Phenotypes and genetic markers of cancer cachexia

xi Chapter 1 – Introduction . 1 1.1 Background . 2 1.2 Definition of cancer cachexia . 2 1.3 Epidemiology and incidence of cancer cachexia . 3 1.4 Staging of cancer cachexia as a spectrum . 6 1.5 Mechanisms of tissue loss . 7 1.5. 1 Anorexia / decreased food intake . 7 1.5. 2 Increased enery expenditure . 9 1.5. 3 Other aspects of abnormal metabolism associated with cachexia . 12 1.6 Mediators of tissue loss . 14 1.6. 1 Changes in muscle and adipose tissue mass . 15 1.7 Fat loss . 23 1.8 Muscle loss . 23 1.9 Biomarkers of cancer cachexia . 24 1.9. 1 Plasma biomarkers of cancer cachexia . 25 1.9. 2 Muscle biomarkers of cancer cachexia . 25 1.9. 3 Urinary biomarkers of cancer cachexia . 26 1.9. 4 Genetic biomarkers of cancer cachexia . 27 1.9. 5 Candidate gene association analysis in cachexia . 29 1.10 Clinical consequences and management of cachexia . 32 1.11 Hypothesis . 36 1.12 Aims . 36 1.13 Plan . 37 Chapter 2 – Muscle wasting in cancer . 39 2.1 Summary . 40 2.2 Introduction . 40 2.3 Heterogeneity . 41 2.4 Skeletal muscle morphology in cancer associated myopenia: murine models . 43 2.4.1 Fibre size and type in cancer associated myopenia . 44 2.4.2 Fibre RNA, DNA, and protein content in cancer associated myopenia . 45 2.4.3 Dystrophin glycoprotein complex (DGC) . 46 2.5 Mediators of cancer associated myopenia: murine models . 46 2.5. 1 Pro-inflammatory mediators . 46 2.5. 2 Catabolic mediators . 48 2.5. 3 Anabolic mediators . 48 2.6 Mechanisms of cancer associated myopenia: murine models . 49 2.6. 1 Protein degradation pathways . 49 2.6. 2 Protein synthesis pathways . 55 2.6. 3 Fat-muscle cross talk . 55 2.7 Skeletal muscle morphology and function in cancer associated myopenia: clinical data . 56 2.8 Mediators of cancer associated myopenia: clinical data . 57 2.8. 1 Systemic mediators of cachexia . 58 2.8. 2 Pro-inflammatory cytokines . 58 2.8. 3 Other circulating mediators . 59 2.9 Mechanisms of cancer associated myopenia: clinical data . 62 2.9. 1 UPP / E3 ubiquitin ligases . 62 2.9. 2 Autophagy / Lysosomal pathway . 64 2.9. 3 Calpains. 64 2.9. 4 Caspases . 64 2.9. 5 Myogenesis . 65 2.10 Future prospects . 65 Chapter 3 – Materials and methods . 66 3.1 Ethical approval . 67 3.2 Study participants . 67 3.2.1 Clinical Classification of Cancer Cachexia: Phenotypic Correlates in Human Skeletal Muscle (Chapter 4) . 67 3.2.2 Genetic signatures associated with susceptibility to cancer cachexia vary according to weight-loss or low muscle mass phenotype (Chapter 6) . 68 3.3 Clinical and pathological data collection (Chapter 4 and 6) . 70 3.4 Nutritional assessment (Chapter 4 and 6) . 70 3.5 Collection of biological samples (Chapter 4 and 6) . 70 3.5. 1 Blood collection (Chapter 4 and 6) . 70 3.5. 2 Rectus abdominis muscle biopsy and storage for biochemical analysis (Chapter 4 and 6) . 71 3.5. 3 Rectus abdominis muscle sample preparation for cryo-section (Chapter 4) . 71 3.6 Blood assays (Chapter 4 and 6) . 71 3.6. 1 Measurement of C-reactive protein (CRP) . 71 3.7 Body composition analysis . 72 3.7. 1 Estimation of total body skeletal and adipose tissue using computed tomography (CT) scans . 72 3.8 Cellular methods (Chapter 4 and 6) . 74 3.8. 1 Immunohistochemistry (Chapter 4) . 74 3.8. 2 Tissue Preparation for DNA, RNA and Protein extractions (Chapter 4) . 75 3.8. 3 DNA and RNA extraction and linearity of the extraction method (Chapter 4) . 75 3.8. 4 Protein extractions (Chapter 4) . 76 3.8. 5 Western blots (Chapter 4) . 76 3.8. 6 DNA extraction from blood (Chapter 6) . 77 3.8. 7 Quantification of DNA (Chapter 6) . 78 3.9 Single nucleotide polymorphism (SNP) genotyping (Chapter 6) . 78 3.10 Microarray analysis (Chapter 6) . 80 3.11 Statistics . 83 3.11.1 General statistics . 83 3.11.2 SNP genotyping statistical analysis (Chapter 6) . 83 3.11.3 Transcriptomic study (Chapter 6) . 84 Chapter 4 – Clinical Classification of Cancer Cachexia: Phenotypic Correlates in Human Skeletal Muscle . 85 4.1 Summary . 86 4.1.1 Aims . 86 4.1.2 Methods . 86 4.1.3 Findings . 86 4.1.4 Conclusion . 87 4.2 Introduction . 87 4.3 Materials and methods . 89 4.3.1 Patient recruitment, identification, consent and ethics . 89 4.3.2 Calculation of weight loss . 89 4.3.3 Classification of cancer cachexia . 89 4.3.4 Rectus abdominis muscle biopsy . 90 4.3.5 Rectus abdominis muscle sample preparation for cryo-sectio