it provides evidence for developing decreased tCho, following IGF1R targeted drugs, as a specific biomarker for inhibition of IGF signaling. Citation Format: Huy T. Donguyen, Joseph C. Weber, Deepali Sachdev. IGF-I and insulin signaling enhances levels of choline kinase in breast cancer cells: Implications for developing noninvasive magnetic resonance spectroscopy of choline containing compounds as a biomarker of response to IGF1R targeted therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1327. doi:10.1158/1538-7445.AM2013-1327

Multimodal Elucidation of Choline Metabolism in a Murine Glioma Model Using Magnetic Resonance Spectroscopy and 11C-Choline Positron Emission Tomography

The metabolites, transporters, and enzymes involved in choline metabolism are regarded as biomarkers for disease progression in a variety of cancers, but their in vivo detection is not ideal. Both magnetic resonance spectroscopy [MRS using chemical shift imaging (CSI) total choline (tCho)] and C-choline positron emission tomography (PET) can probe this pathway, but they have not been compared side by side. In this study, we used the spontaneous murine astrocytoma model SMA560 injected intracranially into syngeneic VM/Dk mice, analyzing animals at various postimplantation time points using dynamic microPET imaging and CSI MRS. We observed an increase in tumor volume and C-choline uptake between days 5 and 18. Similarly, tCho levels decreased at days 5 to 18. We found a negative correlation between the tCho and PET results in the tumor and a positive correlation between the tCho tumor-to-brain ratio and choline uptake in the tumor. PCR results confirmed expected increases in expression levels for most of the transporters and enzymes. Using MRS quantification, a good agreement was found between CSI and C-choline PET data, whereas a negative correlation occurred when CSI was not referenced. Thus, C-choline PET and MRS methods seemed to be complementary in strengths. While advancing tumor proliferation caused an increasing C-choline uptake, gliosis and inflammation potentially accounted for a high peritumoral tCho signal in CSI, as supported by histology and secondary ion mass spectrometry imaging. Our findings provide definitive evidence of the use of MRS, CSI, and PET for imaging tumors in vivo.

Abstract WP437: The Relationship Between Cholesterol And Cerebral Vasospasm In A Rat Model Of Subarachnoid Hemorrhage: A Mechanistic Role For Lipid Rafts

Objective: Rho-kinase (ROK)-mediated vascular smooth muscle (VSM) contraction plays a pivotal role in cerebral vasospasm (CV). Sphingosylphosphorylcholine (SPC), a sphingolipid, induces ROK-mediated VSM contraction, and hypercholesterolemia increases SPC-ROK-mediated VSM contraction. Lipid rafts are cholesterol-enriched membrane microdomains that influence signal transduction. In this study, we report the effect of cholesterol and lipid rafts on CV. Methods: Adult male Sprague-Dawley rats (400-500g, n=53) were fed control, 1% cholesterol or 1% cholesterol +5% β-cyclodextrin (β-CD), which depletes VSM cholesterol, diets for 8 weeks. We used a double-hemorrhage rat subarachnoid hemorrhage (SAH) model, and investigated CV of the basilar artery (BA) at Day5 using a cranial window. T-Cho levels in the internal carotid artery (ICA) were measured using Gas Chromatography. The expression of Flotillin-1, a raft marker, and Flotillin-1 mRNA in the ICA were analysed by Western blot and real-time RT-PCR, respectively. Results: The BA diameters were 358.94±32.05μm and 302.21±38.36μm in control diet (non-treated) and SAH rats, respectively (n=9/group, p<0.05 vs. non-treated). SAH-induced BA contraction was markedly reversed by intracisternal infusion of 10μM Y27632 (n=4, p<0.05), a ROK inhibitor. The BA diameter in SAH rats fed the cholesterol diet was 248.11±34.56μm (n=6, p<0.05 vs. control SAH-rats), compared with 305±25.28μm in SAH rats fed a β-CD diet (n=6, p<0.05 vs. SAH-rat fed a cholesterol diet). T-Cho levels in the ICA were significantly higher in rats fed a cholesterol diet compared with the control diet (0.82±0.24 vs. 0.49±0.09mg/g, n=5/group, p=0.028) or the β-CD diet (0.47±0.06 mg/g, n=5, p=0.016 vs. cholesterol diet). Western blot analysis and real-time RT-PCR revealed increases in Flotillin-1 and Flotillin-1 mRNA expression in the ICA from rats fed a cholesterol diet, whereas Flotillin-1 and Flotillin-1 mRNA expression were not altered in the ICA from rats fed the β-CD diet. Conclusions: These results indicate that cholesterol potentiates SAH-induced CV and are compatible with a role for lipid rafts in this process.

