Total Choline-containing Compounds Research Articles

Elevated choline drives KLF5-dominated transcriptional reprogramming to facilitate liver cancer progression.

An increase in the total choline-containing compound content is a common characteristic of cancer cells, and aberrant choline metabolism in cancer is closely associated with malignant progression. However, the potential role of choline-induced global transcriptional changes in cancer cells remains unclear. In this study, we reveal that an elevated choline content facilitates hepatocellular carcinoma (HCC) cell proliferation by reprogramming Krüppel-like factor 5 (KLF5)-dominated core transcriptional regulatory circuitry (CRC). Mechanistically, choline administration leads to elevated S-adenosylmethionine (SAM) levels, inducing the formation of H3K4me1 within the super-enhancer (SE) region of KLF5 and activating its transcription. KLF5, as a key transcription factor (TF) of CRC established by choline, further transactivates downstream genes to facilitate HCC cell cycle progression. Additionally, KLF5 can increase the expression of choline kinase-α (CHKA) and CTP:phosphocholine cytidylyltransferase (CCT) resulting in a positive feedback loop to promote HCC cell proliferation. Notably, the histone deacetylase inhibitor (HDACi) vorinostat (SAHA) significantly suppressed KLF5 expression and liver tumor growth in mice, leading to a prolonged lifespan. In conclusion, these findings highlight the epigenetic regulatory mechanism of the SE-driven key regulatory factor KLF5 conducted by choline metabolism in HCC and suggest a potential therapeutic strategy for HCC patients with high choline content.

Spectroscopic abnormalities in the pregenual anterior cingulate cortex in obsessive-compulsive disorder using proton magnetic resonance spectroscopy: a controlled study

BackgroundThe main aim of the present study is to determine the role of metabolites observed using proton magnetic resonance spectroscopy (1H-MRS) in obsessive-compulsive disorder (OCD). As the literature describing biochemical changes in OCD yields conflicting results, we focused on accurate metabolite quantification of total N-acetyl aspartate (tNAA), total creatine (tCr), total choline-containing compounds (tCh), and myo-inositol (mI) in the anterior cingulate cortex (ACC) to capture the small metabolic changes between OCD patients and controls and between OCD patients with and without medication.MethodsIn total 46 patients with OCD and 46 healthy controls (HC) matched for age and sex were included in the study. The severity of symptoms in the OCD was evaluated on the day of magnetic resonance imaging (MRI) using the Yale-Brown Obsessive-Compulsive Scale (YBOCS). Subjects underwent 1H-MRS from the pregenual ACC (pgACC) region to calculate concentrations of tNAA, tCr, tCho, and mI. Twenty-eight OCD and 28 HC subjects were included in the statistical analysis. We compared differences between groups for all selected metabolites and in OCD patients we analyzed the relationship between metabolite levels and symptom severity, medication status, age, and the duration of illness.ResultsSignificant decreases in tCr (U = 253.00, p = 0.022) and mI (U = 197.00, p = 0.001) in the pgACC were observed in the OCD group. No statistically significant differences were found in tNAA and tCho levels; however, tCho revealed a trend towards lower concentrations in OCD patients (U = 278.00, p = 0.062). Metabolic concentrations showed no significant correlations with the age and duration of illness. The correlation statistics found a significant negative correlation between tCr levels and YBOCS compulsions subscale (cor = -0.380, p = 0.046). tCho and YBOCS compulsions subscale showed a trend towards a negative correlation (cor = -0.351, p = 0.067). Analysis of subgroups with or without medication showed no differences.ConclusionsPatients with OCD present metabolic disruption in the pgACC. The decrease in tCr shows an important relationship with OCD symptomatology. tCr as a marker of cerebral bioenergetics may also be considered as a biomarker of the severity of compulsions. The study failed to prove that metabolic changes correlate with the medication status or the duration of illness. It seems that a disruption in the balance between these metabolites and their transmission may play a role in the pathophysiology of OCD.

