Project description:We performed whole-exome sequencing of tumour bulks from opposite side of the neoplasm (A/B). From each we selected a panel of sub-clonal mutations and profiled multiple single tumour glands from the same neoplasm using high depth targeted re-sequencing. The aim was to infer tumour evolutionary dynamics and reconstruct the timeline of progression
Project description:Non-invasive prognostic markers are needed to improve survival of colorectal cancer (CRC) patients. Towards this goal, we here apply integrative systems glycobiology approaches to tumour tissues and PBMCs from CRC patients and matching controls as well as a CRC patient-derived cell line. The untargeted -omics-driven approaches revealed that non-canonical paucimannosidic proteins from monocytic and cancer cell origins are prominent signatures in CRC tumour tissues, and that their expression associates with CRC progression. Guided by these novel relationships, we then show in vitro that N-acetyl-β-D-hexosaminidase (Hex) drives paucimannosidic protein biosynthesis in CRC cells, and is intimately involved in processes underpinning CRC metastasis (adhesion, migration, invasion). Importantly, Hex activity was elevated in PBMCs and plasma from patients with advanced CRC relative to those with early-stage disease. Notably, we show that plasma Hex activity accurately informs on CRC patient survival. Our study opens new avenues for effective prognostication and therapeutic intervention in CRC.
Project description:Identification of unknown peaks in gas chromatography/mass spectrometry (GC/MS)-based discovery metabolomics is challenging, and remains necessary to permit discovery of novel or unexpected metabolites that may elucidate disease processes and/or further our understanding of how genotypes relate to phenotypes. Here, we introduce two new technologies and an analytical workflow that can facilitate the identification of unknown peaks. First, we report on a GC/Quadrupole-Orbitrap mass spectrometer that provides high mass accuracy, high resolution, and high sensitivity analyte detection. Second, with an "intelligent" data-dependent algorithm, termed molecular-ion directed acquisition (MIDA), we maximize the information content generated from unsupervised tandem MS (MS/MS) and selected ion monitoring (SIM) by directing the MS to target the ions of greatest information content, that is, the most-intact ionic species. We combine these technologies with (13)C- and (15)N-metabolic labeling, multiple derivatization and ionization types, and heuristic filtering of candidate elemental compositions to achieve (1) MS/MS spectra of nearly all intact ion species for structural elucidation, (2) knowledge of carbon and nitrogen atom content for every ion in MS and MS/MS spectra, (3) relative quantification between alternatively labeled samples, and (4) unambiguous annotation of elemental composition.
Project description:Fatty acids (FAs) play critical roles in health and disease. The detection of FA imbalances through metabolomics can provide an overview of an individual's health status, particularly as regards chronic inflammatory disorders. In this study, we aimed to establish sensitive reference value ranges for targeted plasma FAs in a well‑defined population of healthy adults. Plasma samples were collected from 159 participants admitted as outpatients. A total of 24 FAs were analyzed using gas chromatography‑mass spectrometry, and physiological values and 95% reference intervals were calculated using an approximate method of analysis. The differences among the age groups for the relative levels of stearic acid (P=0.005), the omega‑6/omega‑3 ratio (P=0.027), the arachidonic acid/eicosapentaenoic acid ratio (P<0.001) and the linoleic acid‑produced dihomo‑gamma‑linolenic acid (P=0.046) were statistically significant. The majority of relative FA levels were higher in males than in females. The levels of myristic acid (P=0.0170) and docosahexaenoic acid (P=0.033) were significantly different between the sexes. The reference values for the FAs examined in this study represent a baseline for further studies examining the reproducibility of this methodology and sensitivities for nutrient deficiency detection and investigating the biochemical background of pathological conditions. The application of these values to clinical practice will allow for the discrimination between health and disease and contribute to early prevention and treatment.
Project description:Colorectal cancer (CRC) is a leading cause of cancer-related death. Diagnosis at an early stage can greatly improve the patient outcome. Therefore, developing sensitive and specific non-invasive screening and diagnostic methods has a tremendous potential to combat the disease.
Project description:Germline mutations in the mismatch repair genes mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2), MSH6, and postmeiotic segregation increased 2 (PMS2) lead to the development of hereditary nonpolyposis colorectal cancer (HNPCC). Diagnosis of HNPCC relies on the compilation of a thorough family history of cancer, documentation of pathological findings, tumor testing for microsatellite instability (MSI) and immunohistochemistry (IHC), and germline mutation analysis of the suspected genes. As a hallmark of HNPCC, microsatellite instability is widely accepted as a primary method for identifying individuals at risk for HNPCC. It serves as an excellent, easy-to-evaluate marker of mismatch repair deficiency. Recent improvements in MSI testing have significantly enhanced the accuracy and reduced its cost. Proficiency testing for MSI is available, and laboratory-to-laboratory reproducibility of such testing can be easily evaluated. In addition, the combination of microsatellite instability testing, MLH1 promoter methylation analysis, and BRAF (V600E) mutation analysis can distinguish a sporadic colorectal cancer from one associated with HNPCC, helping to avoid costly molecular genetic testing for germline mutations in mismatch repair genes. In this article, we discuss the development of MSI markers used for HNPCC screening and focus on the advantages and disadvantages of MSI testing in screening for HNPCC patients. We conclude that MSI is as sensitive and specific as IHC, given its excellent reproducibility and its potential capability to indicate mutations not be detected by IHC. MSI has been used and will continue to prevail as the primary screening tool for identifying HNPCC patients.
Project description:We investigated a panel of 21 genes by parallel sequencing on the Ion Torrent Personal Genome Machine platform. We sequenced 65 CRCs that were treated with cetuximab or panitumumab ( 37 samples were responsive and 28 were resistant).
Project description:In order to determine whether dis-regulation of a genetic pathway could explain the increased apoptosis of parp-2-/- double positive thymocytes, the gene expression profiles in double positive thymocytes derived from wild-type and parp-2-/- mice were analysed using Affymetrix oligonucleotide chips (mouse genome 430 2.0).