Effectiveness of Crowberry on Plasma Total Antioxidant Status, Lipid Profile and Homocysteine

The objective of this study was to assess whether the consumption of crowberry could affect serum lipid profile, homocysteine (Hcy) level, and antioxidant status of healthy subjects. Out of 55 healthy volunteered subjects, 51 completed this investigation to final analysis. Each subject consumed 2 grams of powdered crowberries everyday for four weeks. Crowberry consumption led to significant increase in total antioxidant status (TAS) and superoxide dismutase (SOD), while it resulted in significant decrease in total cholesterol and low-density lipoprotein (LDL) levels. The differences in the levels of antioxidant markers and lipid profiles taken before and after crowberry intake were most significant. The levels of Hcy, catalase, T-cho, triglyceride, and LDL in the higher T-cho group decreased significantly after crowberry intake. Furthermore, this study revealed a significant positive correlation between Hcy level and body weight index (BMI). Crowberry intake improved lipid profile as demonstrated by having decreased T-cho, TG and LDL; increased antioxidative reactions (such as TAS or SOD enzymatic activities) and decreased Hcy levels in healthy subjects.

Malignant transformation: The role of MRS

Magnetic Resonance Spectroscopy (MRS) has been instrumental in elucidating the metabolic changes that arise as a consequence of malignant transformation. The metabolic phenotype of cancers is characterized by high glucose uptake, high glycolytic activity and lactate production, low mitochondrial activity, low bioenergetic status, and abnormal phospholipid metabolism [1,2]. As shown in Fig. 1, noninvasive H MRS is able to detect elevated lactate and increased total choline (tCho) levels in breast (and other) cancers, which primarily arise from oncogenic signaling pathways in cancer cells as indicated by green boxes in Fig. 1A. The increased levels of lactate and tCho provide noninvasive MR biomarkers of transformation, staging, and response to therapy. Enzymes in glucose and choline metabolism present targets for image-guided cancer therapy.

MR-Spectroscopy at 1.5 tesla and 3 tesla. Useful? A systematic review and meta-analysis

Dynamic contrast enhanced magnetic resonance imaging is the most sensitive method for detection of breast cancer [1,2]. These T1-weighted imaging studies measure the extracellular distribution of paramagnetic contrast agents which are mainly affected by vascular properties of the tissue investigated. Although cancers show a characteristically early and strong enhancement, substantial overlaps of enhancement characteristics between benign and malignant breast lesions have been described. Consequently, for lesion classification in clinical practice, a combination of morphologic criteria and dynamic enhancement pattern analysis is applied [3]. Such an approach is mainly subjective and thus prone to experience related mistakes and interobserver variation. Proton magnetic resonance spectroscopy (H MR spectroscopy, MRS) is a noninvasive examination technique for the assessment of biochemical tissue properties. Presence of a compound resonance around 3.23 ppm is attributed to choline metabolites such as choline, phosphocholine and glycerophosphocholine and simply referred to as total choline (tCho). Increased levels of tCho have been detected in malignant cancers and are ascribed to an increased cellular membrane turnover [4–6]. In vivo qualitative and quantitative tCho measurements have been used as a diagnostic test in the workup of neoplastic breast lesions. The present systematic review and meta-analysis aims to investigate the diagnostic capability of tCho measurements for the differentiation of breast lesions focusing on state of the art field strengths for clinical breast imaging of 1.5 and 3 tesla.

