Panagiotis A. Vorkas

Clinical evaluation of microRNA expression profiling in non small cell lung cancer

Deregulation of miRNAs expression levels has been detected in many human tumor types, and recent studies have demonstrated the critical roles of miRNAs in cancer pathogenesis. Numerous recent studies have shown that miRNAs are rapidly released from tissues into the circulation in many pathological conditions. The high relative stability of miRNAs in biofluids such as plasma and serum, and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNAs as promising non-invasive new tumor biomarkers. In this study, we used liquid bead array technology (Luminex) to profile the expression of 320 mature miRNAs in a pilot testing group of 19 matched fresh frozen cancerous and non-cancerous tissues from NSCLC patients. We further validated our results by RT-qPCR for differentially expressed miRNAs in an independent group of 40 matched fresh frozen tissues, 37 plasma samples from NSCLC patients and 28 healthy donors. We found that eight miRNAs (miR-21, miR-30d, miR-451, miR-10a, miR-30e-5p and miR-126*, miR-126, miR-145) were differentially expressed by three different statistical analysis approaches. Two of them (miR-10a and miR-30e-5p) are reported here for the first time. Bead-array results were further verified in an independent group of 40 matched fresh frozen tissues by RT-qPCR. According to RT-qPCR miR-21 was significantly up-regulated (P = 0.010), miR-126* (P = 0.002), miR-30d (P = 0.012), miR-30e-5p (P < 0.001) and miR-451 (P < 0.001) were down-regulated, while miR-10a was not differentiated (P = 0.732) in NSCLC tissues. However, in NSCLC plasma samples, only three of these miRNAs (miR-21, miR-10a, and miR-30e-5p) displayed differential expression when compared to plasma of healthy donors. High expression of miR-21 was associated with DFI and OS both in NSCLC tissues (P = 0.022 and P = 0.037) and plasma (P = 0.045 and P = 0.065), respectively. Moreover, we report for the first time that low expression of miR-10a in NSCLC plasma samples was associated with worse DFI (P = 0.050) and high expression of miR-30e-5p was found to be associated with shorter OS (P = 0.048). In conclusion, circulating miR-21, miR-10a and miR-30e-5p in plasma should be further evaluated as potential non-invasive biomarkers in NSCLC.

Untargeted UPLC-MS Profiling Pipeline to Expand Tissue Metabolome Coverage: Application to Cardiovascular Disease

Metabolic profiling studies aim to achieve broad metabolome coverage in specific biological samples. However, wide metabolome coverage has proven difficult to achieve, mostly because of the diverse physicochemical properties of small molecules, obligating analysts to seek multiplatform and multimethod approaches. Challenges are even greater when it comes to applications to tissue samples, where tissue lysis and metabolite extraction can induce significant systematic variation in composition. We have developed a pipeline for obtaining the aqueous and organic compounds from diseased arterial tissue using two consecutive extractions, followed by a different untargeted UPLC-MS analysis method for each extract. Methods were rationally chosen and optimized to address the different physicochemical properties of each extract: hydrophilic interaction liquid chromatography (HILIC) for the aqueous extract and reversed-phase chromatography for the organic. This pipeline can be generic for tissue analysis as demonstrated by applications to different tissue types. The experimental setup and fast turnaround time of the two methods contributed toward obtaining highly reproducible features with exceptional chromatographic performance (CV % < 0.5%), making this pipeline suitable for metabolic profiling applications. We structurally assigned 226 metabolites from a range of chemical classes (e.g., carnitines, α-amino acids, purines, pyrimidines, phospholipids, sphingolipids, free fatty acids, and glycerolipids) which were mapped to their corresponding pathways, biological functions and known disease mechanisms. The combination of the two untargeted UPLC-MS methods showed high metabolite complementarity. We demonstrate the application of this pipeline to cardiovascular disease, where we show that the analyzed diseased groups (n = 120) of arterial tissue could be distinguished based on their metabolic profiles.

Chemical mapping of the colorectal cancer microenvironment via MALDI imaging mass spectrometry (MALDI-MSI) reveals novel cancer-associated field effects

Matrix‐assisted laser desorption ionisation imaging mass spectrometry (MALDI‐MSI) is a rapidly advancing technique for intact tissue analysis that allows simultaneous localisation and quantification of biomolecules in different histological regions of interest. This approach can potentially offer novel insights into tumour microenvironmental (TME) biochemistry. In this study we employed MALDI‐MSI to evaluate fresh frozen sections of colorectal cancer (CRC) tissue and adjacent healthy mucosa obtained from 12 consenting patients undergoing surgery for confirmed CRC. Specifically, we sought to address three objectives: (1) To identify biochemical differences between different morphological regions within the CRC TME; (2) To characterise the biochemical differences between cancerous and healthy colorectal tissue using MALDI‐MSI; (3) To determine whether MALDI‐MSI profiling of tumour‐adjacent tissue can identify novel metabolic ‘field effects’ associated with cancer. Our results demonstrate that CRC tissue harbours characteristic phospholipid signatures compared with healthy tissue and additionally, different tissue regions within the CRC TME reveal distinct biochemical profiles. Furthermore we observed biochemical differences between tumour‐adjacent and tumour‐remote healthy mucosa. We have referred to this ‘field effect’, exhibited by the tumour locale, as cancer‐adjacent metaboplasia (CAM) and this finding builds on the established concept of field cancerisation.

