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The use of multiple platform “omics” datasets to define new biomarkers in oral cancer and to determine biological processes underpinning heterogeneity of the disease Anas A M Saeed Thesis submitted for the degree of Doctor of Philosophy Edinburgh Dental Institute College of Medicine and Veterinary Medicine University of Edinburgh April 2013 Contents Table of Contents Declaration ................................................................................................................. xi Acknowledgements ................................................................................................... xii List of Abbreviations .............................................................................................. xiii Abstract .................................................................................................................... xix Chapter 1: Introduction ............................................................................................ 1 1.1 Oral cancer: Disease and diagnosis .................................................................... 1 1.2 Oral cancer pathology (development and metastasis) ........................................ 4 1.3 Aetiology and risk factors of oral cancer ........................................................... 6 1.4 Multiplicity of gene expression in human .......................................................... 8 1.5 Tumour heterogeneity of the oral cavity ............................................................ 9 1.5.1 Oral cancer in the UK and western countries ............................................. 9 1.5.2 Oral cancer in Sri Lanka and Indo-Asian sub-continent ............................. 9 1.6 Cancer as a metabolic disease .......................................................................... 10 1.7 Biomarkers as diagnostic tool for HNSCC and OSCC .................................... 13 1.8 Metabolomics as tumour biomarkers ............................................................... 14 1.9 Microarray technology ..................................................................................... 17 1.10 Transcriptomic analysis ................................................................................. 18 1.11 Selection of bioinformatics approaches ......................................................... 19 1.11.1 Bioconductor ........................................................................................... 21 1.11.2 Data filtering and normalization ............................................................. 21 1.11.3 Corrections of batch effects .................................................................... 22 1.11.4 Differential gene expression analyses ..................................................... 22 1.11.5 Biological annotations of significant genes and metabolites .................. 23 1.12 Metabolomics and metabonomics .................................................................. 24 1.12.1 NMR metabolomics/metabonomics techniques ...................................... 29 1.12.2 Points to be considered using NMR spectroscopy for metabolomic analysis ............................................................................................................... 33 1.12.3 Data analysis and interpretation of metabolomics .................................. 35 1.13 Integrative Analysis of transcriptomics and metabolomics ........................... 36 Chapter 2: Common gene expression profiling reveals biological alterations involved in early tumourigenesis of Oral Squamous Cell carcinomas ................ 42 i 2.1 Introduction ...................................................................................................... 42 2.2 Aim of the study ............................................................................................... 43 2.3 Materials and methods ..................................................................................... 43 2.3.1 Selection of study samples ........................................................................ 43 2.3.2 Construction of the list of common genes ................................................ 44 2.3.3 Exploration of the common genes expression in the raw data .................. 45 2.3.4 Biological Annotations of significant common genes .............................. 45 2.4 Results .............................................................................................................. 46 2.4.1 Gene expression profile of OSCC versus normal oral mucosa ................. 49 2.4.2 Exploring the set of common genes in a raw data .................................... 53 2.4.3 Biological interpretation of the common expressed genes ....................... 55 2.5 Discussion ........................................................................................................ 63 Chapter 3: Detection of a robust gene signature for oral squamous cell carcinoma by integrating multiple microarray datasets ...................................... 67 3.1 Introduction ...................................................................................................... 67 3.2 Aims and objectives of the study ..................................................................... 68 3.3 Materials and Methods ..................................................................................... 68 3.3.1 Selection of Data Sets ............................................................................... 69 3.3.2 Data processing and analysis .................................................................... 71 3.3.3 Integration of datasets and batch correction ............................................. 71 3.3.4 Differential gene expression analyses ....................................................... 72 3.3.5 Biological annotations of significant genes .............................................. 73 3.4 Results .............................................................................................................. 73 3.4.1 Filtering and processing of the datasets .................................................... 73 3.4.2 Integration of datasets and correction of batch effect ............................... 76 3.4.3 Verification of data integration ................................................................. 77 3.4.4 Gene expression profile of OSCC versus normal oral mucosa ................. 77 3.4.5 Prediction models ...................................................................................... 86 3.4.6 Biological interpretation of the differentially expressed meta-genes ....... 96 3.4.7 Comparison between meta-genes and common genes of the review study .......................................................................................................................... 106 3.5 Discussion ...................................................................................................... 109 ii Chapter 4: Gene expression profiling reveals biological pathways responsible for phenotypic heterogeneity between UK and Sri Lankan oral squamous cell carcinomas .............................................................................................................. 115 4.1 Introduction .................................................................................................... 115 4.2 Aims and objectives of the study ................................................................... 115 4.3 Materials and Methods ................................................................................... 116 4.3.1 Sample characteristics and biopsy specimens ......................................... 116 4.3.2 RNA extraction / cDNA synthesis, processing, labeling, and hybridization to microarray chip ............................................................................................ 120 4.3.3 Data quality control, pre-processing and analysis .................................. 120 4.3.4 Quality checks of microarray GeneChip results (QC) ............................ 121 4.3.5 Differential gene expression analyses ..................................................... 123 4.3.6 Biological interpretation of the expressed genes .................................... 124 4.3.7 Quantitative real-time PCR analysis (validation of gene expression level) .......................................................................................................................... 125 4.4 Results ............................................................................................................ 125 4.4.1 Study populations .................................................................................... 125 4.4.2 Report of microarray quality control .....................................................