Localized Hypoxia Results in Spatially Heterogeneous Metabolic Signatures in Breast Tumor Models

Tumor hypoxia triggers signaling cascades that significantly affect biologic outcomes such as resistance to radiotherapy and chemotherapy in breast cancer. Hypoxic regions in solid tumor are spatially heterogeneous. Therefore, delineating the origin and extent of hypoxia in tumors is critical. In this study, we have investigated the effect of hypoxia on different metabolic pathways, such as lipid and choline metabolism, in a human breast cancer model. Human MDA-MB-231 breast cancer cells and tumors, which were genetically engineered to express red fluorescent tdTomato protein under hypoxic conditions, were used to investigate hypoxia. Our data were obtained with a novel three-dimensional multimodal molecular imaging platform that combines magnetic resonance (MR) imaging, MR spectroscopic imaging (MRSI), and optical imaging of hypoxia and necrosis. A higher concentration of noninvasively detected total choline-containing metabolites (tCho) and lipid CH3 localized in the tdTomato-fluorescing hypoxic regions indicated that hypoxia can upregulate tCho and lipid CH3 levels in this breast tumor model. The increase in tCho under hypoxia was primarily due to elevated phosphocholine levels as shown by in vitro MR spectroscopy. Elevated lipid CH3 levels detected under hypoxia were caused by an increase in mobile MR-detectable lipid droplets, as demonstrated by Nile Red staining. Our findings demonstrate that noninvasive MRSI can help delineate hypoxic regions in solid tumors by means of detecting the metabolic outcome of tumor hypoxia, which is characterized by elevated tCho and lipid CH3.

Quantitative mapping of total choline in healthy human breast using proton echo planar spectroscopic imaging (PEPSI) at 3 Tesla

To quantitatively measure tCho levels in healthy breasts using Proton-Echo-Planar-Spectroscopic-Imaging (PEPSI). The two-dimensional mapping of tCho at 3 Tesla across an entire breast slice using PEPSI and a hybrid spectral quantification method based on LCModel fitting and integration of tCho using the fitted spectrum were developed. This method was validated in 19 healthy females and compared with single voxel spectroscopy (SVS) and with PRESS prelocalized conventional Magnetic Resonance Spectroscopic Imaging (MRSI) using identical voxel size (8 cc) and similar scan times (∼7 min). A tCho peak with a signal to noise ratio larger than 2 was detected in 10 subjects using both PEPSI and SVS. The average tCho concentration in these subjects was 0.45 ± 0.2 mmol/kg using PEPSI and 0.48 ± 0.3 mmol/kg using SVS. Comparable results were obtained in two subjects using conventional MRSI. High lipid content in the spectra of nine tCho negative subjects was associated with spectral line broadening of more than 26 Hz, which made tCho detection impossible. Conventional MRSI with PRESS prelocalization in glandular tissue in two of these subjects yielded tCho concentrations comparable to PEPSI. The detection sensitivity of PEPSI is comparable to SVS and conventional PRESS-MRSI. PEPSI can be potentially used in the evaluation of tCho in breast cancer. A tCho threshold concentration value of ∼0.7 mmol/kg might be used to differentiate between cancerous and healthy (or benign) breast tissues based on this work and previous studies.

Abstract 2501: Inhibition of the PI3K pathway potentiates temozolomide effects in pediatric glioblastoma and results in alterations in glucose and choline metabolism detected by MRS