The distribution of dietary choline intake and serum choline levels in Australian women during pregnancy and associated early life factors

BackgroundMaternal dietary choline has a central role in foetal brain development and may be associated with later cognitive function. However, many countries are reporting lower than recommended intake of choline during pregnancy.MethodsDietary choline was estimated using food frequency questionnaires in pregnant women participating in population-derived birth cohort, the Barwon Infant Study (BIS). Dietary choline is reported as the sum of all choline-containing moieties. Serum total choline-containing compounds (choline-c), phosphatidylcholine and sphingomyelin were measured using nuclear magnetic resonance metabolomics in the third trimester. The main form of analysis was multivariable linear regression.ResultsThe mean daily dietary choline during pregnancy was 372 (standard deviation (SD) 104) mg/day. A total of 236 women (23%) had adequate choline intake (440 mg/day) based on the Australian and New Zealand guidelines, and 27 women (2.6%) took supplemental choline (ge 50 mg/dose) daily during pregnancy. The mean serum choline-c in pregnant women was 3.27 (SD 0.44) mmol/l. Ingested choline and serum choline-c were not correlated (R2) = − 0.005, p = 0.880. Maternal age, maternal weight gain in pregnancy, and a pregnancy with more than one infant were associated with higher serum choline-c, whereas gestational diabetes and environmental tobacco smoke during preconception and pregnancy were associated with lower serum choline-c. Nutrients or dietary patterns were not associated with variation in serum choline-c.ConclusionIn this cohort, approximately one-quarter of women met daily choline recommendations during pregnancy. Future studies are needed to understand the potential impact of low dietary choline intake during pregnancy on infant cognition and metabolic intermediaries.

ChoK-Full of Potential: Choline Kinase in B Cell and T Cell Malignancies

Aberrant choline metabolism, characterized by an increase in total choline-containing compounds, phosphocholine and phosphatidylcholine (PC), is a metabolic hallmark of carcinogenesis and tumor progression. This aberration arises from alterations in metabolic enzymes that control PC biosynthesis and catabolism. Among these enzymes, choline kinase α (CHKα) exhibits the most frequent alterations and is commonly overexpressed in human cancers. CHKα catalyzes the phosphorylation of choline to generate phosphocholine, the first step in de novo PC biosynthesis. CHKα overexpression is associated with the malignant phenotype, metastatic capability and drug resistance in human cancers, and thus has been recognized as a robust biomarker and therapeutic target of cancer. Of clinical importance, increased choline metabolism and CHKα activity can be detected by non-invasive magnetic resonance spectroscopy (MRS) or positron emission tomography/computed tomography (PET/CT) imaging with radiolabeled choline analogs for diagnosis and treatment monitoring of cancer patients. Both choline-based MRS and PET/CT imaging have also been clinically applied for lymphoid malignancies, including non-Hodgkin lymphoma, multiple myeloma and central nervous system lymphoma. However, information on how choline kinase is dysregulated in lymphoid malignancies is very limited and has just begun to be unraveled. In this review, we provide an overview of the current understanding of choline kinase in B cell and T cell malignancies with the goal of promoting future investigation in this area.

Proton magnetic resonance spectroscopy changes in the brainstem in patients after mild traumatic brain injury with loss of consciousness.

Loss of consciousness (LOC) is used as a diagnostic feature of mild traumatic brain injury (MTBI). However, only 10% of concussions result in LOC. There are only a limited number of in-vivo studies dealing with unconsciousness and structural and functional integrity of the brainstem in patients with MTBI. The aim of our pilot study was to assess the sensitivity of proton magnetic resonance spectroscopy (1H-MRS) to detect metabolic changes in the brainstem in patients after MTBI with unconscioussness. Twenty-four patients (12 with LOC, and 12 without LOC) within 3 days of MTBI and 19 healthy controls were examined. All subjects underwent single-voxel 1H-MRS examination of the upper brainstem. Spectra were evaluated using LCModel software. Ratios of total N-acetylaspartate (tNAA), total choline-containing compounds (tCho) and glutamate plus glutamine (Glx) to total creatine (tCre) were used for calculations. We found a significant decrease in tNAA/tCre and tCho/tCre ratios in the patient group with LOC when compared with the control group of healthy volunteers (P=0.002 and P=0.041, respectively), and a significant decrease in the tNAA/tCre ratio in the LOC group when compared with patients without LOC (P=0.04). Other metabolite ratios in the brainstem did not show any significant group differences. Our findings indicate that decrease of tNAA/tCre ratio in the upper brainstem using single-voxel 1H-MRS may provide a potential biomarker for MTBI associated with LOC.