Implementation of molecular phenotyping approaches in the personalized surgical patient journey.

Objective:The present review describes commonly employed metabolic profiling platforms and discusses the current and likely future application of these technologies in surgery. Background:The metabolic adaptations that occur in response to surgical illness and trauma are incompletely understood. Evaluating these will be critical to the development of personalized surgical health solutions. Metabonomics is an advancing field in systems biology, which provides a means of interrogating these metabolic shifts. Methods:Recent literature regarding metabolic profiling technologies and their applications in surgical practice are discussed. Future strategies are proposed for the incorporation of these and next-generation technologies in the evaluation of all steps in the patient surgical pathway. Results:Metabolite-based profiling has provided valuable insights into the metabolic irregularities that occur in cancer development and progression across a variety of cancer subclasses including colorectal, breast, prostate, and lung cancers. In addition, metabolic modeling has shown considerable promise in other surgical conditions including trauma and sepsis and in the assessment of pharmacotherapeutic efficacy. Discussion:Metabonomics offers a posttranscriptional view of system activity providing functional information downstream of the genome and proteome. Information at this level will provide the surgeon with a novel means of evaluating major socioeconomic problems such as cancer and sepsis. In addition, the rapid nature of emerging next generation profiling platforms provides a viable means of “real-time” perioperative metabolic assessment and optimization.

Metabolic Phenotyping of Atherosclerotic Plaques Reveals Latent Associations between Free Cholesterol and Ceramide Metabolism in Atherogenesis

Current optimum medical treatments have had limited success in the primary prevention of cardiovascular events, underscoring the need for new pharmaceutical targets and enhanced understanding of mechanistic metabolic dysregulation. Here, we use a combination of novel metabolic profiling methodologies, based on ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) followed by chemometric modeling, data integration, and pathway mapping, to create a systems-level metabolic atlas of atherogenesis. We apply this workflow to compare arterial tissue incorporating plaque lesions to intimal thickening tissue (immediate preplaque stage). We find changes in several metabolite species consistent with well-established pathways in atherosclerosis, such as the cholesterol, purine, pyrimidine, and ceramide pathways. We then illustrate differential levels of previously unassociated lipids to atherogenesis, namely, phosphatidylethanolamine-ceramides (t-test p-values: 3.8 × 10(-6) to 9.8 × 10(-12)). Most importantly, these molecules appear to be interfacing two pathways recognized for their involvement in atherosclerosis: ceramide and cholesterol. Furthermore, we show that β-oxidation intermediates (i.e., acylcarnitines) manifest a pattern indicating truncation of the process and overall dysregulation of fatty acid metabolism and mitochondrial dysfunction. We develop a metabolic framework that offers the ability to map significant statistical associations between detected biomarkers. These dysregulated molecules and consequent pathway modulations may provide novel targets for pharmacotherapeutic intervention.

Systems Biology of Human Atherosclerosis

Systems biology describes a holistic and integrative approach to understand physiology and pathology. The “omic” disciplines include genomics, transcriptomics, proteomics, and metabolic profiling (metabonomics and metabolomics). By adopting a stance, which is opposing (yet complimentary) to conventional research techniques, systems biology offers an overview by assessing the “net” biological effect imposed by a disease or nondisease state. There are a number of different organizational levels to be understood, from DNA to protein, metabolites, cells, organs and organisms, even beyond this to an organism’s context. Systems biology relies on the existence of “nodes” and “edges.” Nodes are the constituent part of the system being studied (eg, proteins in the proteome), while the edges are the way these constituents interact. In future, it will be increasingly important to collaborate, collating data from multiple studies to improve data sets, making them freely available and undertaking integrative analyses.

In-vitro Identification of Distinctive Metabolic Signatures of Intact Varicose Vein Tissue via Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

OBJECTIVES Nuclear magnetic resonance (NMR) spectroscopy is an established tool for metabolic profiling of tissues or biofluids with utility in identifying disease biomarkers and changes in enzymatic or gene expression. This pilot study aims to compare the metabolic profiles of intact varicose and non-varicose vein tissue via magic angle spinning (MAS) NMR spectroscopy with a view to promoting the understanding of the pathogenesis of varicose vein formation. METHODS Varicose vein tissue (n = 8) was collected from patients undergoing varicose veins surgery. Control non-varicose great saphenous vein samples were collected from patients undergoing lower limb amputation (n = 3) and peripheral arterial bypass surgery (n = 5). Intact tissue samples (average weight 10.33 ± 0.8 mg) from each vein segment were analysed using 1D MAS (1)H NMR (600 MHz) spectroscopy. For selected vein samples, two-dimensional (2D) NMR experiments were performed. Differences between spectra from varicose and non-varicose tissues were elucidated using a variety of multivariate statistical analyses. RESULTS The metabolic profiles of varicose veins samples were clearly differentiated from non-varicose veins samples. Lipid metabolites were present at a higher concentration in the non-varicose veins group whilst creatine, lactate and myo-inositol metabolites were more characteristic of the varicose veins group. CONCLUSION We demonstrate differential metabolic profiles between varicose veins and non-varicose veins. Elucidating the metabolic signature underlying varicose veins can further improve our understanding of the biological mechanisms of disease initiation, progression, and aid in identifying putative therapeutic targets.