Abstract Introduction: Children with high grade-glioma have a poor outcome with a 5-year survival rate of less than 20%. The use of adjuvant temozolomide (TMZ) has now been well accepted as a standard of care in glioblastoma. However, it does not seem to improve survival in children. We have recently shown that resistance to TMZ in pediatric glioma cell lines can be reversed by PI3K pathway inhibition. The aim of this study was to identify whether combination of PI3K pathway inhibition with TMZ would result in metabolic changes detectable by magnetic resonance spectroscopy (MRS), and potentially can be used as a noninvasive method of monitoring response in early stage clinical trials in children with glioma. Methods: The MGMT-independent TMZ-resistant pediatric glioblastoma cell line KNS42 was treated for 48h with dual PI3K/mTOR inhibitor PI-103 (2xGI50), TMZ (2xGI50) or with the combination of both (each at 2xGI50). Control and treated cells were extracted and the aqueous fractions were analyzed by 1H MRS. Results and Discussion: The number of treated KNS42 cells was significantly reduced relative to their controls, to 79±8% and 65±10% (P<0.02) following treatment with PI-103 and TMZ respectively. Combination of PI-103 and TMZ further reduced the number of treated vs. control cells (down to 54±6%, P<0.01). 1H MRS analysis showed that PI-103 treatment caused a decrease in lactate, phosphocholine (PC) and total choline (tCho) levels down to 48±17%, 52±11%, 64±14% (P<0.02) respectively. In contrast, treament with TMZ resulted in an increase in glycerolphosphocholine (GPC) up to 176±43% (P=0.05). Combination of PI-103 with TMZ retained the combined metabolic effects of both agents including a decrease in the levels of lactate (49±12%, P<0.01) and PC (71±11%, P<0.02) while an increase in GPC (190±40%, P<0.03) levels. tCho was normalized by the opposing changes in PC and GPC and hence was similar to controls. To explore potential mechanisms underlying the detected metabolic changes, immunoblotting was used to assess treatment effects on protein levels of enzymes involved in glycolysis and choline metabolism. The decrease in PC levels was associated with a decrease in the protein levels of Choline kinase (ChoK), the enzyme responsible for choline phosphorylation into PC. ChoK was not affected by TMZ single treatment leading to no change in PC levels. Furthermore, a decrease in hexokinase II protein was detected following PI-103 treatment and the combination, but not with TMZ alone, suggesting a mechanism for depletion of lactate. Conclusions: Our results show that using MRS we can detect distinct biomarkers following treatment with PI-103 compared to TMZ. Choline metabolites and lactate are potential noninvasive biomarkers for monitoring response to PI3K/mTOR inhibitors both as single agents and in combination with cytotoxic therapy in children with glioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2501. doi:1538-7445.AM2012-2501

Long term intensive exercise training leads to a higher plasma malate/lactate dehydrogenase (M/L) ratio and increased level of lipid mobilization in horses

Continuous high intensity training may induce alterations to enzyme activities related to glucose and lipid metabolism in horses. In our study, five Thoroughbred race horses (3 male and 2 female, avg age=5 yrs old) were compared against five riding horses (1 male, 1 female, 3 gelding, avg age=13 yrs old) in terms of energy metabolism, by examining plasma malate (MDH) and lactate (LDH) dehydrogenase activities and M/L ratio. MDH is involved in NADH and ATP generation, whereas LDH can convert NADH back into NAD(+) for ATP generation. An increase in plasma M/L ratio can reflect heightened energy metabolism in the liver and skeletal muscle of horses adapted to continuous intensive exercise. Moreover, plasma lipid metabolism analytes (adiponectin, NEFA, total cholesterol (T-Cho), and triglycerides (TG)) can reflect changes to lipolysis rate, which can also indicate a change in energy metabolism. Overall, race horses demonstrated increased MDH and LDH activity in plasma (4x and 2x greater, respectively), in addition to a plasma M/L ratio twice as high as that of riding horses (2.0 vs 1.0). In addition, race horses also demonstrated significantly higher levels of plasma NEFA (50% greater), TG (2x greater), and T-Cho (20% greater) as compared to riding horses. Therefore, race horse muscles may have adapted to prolonged high intensity endurance exercise by gaining a higher oxidative capacity and an increased capacity for fat utilization as an energy source, resulting in heightened energy metabolism and increased rate of lipid mobilization.

Diagnostic Value of Breast Proton Magnetic Resonance Spectroscopy at 1.5T in Different Histopathological Types

The purpose of this study was to investigate the usefulness of quantitative proton magnetic resonance spectroscopy (1H-MRS) for characterizing breast lesions at 1.5T, and to evaluate the diagnostic performance of in vivo breast 1H-MRS using receiver operating characteristics (ROC) analysis. 112 patients (99 malignant and 13 benign tumors) who were scanned with the MRI/MRS protocol were included in this study. Choline-containing compounds (tCho) levels were measured and compared with histological findings. The measured tCho levels in this work had range of 0.08–9.99 mmol/kg from 65 (66%) of 99 patients with malignant tumors. Of the 13 benign lesions, 1H-MRS detected one as false positive, with tCho level of 0.66 mmol/kg. The optimal tCho level cutoff point that yielded the highest accuracy was found to be >0.0 mmol/kg. The resulting sensitivity was 66% and specificity 92% for distinguishing benign from malignant lesions. The tCho levels were found to be higher in invasive cancer compared to ductal carcinoma in situ or benign lesions, possibly associated with more aggressive behavior or faster cell replication in invasive cancer. Quantitative in vivo 1H-MRS may provide useful information for characterizing histopatholoigical types in breast cancer.

