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Epithelial Ovarian Cancer (EOC) "cholinic phenotype", characterized by increased intracellular phosphocholine content sustained by over-expression/activity of choline kinase-alpha (ChoKα/CHKA), is a metabolic cellular reprogramming involved in chemoresistance with still unknown mechanisms.By stable CHKA silencing and global metabolic profiling here we demonstrate that CHKA knockdown hampers growth capability of EOC cell lines both in vitro and in xenotransplant in vivo models. It also affected antioxidant cellular defenses, decreasing glutathione and cysteine content while increasing intracellular levels of reactive oxygen species, overall sensitizing EOC cells to current chemotherapeutic regimens. Natural recovering of ChoKα expression after its transient silencing rescued the wild-type phenotype, restoring intracellular glutathione content and drug resistance. Rescue and phenocopy of siCHKA-related effects were also obtained by artificial modulation of glutathione levels. The direct relationship among CHKA expression, glutathione intracellular content and drug sensitivity was overall demonstrated in six different EOC cell lines but notably, siCHKA did not affect growth capability, glutathione metabolism and/or drug sensitivity of non-tumoral immortalized ovarian cells. The "cholinic phenotype", by recapitulating EOC addiction to glutathione content for the maintenance of the antioxidant defense, can be therefore considered a unique feature of cancer cells and a suitable target to improve chemotherapeutics efficacy.

Magnetic resonance imaging (MRI) and spectroscopy (MRS) offer powerful approaches for detecting physiological and metabolic alterations in malignancies and help investigate underlying molecular mechanisms. Research on epithelial ovarian carcinoma (EOC), the gynaecological malignancy with the highest death rate characterised by frequent relapse and onset of drug resistance, could benefit from application of these molecular imaging approaches. In this study, MRI/MRS were used to characterise solid tumour models obtained by subcutaneous (s.c.) or intraperitoneal (i.p.) implantation of human SKOV3.ip cells in severe combined immunodeficiency (SCID) mice. In vivo MRI/MRS, ex vivo magic-angle-spinning (MAS), and in vitro (1)H-NMR measurements were carried out at 4.7 T, 9.4 T, and 9.4/16.5 T, respectively. MRI evaluation was performed by T1-, T2-, and diffusion-weighted (DW) multislice spin-echo imaging. The in vivo (1)H spectra of all tumour models showed a prominent resonance of total choline-containing metabolites (tCho). Quantitative in vivo MRS of both i.p. and s.c. SKOV3.ip xenografts showed that the mean tCho content was in the 2.9-4.5 mM range, with a mean PCho/tCho ratio of 0.99 ± 0.01 [23 examinations, 14-34 days post injection (dpi)], in good agreement with ex vivo and in vitro analyses. Myo-inositol ranged between 11.7 and 17.0 mM, with a trend towards higher values in i.p. xenografts at 14-16 dpi. The average apparent diffusion coefficient (ADC) values of SKOV3.ip xenografts [1.64 ± 0.11 (n = 9, i.p.) and 1.58 ± 0.03 x10(-3) mm(2)/s (n = 7, s.c.)] were in agreement with values reported for tumours from patients with EOC, while the mean vascular signal fraction (VSF) was lower (≤ 4%), probably due to the more rapid growth of preclinical models. Both s.c. and i.p. xenografts are valuable preclinical models for monitoring biochemical and physiopathological changes associated with in vivo EOC tumour growth and response to therapy, which may serve as the basis for further clinical development of noninvasive MR approaches., (Copyright © 2011 John Wiley & Sons, Ltd.)

Objectives: To assess the clinical feasibility of 3-dimensional (3D) proton magnetic resonance spectroscopy (MRS) of ovarian masses at 1.5 T., Materials and Methods: We prospectively evaluated 16 patients with 23 ovarian masses using contrast-enhanced magnetic resonance imaging and 3D chemical shift imaging MRS (time of reception/time of echo = 700/135 ms, number of excitations = 6, interpolated voxel = 5 × 5 × 5 mm(3), water and fat suppression). Spectral editing consisted of water reference, filtering, zero-filling, Fourier transformation, frequency shift, automatic baseline and phase correction, and curve fitting. The volume of interest was placed to encompass both solid and cystic tumor components as well as apparently healthy pelvic tissues. The presence of a choline peak at 3.14 to 3.34 ppm was considered as a marker of malignancy. All patients underwent surgery and histopathological evaluation., Results: Of 23 masses, 19 were malignant and the remaining 4 benign lesions were a fibrothecoma, an endometriosis, a cyst, and a cystadenofibroma. A choline peak was detected in 17/19 malignant tumors (sensitivity 89%), absent in 2 G1 tumors. It was visible in 16 solid components of 19 malignant tumors (in one of them, a choline peak was detected only in the cystic component, in 6 in both solid and cystic components). The choline peak was absent in 20/21 apparently healthy pelvic tissues, with a very low choline peak being detected in one intraperitoneal fluid collection with malignant cells at cytologic analysis; 3/4 benign tumors showed a choline peak (overall specificity 21/25 = 84%). A significant difference between the mean choline peak integral detected within the solid component and that within the cystic component was observed (P = 0.002). No correlation between the choline peak integral and the tumor size was found (r = 0.120, P = 0.615)., Conclusions: 3D MRS of ovarian masses is clinically feasible at 1.5 T. This opens new research strategies for early diagnosis of ovarian cancer.