A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples

BackgroundCST6 promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. The aim of our study was to develop a Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) assay for the investigation of CST6 promoter methylation.MethodsWe designed primers that amplify both methylated and unmethylated CST6 sequences after sodium bisulfate (SB) treatment and used spiked control samples of fully methylated to unmethylated SB converted genomic DNA to optimize the assay. We first evaluated the assay by analyzing 36 samples (pilot training group) and further analyzed 80 FFPES from operable breast cancer patients (independent group). MS-HRMA assay results for all 116 samples were compared with Methylation-Specific PCR (MSP) and the results were comparable.ResultsThe developed assay is highly specific and sensitive since it can detect the presence of 1% methylated CST6 sequence and provides additionally a semi-quantitative estimation of CST6 promoter methylation. CST6 promoter was methylated in 39/80 (48.75%) of FFPEs with methylation levels being very different among samples. MS-HRMA and MSP gave comparable results when all samples were analyzed by both assays.ConclusionsThe developed MS-HRMA assay for CST6 promoter methylation is closed tube, highly sensitive, cost-effective, rapid and easy-to-perform. It gives comparable results to MSP in less time, while it offers the advantage of additionally providing an estimation of the level of methylation.

Mutation scanning of exon 20 of the BRCA1 gene by high-resolution melting curve analysis.

OBJECTIVES 5382insC frameshift mutation along with 5331G>A (G1738R) missense mutation, both found in exon 20 of the BRCA1 gene, are relatively frequent among the Greek breast and ovarian cancer population (46%). Our goal was to develop a novel, reliable and rapid genotyping/scanning method for mutation detection of the exon 20 of the BRCA1 gene, using high-resolution melting curve analysis. DESIGN AND METHODS The developed methodology was based on real-time PCR and high-resolution melting curve analysis in the presence of LCGreen I dye. Two amplicons on the exon 20 of BRCA1 gene were designed (157 bp and 100 bp), one flanking the exons boundaries, and one embracing the 5382insC mutation. Our methodology was first optimized and validated by using genomic DNA samples with the 5382insC and 5331G>A (G1738R) mutations and wild-type. In total, the developed methodology was applied on 90 peripheral blood and 127 formalin-fixed paraffin-embedded breast tissue samples. RESULTS Sensitivity studies with gDNA isolated from peripheral blood showed that mutated DNA could be reliably detected in the presence of wild-type DNA at 5% and 0.5% ratio with the larger and the smaller amplicon, respectively. By using the developed methodology we successfully identified 5382insC, 5331G>A and 5370C>T (R1751X) mutations, in genomic DNA isolated from peripheral blood samples and 5382insC mutation in two breast tumors, as verified by DNA sequencing. CONCLUSIONS The combination of real-time PCR and high-resolution melting curve analysis provides a cost-efficient, simple and rapid approach to successfully scan exon 20 of BRCA1 gene for these clinically important and frequent mutations.

Perturbations in fatty acid metabolism and apoptosis are manifested in calcific coronary artery disease: An exploratory lipidomic study.

BACKGROUND Controversy exists concerning the beneficial or harmful effects of the presence of ectopic calcification in the coronary arteries. Additionally, further elucidation of the exact pathophysiological mechanism is needed. In this study, we sought to identify metabolic markers of vascular calcification that could assist in understanding the disease, monitoring its progress and generating hypotheses describing its pathophysiology. METHODS Untargeted lipid profiling and complementary modeling strategies were employed to compare serum samples from patients with different levels of calcific coronary artery disease (CCAD) based on their calcium score (CS). Subsequently, patients were divided into three groups: no calcification (NC; CS=0; n=26), mild calcification (MC; CS:1-250; n=27) and severe (SC; CS>250; n=17). RESULTS Phosphatidylcholine levels were found to be significantly altered in the disease states (p=0.001-0.04). Specifically, 18-carbon fatty acyl chain (FAC) phosphatidylcholines were detected in lower levels in the SC group, while 20:4 FAC lipid species were detected in higher concentrations. A statistical trend was observed with phosphatidylcholine lipids in the MC group, showing the same tendency as with the SC group. We also observed several sphingomyelin signals present at lower intensities in SC when compared with NC or MC groups (p=0.000001-0.01). CONCLUSIONS This is the first lipid profiling study reported in CCAD. Our data demonstrate dysregulations of phosphatidylcholine lipid species, which suggest perturbations in fatty acid elongation/desaturation. The altered levels of the 18-carbon and 20:4 FAC lipids may be indicative of disturbed inflammation homeostasis. The marked sphingomyelin dysregulation in SC is consistent with profound apoptosis as a potential mechanism of CCAD.