P5-13-05: Non-Invasive In Vivo 1H Magnetic Resonance Spectroscopy (MRS) Monitoring of Breast Tumor Response to IGF1R Targeted Therapy.

Abstract Drugs targeting IGF1R are in clinical trials for breast cancer therapy. To date no suitable biomarkers of response have been identified for this therapy. It is important to select patients with tumors sensitive to disruption of IGF1R signaling. In addition, predicting response in patients quickly following initial treatment would also assist in the development of these strategies. While expression of IGF1R must be a minimum requirement for enrolling patients in trials with IGF1R agents, the mere expression of IGF1R is not necessarily sufficient for IGF-driven biology. We have previously shown that monitoring levels of total choline-containing compounds (tCho) non-invasively in breast cancer patients with locally advanced breast cancer is a useful predictor of response to primary systemic therapy. The tCho measured in these studies included choline, phosphocholine (pCho) and glycerophosphocholine (GPC). In this study, we examined if changes in tCho could be used as a marker of response to anti-IGF1R therapy. We tested the effect of two antibodies against IGF1R on intratumoral tCho levels in vivo. Mice bearing MCF-7 xenografts were subjected to MR imaging (MRI) and spectroscopy (MRS) to measure baseline tCho levels. MRI was performed at 4.7 Tesla (200 mHz) with a transceive surface coil. Single-voxel 1H spectroscopy was performed using water suppression and TE-averaging. A fully relaxed, unsuppressed water spectrum from the same voxel was acquired and tCho signal was referenced against the tissue water signal and expressed as mmol/kg water. The next day, mice were treated with 500 μg of scFv-Fc or phosphate buffered saline, or 800 μg EM164 or isotype-matched control antibody. 24 hours post-treatment, tCho was measured. The percent change in tCho 24h after treatment (% tCho24) was determined using the formula 100[(tCho24-tCho0)/tCho0]. In 9 out 10 mice treated with scFv-Fc, % tCho24 ranged from −26 to −81% of baseline levels while in mice treated with PBS the change was from 0% to +46%. EM164 treatment also caused decrease in tCho levels in 3 out of 3 mice while the control antibody did not. tCho was also monitored for a period of 2 weeks during the course of treatment with EM164 or control antibody; EM164 caused a decrease in tCho with % tCho of −35% while the control antibody treated mice showed % tCho of +43% over the two-week treatment. To further delineate the precise choline species regulated by IGF signaling, MCF-7 cells were treated in vitro with IGF-I for 24h and cell extracts subjected to MRS at 14.1 Tesla using a NMR scanner. IGF-I enhanced levels of pCho compared to choline and GPC. We next investigated if there is a mechanistic link between IGF signaling and choline metabolism. MCF-7 cells were treated with IGF-I for 10 minutes or 24h and levels of choline kinase (ChoK), the rate-limiting enzyme that converts choline to pCho and then phosphatidylcholine were examined. Surprisingly, IGF-I signaling enhanced ChoK levels. Our data show that decrease in tCho could be a surrogate marker of early response to anti-IGF1R therapy. Thus, MRS to measure tCho levels could be included in the design of clinical trials with such reagents to quickly identify patients whose tumors are sensitive to inhibition of IGF1R. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-13-05.

