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Epigenetic Study of Plasma Circulating DNA in Prostate Cancer

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Epigenetic Study of Plasma Circulating DNA in Prostate Cancer Epigenetic Study of Plasma Circulating DNA in Prostate Cancer by Andrew Kwan A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Pharmacology and Toxicology University of Toronto © Copyright by Andrew Kwan (2015) Epigenetic Study of Plasma Circulating DNA in Prostate Cancer Andrew Kwan Master of Science Graduate Department of Pharmacology and Toxicology University of Toronto 2015 Abstract Malignant cells exhibit epigenetic differences compared to normal cells, which can also be detected in the cell-free circulating DNA (cirDNA) of bodily fluids. DNA modification studies in cirDNA may lead to the development of non-invasive cancer biomarkers. This thesis investigates plasma cirDNA modifications of prostate cancer patients, benign prostate hyperplasia patients, and men without any known prostate disease. A modification-sensitive restriction enzyme approach was used to enrich the modified DNA fractions, which were interrogated on CpG island- and Affymetrix tiling microarrays. Our initial discovery screen identified a number of differentially modified genes and non-coding loci between PCa patients and controls; a subset of candidate loci were verified using bisulfite pyrosequencing. We applied machine-learning techniques to develop a multi-locus biomarker set, which exhibited promising predictive performance in distinguishing cancer patients from unaffected controls. Our results suggest that epigenomics of cirDNA is a promising source for diagnostic non-invasive biomarkers of solid tumors. ii Acknowledgments I would like to start by expressing my gratitude to my supervisor, Dr. Arturas Petronis, for giving me the opportunity to work under his mentorship and guidance for the past few years. It is under his tutelage that I have grown both as a person and a scientist. I will also be forever indebted to the supportive post-doctoral fellows that I have had the chance to work with, Dr. Rene Cortese and Dr. Tarang Khare. Their friendship and care for me – extending well outside of the lab setting – have inspired me to develop mentorship skills and has truly made the lab feel like a second home. To each member of the Krembil Epigenetics Laboratory, the memories have been truly unforgettable and I thank each of you for your friendship on this journey. My thanks also go out to my committee members, Dr. Albert Wong and Dr. Jose Nobrega for their invaluable advice throughout my program. Further, I would like to acknowledge our collaborators, Dr. Paul Boutros, Emilie Lalonde, and Cindy Yao for performing the bioinformatics on our experimental work. I would also like to acknowledge Vanier CGS for providing me with the financial support to pursue my degree. Last but certainly not least, I owe all of my successes to my family and friends for their love and unending support throughout these years. I would not have been able to accomplish any of this without all of you. iii Table of Contents Acknowledgments ........................................................................................................................ iii Table of Contents ......................................................................................................................... iv List of Tables ............................................................................................................................... vii List of Figures ............................................................................................................................. viii List of Appendices ........................................................................................................................ ix Abbreviations ................................................................................................................................ x Chapter 1 Introduction ................................................................................................................. 1 1 Introduction ................................................................................................................................ 1 1.1 Statement of research problem ............................................................................................ 1 1.2 Review of Literature ........................................................................................................... 2 1.2.1 Epigenetics .............................................................................................................. 2 1.2.2 Cancer biomarkers .................................................................................................. 6 1.2.3 Circulating DNA (cirDNA) .................................................................................... 8 1.2.4 Prostate cancer ...................................................................................................... 17 1.3 Study objectives and rationale for hypotheses .................................................................. 25 Chapter 2 Materials and Methods ............................................................................................. 26 2 Materials and Methods ............................................................................................................. 26 2.1 Samples ............................................................................................................................. 26 2.1.1 Prostate cancer pilot study .................................................................................... 26 2.1.2 Prostate cancer tiling microarray study ................................................................. 26 2.2 DNA extraction ................................................................................................................. 27 2.3 DNA modification amplicon preparation ......................................................................... 27 2.3.1 Principle of DNA modification detection ............................................................. 27 2.3.2 DNA blunting ........................................................................................................ 29 iv 2.3.3 Adaptor ligation .................................................................................................... 29 2.3.4 DNA modification-sensitive restriction enzyme digestion ................................... 29 2.3.5 Adaptor-mediated PCR ......................................................................................... 30 2.4 Microarray experiments and data analysis ........................................................................ 31 2.4.1 Prostate cancer pilot study .................................................................................... 31 2.4.2 Prostate cancer tiling microarray study ................................................................. 39 Chapter 3 Results ........................................................................................................................ 44 3 Results ...................................................................................................................................... 44 3.1 Prostate cancer pilot study ................................................................................................ 44 3.1.1 Differentially modified cirDNA in prostate cancer .............................................. 44 3.1.2 Microarray verification of candidate genes by pyrosequencing ........................... 48 3.1.3 Predictive value of DNA modification of RNF219 .............................................. 54 3.1.4 Analysis of pericentromeric DNA repeats in chromosome 10 ............................. 56 3.1.5 Epigenetic signature of PCa using machine-learning analyses ............................. 58 3.1.6 Differentially modified and differentially expressed genes in prostate tumor ..... 58 3.2 CirDNA analysis in prostate cancer using high density tiling microarrays ...................... 63 3.2.1 Microarray based epigenetic profiling of cirDNA in prostate cancer patients and controls ........................................................................................................... 63 3.2.2 Functional Analysis .............................................................................................. 67 3.2.3 Performance of an epigenetic signature as a diagnostic biomarker for PCa ......... 68 3.2.4 Performance of an epigenetic signature as prognostic biomarker for PCa ........... 69 Chapter 4 Discussion .................................................................................................................. 72 4 Discussion ................................................................................................................................ 72 4.1 Prostate cancer pilot study of DNA modification differences in plasma cirDNA ............ 72 4.1.2 Predictive value of DNA modification of RNF219 .............................................. 75 4.1.3 Improved accuracy with biomarker panels ........................................................... 76 v 4.1.4 Changes in repetitive DNA elements in cancer .................................................... 77 4.1.5 Primary PCa tumor analysis .................................................................................. 78 4.2 PCa tiling microarray study .............................................................................................. 79 4.2.1 DNA modification profiling on high resolution tiling arrays ............................... 79 4.2.2 Predictive performance of epigenetic signature
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