Abstract 2546: The immune checkpoint PD-L1 alters choline kinase expression and metabolism in triple negative breast cancer cells

Abstract Introduction Cancer immunotherapy is designed to activate the immune system against cancer cells. This field has been revitalized with the discovery of immune checkpoints such as programmed cell death protein-1 (PD-1) and its ligand (PD-L1). PD-L1 has been exploited for immune therapy with varying success depending on the tumor type, with several studies pointing towards cancer metabolism as one of the possible causes of this variability. One of the most common tumor associated metabolic alteration is increased expression of choline kinase-α (Chk-α), characterized by increased phosphocholine (PC) and total choline-containing compounds. Since the presence of PC on proteins has been related to escape from immune surveillance, here, we investigated the relationship between Chk-α, PC and PD-L1. Methods Experiments were performed using triple negative breast MDA-MB-231 cells transiently transfected with small interfering RNA (siRNA) against Chk-α or PD-L1 following standard protocols. Total RNA was isolated, complementary cDNA synthesised and quantitative real-time PCR performed using IQ SYBR Green supermix and gene specific primers. For high resolution 1H Magnetic Resonance Spectroscopy (MRS), cell extracts were obtained using a dual-phase extraction method. 1H MRS was recorded on a Bruker Biospin Avance-III 750 MHz spectrometer. Integrals of the metabolites of interest were determined and normalized to the TSP reference and the number of cells. Metabolites were estimated from at least three experimental samples. Statistical significance was evaluated using the Student t-test. Results and Discussion We observed that silencing Chk-α resulted in a significant increase of PD-L1 expression and silencing PD-L1 resulted in a significant increase of Chk-α expression. Consistent with the molecular changes, a significant increase of PC was observed in cells transfected with PD-L1 siRNA. Metabolic changes, however, extended well beyond the choline containing compounds. We identified changes in amino acids, organic acids, nucleotides and other compounds. Furthermore, we found that PD-L1 downregulation profoundly altered the lipid profile of MDA-MB-231 cells. Metabolic characterization showed that cells with downregulated Chk-α levels shifted towards a more immunosuppressive profile through metabolic reprogramming, increasing the production of metabolites that have been linked to decreased effectiveness of T-cells. Our data also suggest that low levels of PD-L1 can skew cancer cell metabolism towards a more immunosuppressive profile by a significant increase of lipid production and changes in the lipid profile. These results provide new insights in the role of PD-L1 in the cancer metabolome that may be exploited to improve the outcome of treatment with immune checkpoint inhibitors. Acknowledgement: This work was supported by NIH R35CA209960 and R01CA82337. JPT was funded by Fundación Alonso Martín Escudero and MSCA. Citation Format: Jesus Pacheco-Torres, Marie-France Penet, Flonne Wildes, Yelena Mironchik, Balaji Krishnamachary, Zaver M. Bhujwalla. The immune checkpoint PD-L1 alters choline kinase expression and metabolism in triple negative breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2546.

MR Spectroscopy of the Cervical Spinal Cord in Chronic Spinal Cord Injury.

Purpose To investigate metabolic changes in chronic spinal cord injury (SCI) by applying MR spectroscopy in the cervical spinal cord. Materials and Methods Single-voxel short-echo spectroscopic data in study participants with chronic SCI and healthy control subjects were prospectively acquired in the cervical spinal cord at C2 above the level of injury between March 2016 and January 2017 and were compared between groups. Concentrations of total N-acetylaspartate (tNAA), myo-inositol (mI), total choline-containing compounds (tCho), creatine, and glutamine and glutamate complex were estimated from the acquired spectra. Participants were assessed with a comprehensive clinical evaluation investigating sensory and motor deficits. Correlation analysis was applied to investigate relationships between observed metabolic differences, lesion severity, and clinical outcome. Results There were 18 male study participants with chronic SCI (median age, 51 years; range, 30-68 years) and 11 male healthy control subjects (median age, 45 years; range, 30-67 years). At cervical level C2, tNAA/mI and tCho/mI ratios were lower in participants with SCI (tNAA/mI: -26%, P = .003; tCho/mI: -18%; P = .04) than in healthy control subjects. The magnitude of difference was greater with the severity of cord atrophy (tNAA/mI: R2 = 0.44, P = .003; tCho/mI: R2 = 0.166, P = .09). Smaller tissue bridges at the lesion site correlated with lower ratios of tNAA/mI (R2 = 0.69, P = .006) and tCho/mI (R2 = 0.51, P = .03) at the C2 level. Lower tNAA/mI and tCho/mI ratios were associated with worse sensory and motor outcomes (P < .05). Conclusion Supralesional metabolic alterations are observed in chronic spinal cord injury, likely reflecting neurodegeneration, demyelination, and astrocytic gliosis in the injured cervical cord. Lesion severity and greater clinical impairment are both linked to the biochemical changes in the atrophied cervical cord after spinal cord injury. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Lin in this issue.

Assessment of Anterior Cingulate Cortex (ACC) and Left Cerebellar Metabolism in Asperger's Syndrome with Proton Magnetic Resonance Spectroscopy (MRS).

PurposeProton magnetic resonance spectroscopy (1H MRS) is a noninvasive neuroimaging method to quantify biochemical metabolites in vivo and it can serve as a powerful tool to monitor neurobiochemical profiles in the brain. Asperger’s syndrome (AS) is a type of autism spectrum disorder, which is characterized by impaired social skills and restrictive, repetitive patterns of interest and activities, while intellectual levels and language skills are relatively preserved. Despite clinical aspects have been well-characterized, neurometabolic profiling in the brain of AS remains to be clear. The present study used proton magnetic resonance spectroscopy (1H MRS) to investigate whether pediatric AS is associated with measurable neurometabolic abnormalities that can contribute new information on the neurobiological underpinnings of the disorder.MethodsStudy participants consisted of 34 children with AS (2–12 years old; mean age 5.2 (±2.0); 28 boys) and 19 typically developed children (2–11 years old; mean age 5.6 (±2.6); 12 boys) who served as the normal control group. The 1H MRS data were obtained from two regions of interest: the anterior cingulate cortex (ACC) and left cerebellum.ResultsIn the ACC, levels of N-acetylaspartate (NAA), total creatine (tCr), total choline-containing compounds (tCho) and myo-Inositol (mI) were significantly decreased in children with AS compared to controls. On the other hand, no significant group differences in any of the metabolites were found in the left cerebellum. Neither age nor sex accounted for the metabolic findings in the regions.ConclusionThe finding of decreased levels of NAA, tCr, tCho, and mI in the ACC but not in left cerebellar voxels in the AS, suggests a lower ACC neuronal density in the present AS cohort compared to controls.

Abstract 1233: In vitro and in vivo pharmacological study of EB-3D: a novel choline kinase inhibitor for breast cancer treatment

Abstract Choline kinase (ChoK) catalyzes the phosphorylation of choline to phosphocholine (PCho) in the first step of the Kennedy pathway for phosphatidylcholine (PtdCho) byosyntesis. PtdCho is the most abundant phospholipid in eukaryotic cell membrane and plays a crucial role for cell division and lipid second messengers production. In mammalian cells, only the ChoKα isoform is fundamental in sustaining PtdCho synthesis, indeed ChoKβ alone cannot compensate this activity. A large number of studies have shown that ChoKα is overexpressed and hyperactivated in many cancers and that correlates not only with increased cancer cell proliferation but also with malignancy, making it a potential prognostic marker. The cholinic phenotype, characterized by increased total choline-containing compound (tCho) and ChoKα upregulation renders therefore ChoKα an attractive new therapeutic target. Indeed, the feasibility of ChoKα inhibition as antitumoral therapy is being pursued through the development of chemical inhibitors of its enzymatic activity. For this reason we evaluated both in vitro and in vivo the activity of EB-3D (1,1’-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))bis(4-(dimethylamino) pyridinium) bromide), a new choline kinase inhibitor endowed with high antiproliferative activity in two human breast cancer cell lines, MCF-7 and MDA-MB-231. EB-3D treatment induced a strong decrease in cell proliferation due to G1/G0 arrest of the cell cycle in a concentration-dependent manner in both tested cell lines, whereas only a slight increase in apoptotic cells was observed. In addition EB-3D strongly synergized with drugs commonly used in protocols for breast cancer. Reverse-phase protein array (RPPA) data revealed the activation of AMPK and the dephosporylation of mTORC1 downstream targets such as 4E-BP1(S65), p70S6K(T389) and RPS6(S235/236), suggesting that ChoKα inhibition may affect protein synthesis. To further examine the antitumorigenic potential of EB-3D in vivo, a syngeneic orthotopical EO771-C57BL/6 mouse model of breast cancer was used. Preliminary results indicated that the compound significantly reduced the tumor size, with no apparent sign of toxicity. The evaluation of EB-3D as in vivo anti-metastatic adjuvant is ongoing, since cell migration and cell invasion ability of the highly metastatic MDA-MB-231 cell line was impaired after in vitro treatment. Taken together our results suggest that EB-3D have a potent cytostatic effect and it could be a promising new anticancer agent, worthy of further development. Citation Format: Elena Mariotto, Roberta Bortolozzi, Roberto Ronca, Luisa C. López-Cara, Benedetta Accordi, Valentina Serafin, Giuseppe Basso, Giampietro Viola. In vitro and in vivo pharmacological study of EB-3D: a novel choline kinase inhibitor for breast cancer treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1233.

Choline Metabolism Alteration: A Focus on Ovarian Cancer.

Compared with normal differentiated cells, cancer cells require a metabolic reprograming to support their high proliferation rates and survival. Aberrant choline metabolism is a fairly new metabolic hallmark reflecting the complex reciprocal interactions between oncogenic signaling and cellular metabolism. Alterations of the involved metabolic network may be sustained by changes in activity of several choline transporters as well as of enzymes such as choline kinase-alpha (ChoK-α) and phosphatidylcholine-specific phospholipases C and D. Of note, the net outcome of these enzymatic alterations is an increase of phosphocholine and total choline-containing compounds, a “cholinic phenotype” that can be monitored in cancer by magnetic resonance spectroscopy. This review will highlight the molecular basis for targeting this pathway in epithelial ovarian cancer (EOC), a highly heterogeneous and lethal malignancy characterized by late diagnosis, frequent relapse, and development of chemoresistance. Modulation of ChoK-α expression impairs only EOC but not normal ovarian cells, thus supporting the hypothesis that “cholinic phenotype” is a peculiar feature of transformed cells and indicating ChoK-α targeting as a novel approach to improve efficacy of standard EOC chemotherapeutic treatments.

Diffusion-Weighted Imaging-guided MR Spectroscopy in Breast Lesions using Readout-Segmented Echo-Planar Imaging.

To investigate the feasibility and effectiveness of diffusion-weighted imaging (DWI)-guided magnetic resonance spectroscopy (MRS) using readout-segmented echo-planar imaging (RS-EPI) to characterise breast lesions. A total of 258 patients with 258 suspicious breast lesions larger than 1cm in diameter were examined using DWI-guided, single-voxel MRS with RS-EPI. The mean total choline-containing compound (tCho) signal-to-noise ratio (SNR) and concentration were used for the interpretation of MRS data. T-tests, χ(2)-tests, receiver operating characteristic (ROC) curve analyses and Pearson correlations were conducted for statistical analysis. Histologically, 183 lesions were malignant, and 75 lesions were benign. Both the mean tCho SNR and concentration of malignant lesions were higher than those of benign lesions (6.23 ± 3.30AU/mL vs. 1.26 ± 1.75AU/mL and 3.17 ± 2.03mmol/kg vs. 0.86 ± 0.83mmol/kg, respectively; P < 0.0001). For a tCho SNR of 2.0AU/mL and a concentration of 1.76mmol/kg, the corresponding areas under the ROC curves were 0.93 and 0.90, respectively. The mean tCho SNR and concentration negatively correlated with apparent diffusion coefficients calculated from RS-EPI, with correlation coefficients of -0.54 and -0.48, respectively. DWI-guided MRS using RS-EPI is feasible and accurate for characterising breast lesions. • The mean tCho SNR and concentration negatively correlated with ADCs. • DWI-guided MRS using RS-EPI is feasible. • DWI-guided MRS using RS-EPI accurately characterises breast lesions.

Neurochemical Changes Observed by In Vivo Proton Magnetic Resonance Spectroscopy in the Mouse Brain Postadministration of Scopolamine

This study is aimed at investigating neurochemical changes in scopolamine (SCP)-induced memory impairment using spatially localized invivo magnetic resonance spectroscopy (MRS) of the hippocampus. Four groups of mice (eight mice per group) were scanned after the injection of different SCP doses: 0, 1, 3, and 5 mg/kg (intraperitoneally). All the animals received (1)H MRS of their hippocampus at two time intervals: 30minutes and 72hours after SCP injection. This work demonstrated that the doses of 3 mg/kg SCP or higher reduce the concentration of total choline-containing compounds, and these levels returned to baseline after 72hours. These results are consistent with observations made by others using more invasive brain dialysis approaches. The levels of glutamate and glutamic compounds (glutamate+glutamine) were slightly changed at 3 and 5mg/kg SCP dose, but the differences were not statistically significant (P>.05). These findings suggest that SCP produces transient, invivo measurable alterations in the cholinergic system in the hippocampus. On this basis, we conclude that invivo MRS is a feasible noninvasive method to probe aspects of the alterations induced by SCP in the cholinergic neurotransmission pathways in both animal models and human studies of memory impairment.

Phospholipase D1 and choline kinase-α are interactive targets in breast cancer

A consistent metabolic hallmark observed in multiple cancers is the increase of cellular phosphocholine (PC) and total choline-containing compounds (tCho), which is closely related to malignant transformation, invasion, and metastasis. Enzymes in choline phospholipid metabolism present attractive targets to exploit for treatment, but require a clear understanding of the mechanisms underlying the altered choline phospholipid metabolism observed in cancer. Choline kinase-α (Chk-α) is an enzyme in the Kennedy pathway that phosphorylates free choline (Cho) to PC, and its upregulation in several cancers is a major contributor to increased PC levels. Similarly, increased expression and activity of phospholipase D1 (PLD1), which converts phosphatidylcholine (PtdCho) to phosphatidic acid (PA) and Cho, has been well documented in gastric, ovarian and breast cancer. Here we report a strong correlation between expression of Chk-α and PLD1 with breast cancer malignancy. Data from patient samples established an association between estrogen receptor (ER) status and Chk-α and PLD1 expression. In addition, these two enzymes were found to be interactive. Downregulation of Chk-α with siRNA increased PLD1 expression, and downregulation of PLD1 increased Chk-α expression. Simultaneous silencing of PLD1 and Chk-α in MDA-MB-231 cells increased apoptosis as detected by the TUNEL assay. These data provide new insights into choline phospholipid metabolism of breast cancer, and support multiple targeting of enzymes in choline phospholipid metabolism as a strategy for treatment.

Feasibility of MR spectroscopy for characterizing malignant breast lesions using a clinical 3-T scanner

To investigate the feasibility of in vivo proton magnetic resonance spectroscopy ((1)H-MRS) for characterizing malignant breast lesions using a clinical 3-T scanner, and to compare the measured total choline-containing compound (tCho) levels in this work with the previously published results at 1.5T and 4T. Twenty-six patients (35-72years old, mean age 53 years), with biopsy-confirmed breast cancer, underwent anatomical imaging, dynamic contrast-enhanced MR imaging, and single-voxel (1)H-MRS. The absolute tCho concentration in malignant tumors was calculated and was expressed as a concentration unit in mmol/kg. All lesions were divided into two groups (mass- and non-mass-type lesions), based on the morphological pattern of enhancement, and analyzed. The measured tCho levels had a range of 0.10-10.1 (mean±SD, 1.62±2.29mmol/kg) in 15 (58%) of 26 malignant breast spectra, which was consistent with the previous in vivo estimates at 1.5T and 4.0T. There were 11 (42%) false-negative cases, which resulted in 58% sensitivity. The sensitivity of the (1)H-MRS in size-dependent subgroups (e.g., <2.0 and ≥2.0cm) increased from 14 to 66%, in a statistically significant manner (P=0.019). In addition, the tCho detection rate was higher in mass-type lesions (14/21, 66%) than in non-mass-type lesions (1/5, 20%), reaching a significant level (P=0.03). The sensitivity based on the dynamic contrast-enhanced kinetics was 100%. Quantitative in vivo (1)H-MRS at 3T may provide useful information for quantifying tCho concentration levels and characterizing tumor types (e.g., mass vs diffuse) of breast cancer. However, its limitation in characterizing small lesions has to be considered.

Metabolomic profile of the adrenal gland: from physiology to pathological conditions

In this study, we i) assessed the metabolic profile of the normal adrenal cortex and medulla of adult human subjects by means of (1)H-high-resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR) spectroscopy; ii) compared the biochemical profile of adenoma (Ad), adrenal cortical carcinoma (ACC), and pheochromocytoma (PCC) samples with that of healthy adrenal tissue samples; and iii) investigated the metabolic differences between ACCs and Ads as well as between ACCs and PCCs. Sixty-six tissue samples (13 adrenal cortical tissue, eight medullary tissue, 13 Ad, 12 ACC, and 20 PCC samples) were analyzed. Adrenaline and noradrenaline were undetectable in cortical samples representing the metabolic signature of the tissue derived from neural crest. Similarity between the metabolic profile of Ads and that of the normal adrenal cortex was shown. Inversely, ACC samples clearly made up a detached group exhibiting the typical stigmata of neoplastic tissue such as choline-containing compounds, biochemical markers of anaerobic processes, and increased glycolysis. Significantly higher levels of lactate, acetate, and total choline-containing compounds played a major role in the differentiation of ACCs from Ads. Moreover, the high fatty acid content of ACCs contributed to the cluster identification of ACCs. Of the 14 sporadic PCC samples, 12 exhibited predominant or exclusive noradrenaline secretion. The noradrenaline:adrenaline ratio was inverted in the normal medullary tissue samples. Multiple endocrine neoplasia type 2- and NF1-related PCC samples exhibited both adrenaline and noradrenaline secretion. In the von Hippel-Lindau disease-related PCC samples, only noradrenaline secretion was detected by HRMAS NMR spectroscopy. This study is one of the first applications of metabolomics to adrenal pathophysiology and it is the largest study to report HRMAS NMR data related to the adrenal cortex and adrenal cortical tumors.

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.

Relationship between fractional anisotropy of cerebral white matter and metabolite concentrations measured using 1H magnetic resonance spectroscopy in healthy adults

Fractional anisotropy (FA) of water diffusion in cerebral white matter (WM), derived from diffusion tensor imaging (DTI), is a sensitive index of microscopic WM integrity. Physiological and metabolic factors that explain intersubject variability in FA values were evaluated in two cohorts of healthy adults of different age spans (N=65, range: 28-50years; and N=25, age=66.6±6.2, range: 57-80years). Single voxel magnetic resonance spectroscopy (MRS) was used to measure N-acetylaspartate (NAA), total choline-containing compounds, and total creatine, bilaterally in an associative WM tract: anterior corona radiata (ACR). FA values were calculated for the underlying, proximal and two distal WM regions. Two-stage regression analysis was used to calculate the proportion of variability in FA values explained by spectroscopy measurements, at the first stage, and subject's age, at the second stage. WM NAA concentration explained 23% and 66% of intersubject variability (p<0.001) in the FA of the underlying WM in the younger and older cohorts, respectively. WM NAA concentration also explained a significant proportion of variability in FA of the genu of corpus callosum (CC), a proximal WM tract where some of the fibers contained within the spectroscopic voxel decussate. NAA concentrations also explained a significant proportion of variability in the FA values in the splenium of CC, a distal WM tract that also carries associative fibers, in both cohorts. These results suggest that MRS measurements explained a significant proportion of variability in FA values in both proximal and distal WM tracts that carry similar fiber-types.