Predicting long‐term survival and treatment response in breast cancer patients receiving neoadjuvant chemotherapy by MR metabolic profiling

This study aimed to evaluate whether MR metabolic profiling can be used for prediction of long-term survival and monitoring of treatment response in locally advanced breast cancer patients during neoadjuvant chemotherapy (NAC). High resolution magic angle spinning (HR MAS) MR spectra of pre- and post-treatment biopsies from 33 patients were acquired. Tissue concentrations of choline-containing metabolites (tCho), glycine and taurine were assessed using electronic reference to access in vivo concentration (ERETIC) of the signal and receiver operating characteristic (ROC) curves was used to define their potential to predict patient survival and treatment response. The metabolite profiles obtained by HR MAS spectroscopy were related to long-term survival and treatment response by genetic algorithm partial least squares discriminant analysis (GA PLS-DA). Different pre-treatment MR metabolic profiles characterized by higher levels of tCho and lower levels of lactate were observed in patients with long-term survival (≥5 years, survivors) compared to patients who died of cancer recurrence (<5 years, non-survivors). A significant decrease in glycerophosphocholine (GPC) post-treatment was associated with long-term survival (p = 0.046) and partial response (p = 0.014) to NAC. Long-term survival was best predicted by GPC using ROC analyses (sens. 66.7%, spec. 62.5%), while taurine had the best predictive value of treatment response (sens. 72.7%, spec. 63.2%). GA PLS-DA multivariate classification models successfully discriminated between survivors and non-survivors, resulting in 82.7% and 90.2% cross-validation (CV) classification accuracy, pre- and post-treatment, respectively. Classification of treatment response using GA PLS-DA was not successful for this patient cohort. Our results demonstrate that HR MAS MR metabolic profiles consisting of important metabolic characteristics of breast cancer tumors could potentially assist the classification and prediction of long-term survival in locally advanced breast cancer patients, in addition to being used as an adjunct for evaluation of treatment response to NAC.

The Influence of Being Overweight on the Relationship Between Shift Work and Increased Total Cholesterol Level

The aim of this study was to clarify the influence of shift work on serum total cholesterol (T-Cho) levels according to body mass index (BMI) at entry in Japanese male workers. A 14-year prospective cohort study was conducted in day workers (n = 4079) and alternating shift workers (n = 2807) who received annual health check-ups between 1991 and 2005 ina Japanese steel company. The association between job schedule and increases in T-Cho was investigated using multivariate pooled logistic regression analyses with age, BMI, lifestyle (smoking habit, drinking habit, habitual exercise), and the results of blood chemistries (creatinine, glycosylated hemoglobin A1c, aspartic aminotransferase, γ-glutamyl transpeptidase, uric acid) serving as covariates in those who were not overweight (BMI < 25 kg/m(2), n = 5082) and overweight (BMI ≥ 25 kg/m(2), n = 1804) at entry, respectively. The endpoints in the study were either a 20%, 25%, 30%, 35%, 40%, or 45% increase in T-Cho during the period of observation compared to T-Cho at entry. In subjects who were not overweight at entry, alternating shift work was associated significantly with five serum T-Cho endpoints (≥20%: odds ratio [OR] = 1.15, 95% confidence interval [CI)]= 1.05, 1.26; ≥25%: OR = 1.17, 95% CI = 1.05, 1.31; ≥35%: OR = 1.24, 95% CI = 1.05, 1.46; ≥40%: OR= 1.30, 95% CI = 1.06, 1.61; ≥45%: OR = 1.31, 95% CI = 1.01, 1.71). However, alternating shift work was not associated with any of the six T-Cho endpoints in subjects who were overweight at entry. In the present study, shift work was shown to be a potential risk factor for increased T-Cho in non-overweight Japanese male workers. However, we did not obtain a consistent association between shift work and an increase in T-Cho levels in overweight subjects. The results suggest that the effect of shift work on lipid regulation may be influenced by BMI.

Age-specific plasma biochemistry reference ranges in &lt;1 year old dogs in Japan

The purpose of this study was to investigate and propose possible reference intervals of plasma biochemical analytes in young dogs (<12 months old) in Japan, using 896 canine plasma samples, collected from an array of veterinary clinics throughout the greater Tokyo metropolis area in Japan. The following biochemical parameters were assessed: albumin (ALB), alanine aminotransferase (ALT), alkaline phosphatase (ALP), amylase, aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CRE), glucose, lipase, total cholesterol (T-Cho), and total protein (TP) were evaluated. Multivariate linear regression analysis indicated that partitioning according to age or gender may be necessary for some plasma analytes. Age appeared to significantly affect ALB, ALT, ALP, BUN, Glucose, Lipase, and Total Protein (P= <0.001, <0.001, <0.001, 0.013, <0.001, 0.025, and <0.001, respectively). On the other hand, gender significantly influenced ALP, Amylase, Lipase, and T-Cho levels (P=0.017, <0.001, <0.001, and <0.001, respectively) whereas it may be borderline significant with ALT (P=0.072).