Aims and Background: In 2007, an Italian Research Network proposed to the Ministry of Health a concerted action aimed at developing a specific pathway for the analytical and clinical validation of new biomarkers for early cancer diagnosis. The action, funded by the Italian Ministry of Health within the Integrated Program in Oncology (PIO) and coordinated by the National Cancer Institute of Bari, started in 2008 involving 37 national research teams., Methods: To monitor the methodological and analytical needs of the studies proposed by the research teams of PIO as well as to explore the plausibility of planning external quality assessment programs for early cancer biomarker detection, the coordinating team developed an ad hoc questionnaire that was submitted to each research team., Results: From the collected data it emerged that about 70% of the biomarkers under investigation were analyzed according to nonroutine laboratory practices. The biological material utilized for biomarker assessment consisted of solid tissue (normal or pathological) in 31% of studies, serum in 21%, urine in 15%, plasma in 15%, and whole blood in 11%. Specific training of personnel directly involved in the program was reported by 18% of the teams. In 2008, only 6% of laboratories involved in PIO participated in both external quality assessment and internal quality control schemes specifically designed for the biomarkers under consideration. Standard operating procedures for the determination of about half (52%) of the biomarkers proved to be lacking in at least one phase of the biomarker assessment process., Conclusions: On the basis of these results, we decided to give priority to the application of a four-phase process for the analytical validation of new potential biomarkers by setting up and applying standard operating procedures and developing external quality assessment and internal quality control schemes as specific steps of the workflow.

In 2007, an Italian cancer research group proposed a specific concerted action aimed at the "analytical and clinica validation of new biomarkers for early diagnosis: Network, resources, methodology, quality control, and data analysis." The proposal united 37 national operative units involved in different biomarker studies and it created a strong coordinative body with the necessary expertise in methodologies, statistical analysis, quality control, and biological resources to perform ad hoc validation studies for new biomarkers of early cancer diagnosis. The action, financed by the Italian Ministry of Health within the Integrated Oncology Program (PIO) coordinated by NCI-Istituto Tumori Bari, started in 2007 and activated 7 projects, each of which focused on disease-specific biomarker studies. Overall, the 37 participating units proposed studies on 50 biomarkers, including analytical and clinical validation procedures. Clusters of units were specifically involved in research of early-detection biomarkers for cancers of the lung, digestive tract, prostate/bladder, and nervous system, as well as female cancers. Furthermore, a cluster involved in biomarkers for bioimaging and infection-related cancers was created. The first investigators' meeting, "Analytical and clinical validation of new biomarkers for early diagnosis," was held on 9 September 2008 in Bari. During this meeting, methodological aspects, scientific programs and preliminary results were presented and discussed.

Objective: The purpose of our study was to use the total choline-containing compound (tCho) peak integral as a marker of malignancy in breast MR spectroscopy (MRS)., Subjects and Methods: Forty-eight single-voxel water- and fat-suppressed 1.5-T MRS measurements were performed in 42 patients, obtaining both absolute tCho peak integral and tCho peak integral normalized for the volume of interest (VOI). Our reference standard was histology for lesions with BI-RADS category 4 and 5 and histology or at least a 2-year follow-up for findings with BI-RADS 2 and 3 and normal glands. Receiver operating characteristic (ROC) analysis, Mann-Whitney U test, and Spearman's rank correlation were used., Results: Three of 48 measurements (6%) failed. Of the remaining 45 spectra, 18 nonmalignant tissues showed no tCho peak, eight nonmalignant tissues showed a tCho peak integral from 0.99 to 9.03 arbitrary units (AU), and 19 malignant lesions showed a tCho peak integral from 1.26 to 19.80 AU. The diameter of nonmalignant tissues was 16.9 +/- 7.4 mm; that of malignant lesions was 15.3 +/- 6.9 mm (p = 0.308). At ROC analysis, the optimal threshold was 1.90 AU for absolute tCho peak, with 0.895 (17/19) sensitivity, 0.923 (24/26) specificity, and an AUC (area under the curve) of 0.917 (95% CI, 0.822-1.000); the optimal threshold was 0.85 AU/mL for the normalized tCho peak integral with 0.842 (16/19) sensitivity, 0.885 (23/26) specificity, and an AUC of 0.941 (0.879-1.000) (p = 0.470). A negative correlation (p = 0.011) was found between the VOI and the normalized tCho peak integral of malignant tissues., Conclusion: Breast MRS using tCho peak integral reaches a high level of diagnostic performance.

Ovarian carcinomas represent a major form of gynaecological malignancies, whose treatment consists mainly of surgery and chemotherapy. Besides the difficulty of prognosis, therapy of ovarian carcinomas has reached scarce improvement, as a consequence of lack of efficacy and development of drug-resistance. The need of different biochemical and functional parameters has grown, in order to obtain a larger view on processes of biological and clinical significance. In this paper we report novel metabolic features detected in a series of different human ovary carcinoma lines, by (1)H NMR spectroscopy of intact cells and their extracts. Most importantly, a new ovarian adenocarcinoma line CABA I, showed strong signals in the spectral region between 3.5 and 4.0 p.p.m., assigned for the first time to the polyol sorbitol (39+/-11 nmol/10(6) cells). (13)C NMR analyses of these cells incubated with [1-(13)C]-D-glucose demonstrated labelled-sorbitol formation. The other ovarian carcinoma cell lines (OVCAR-3, IGROV 1, SK-OV-3 and OVCA432), showed, in the same spectral region, intense resonances from other metabolites: glutathione (up to 30 nmol/10(6) cells) and myo-inositol (up to 50 nmol/10(6) cells). Biochemical and biological functions are suggested for these compounds in human ovarian carcinoma cells, especially in relation to their possible role in cell detoxification mechanisms during tumour progression.