Prediction of pathologic response to neoadjuvant chemotherapy in patients with breast cancer using diffusion‐weighted imaging and MRS

The aim of this study was to determine whether tumor size, MRS parameters and apparent diffusion coefficient (ADC) measurements could be applied to predict pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC). Ninety patients with breast cancer (median size, 4.5 cm; range, 1.6-9.5 cm) were evaluated with single-voxel ¹H MRS and dynamic contrast-enhanced MRI. Diffusion-weighted imaging was performed in 41 of these patients using a 1.5-T scanner before and after completion of NAC. Pre- and post-treatment measurements and changes in tumor size, MRS parameters [absolute and normalized total choline-containing compound (tCho) integral and tCho signal-to-noise ratio (SNR)] and ADCs in pCR versus non-pCR were compared using the nonparametric Mann-Whitney test. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic performance of each parameter. After NAC, 30 patients (33%) showed pCR and 60 (67%) showed non-pCR. At pretreatment, ADC was the only significant parameter in differentiating between pCR and non-pCR [(0.83 ± 0.05) × 10⁻³ versus (0.97 ± 0.14) × 10⁻³ mm²/s] (p = 0.014). Post-treatment measurements after completion of NAC and changes in tumor size (both p < 0.001), MRS parameters (p = 0.027 and p = 0.020 for absolute tCho integral, p = 0.036 and p = 0.023 for normalized tCho integral, and p = 0.032 and p = 0.061 for tCho SNR) and ADC (p = 0.003 and p < 0.001) were significantly different between the pCR and non-pCR groups, except for changes in tCho SNR. In ROC analysis, the areas under the ROC curve (AUCs) of 0.63-0.73 were obtained for tumor size and MRS parameters. AUCs for pre- and post-treatment ADC and changes in ADC were 0.75, 0.80 and 0.96, respectively. The optimal cut-off of the percentage change in ADC for predicting pCR was 40.7%, yielding 100% sensitivity and 91% specificity. Patients with pCR showed significantly lower pretreatment ADCs than those with non-pCR. The change in ADC after NAC was the most accurate predictor of pCR.

Evaluation of Breast Cancer Using Proton MR Spectroscopy: Total Choline Peak Integral and Signal-to-Noise Ratio as Prognostic Indicators

The purpose of this article is to determine whether the peak integral and signal-to-noise ratio (SNR) of total choline-containing compounds obtained by MR spectroscopy (MRS) correlate with histologic biomarkers currently used for predicting prognosis in patients with breast cancer. Single-voxel proton MRS using a 1.5-T scanner was performed in 184 patients (mean age, 48 years; range, 28-72 years) with breast cancer. We obtained absolute total choline-containing compound peak integral, total choline-containing compound peak integral normalized for the volume of interest, and SNR after MRI. On surgical pathology, pathologic subtype and prognostic factors such as nuclear grade, histologic grade, estrogen receptor (ER), HER-2≠neu, extensive intraductal component (EIC), lymphovascular invasion, and lymph node metastasis were also evaluated. Statistical analysis was performed using Mann-Whitney U test and Spearman rank correlation. The total choline-containing compound SNR, absolute total choline-containing compound peak integral, and normalized total choline-containing compound integral were significantly higher for invasive ductal carcinoma, cancer of high nuclear or histologic grade, and EIC-negative cancer (p < 0.001) than for in situ or other invasive carcinomas (p = 0.005), cancer of low nuclear or histologic grade (p = 0.009), and EIC-positive cancer (p = 0.017). There was a significant difference in the total choline-containing compound SNR between ER-positive and -negative groups (p = 0.007) and between triple-negative and non-triple-negative groups (p = 0.002). A positive correlation was found between the volume of interest (p < 0.001), tumor size (p = 0.011), and three MRS parameters (p = 0.003). Our study suggests that proton MRS can play a role in predicting prognostic indicators of tumor aggressiveness in patients with newly diagnosed 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.