In vivo1H MRS in the assessment of the therapeutic response of breast cancer patients

MRI and in vivo MRS have rapidly evolved as sensitive tools for diagnosis and therapeutic monitoring in cancer research. In vivo MRS provides information on tumor metabolism, which is clinically valuable in the diagnosis and assessment of tumor response to therapy for the management of women with breast diseases. Several centers complement breast MRI studies with (1)H MRS to improve the specificity of diagnosis. Malignant breast tissues show elevated water-to-fat ratio and choline-containing compounds (total choline, tCho), and any effect of therapy on tissue viability or metabolism will be manifested as changes in these levels. Sequential (1)H MRS studies have shown significantly reduced tCho levels during the course of therapy in patients who were responders. However, there are challenges in using in vivo MRS because of the relatively low sensitivity in detecting the tCho resonance with decreased lesion size or significant reduction in the tumor volume during therapy. MRS is also technically challenging because of the low signal-to-noise ratio and heterogeneous distribution of fat and glandular tissues in the breast. MRS is best utilized for the diagnosis of focal masses, most commonly seen in patients with ductal-type neoplasms; however, it has limitations in detecting nonfocal masses, such as the linear pattern of tumors seen in invasive lobular carcinoma. Further work is required to assess the clinical utility of quantitative MRS, with the goal of automation, which will reduce the subjectivity currently inherent in both qualitative and semi-quantitative MRS.

Multimodal Mass Spectrometric Imaging of Small Molecules Reveals Distinct Spatio-Molecular Signatures in Differentially Metastatic Breast Tumor Models

Phosphocholine (PC) and total choline (tCho) are increased in malignant breast tumors. In this study, we combined magnetic resonance spectroscopic imaging (MRSI), mass spectrometry (MS) imaging, and pathologic assessment of corresponding tumor sections to investigate the localization of choline metabolites and cations in viable versus necrotic tumor regions in the nonmetastatic MCF-7 and the highly metastatic MDA-MB-231 breast cancer xenograft models. In vivo three-dimensional MRSI showed that high tCho levels, consisting of free choline (Cho), PC, and glycerophosphocholine (GPC), displayed a heterogeneous spatial distribution in the tumor. MS imaging performed on tumor sections detected the spatial distributions of individual PC, Cho, and GPC, as well as sodium (Na+) and potassium (K+), among many others. PC and Cho intensity were increased in viable compared with necrotic regions of MDA-MB-231 tumors, but relatively homogeneously distributed in MCF-7 tumors. Such behavior may be related to the role of PC and PC-related enzymes, such as choline kinase, choline transporters, and others, in malignant tumor growth. Na+ and K+ colocalized in the necrotic tumor areas of MDA-MB-231 tumors, whereas in MCF-7 tumors, Na+ was detected in necrotic and K+ in viable tumor regions. This may be attributed to differential Na+/K+ pump functions and K+ channel expressions. Principal component analysis of the MS imaging data clearly identified different tumor microenvironmental regions by their distinct molecular signatures. This molecular information allowed us to differentiate between distinct tumor regions and tumor types, which may, in the future, prove clinically useful in the pathologic assessment of breast cancers.

The Phosphoinositide 3-Kinase Inhibitor PI-103 Downregulates Choline Kinase α Leading to Phosphocholine and Total Choline Decrease Detected by Magnetic Resonance Spectroscopy

The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus ((31)P)- and proton ((1)H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103-induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase alpha (ChoK(alpha)), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r(2) = 0.9, P = 0.009) was found only between PC concentrations and ChoK(alpha) expression but not with FAS or pACL. This study identified inhibition of ChoK(alpha) as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of (1)H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with (31)P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors.