Genomic and Transcriptome Profiling of Serous Epithelial Ovarian Cancer By Rebecca Joanne Zoe Menzies B.Sc. A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Medical Biophysics University of Toronto © Copyright Rebecca Joanne Zoe Menzies (2009) Genomic and Transcriptome Profiling of Serous Epithelial Ovarian Cancer Master of Science 2009 Rebecca Joanne Zoe Menzies Department of Medical Biophysics University of Toronto Abstract Epithelial ovarian cancer is the leading cause of death by gynaecological malignancy. Elucidation of the driver genes of ovarian cancer will lead to treatment targets and tailored therapy for this disease. The Affymetrix Genome-Wide SNP Array 6.0 was used to study 100 serous ovarian samples and 10 normal ovarian samples to identify loci and driver genes. The ovarian cancer genome was found to have high overall genomic instability across all chromosomes and key known genes in this disease were identified in the dataset. Aberrant regions of copy number gain were located in “blocks” of constant copy number at 1p, 1q, 8q, 12p, 19q and 20q. The range in copy number for gains was 4.2 to 5.1. The “blocks” of genes were located at 8p and 5p for copy number losses. The range for copy number loss was 0.6 to 0.9. ii Acknowledgements I would like to acknowledge the support of my supervisor Dr. Tak W. Mak. His mentorship and guidance has been truly inspirational. I would also like to thank the members of my supervisory committee, Dr. Igor Jurisica and Dr. Ben Neel for their helpful comments, feedback and insights. Thanks to Nancy Ng and Yury Bukhman for their technical support with running the arrays. I would like to thank Dr. Elisabeth Tillier for her guidance, discussions and help with this project. I would also like to acknowledge our collaborators for the ovarian cancer project, Dr. Denis Slamon and Dr. Patricia Shaw. Thank you to all the members of the Mak laboratory for their helpful discussions, input and collegiality. During the course of my graduate studies I received funding from the Canadian Institute for Health Research (CIHR). I would finally like to thank my parents, Dr. Teresa Menzies and Dr. John Menzies, my two sisters, Erica Menzies and Dr. Fiona Menzies and my boyfriend Jonathan Covato. They, as always, provide the support and love I need to allow me to fulfill my goals. iii TABLE OF CONTENTS Abstract ............................................................................................................ ii Acknowledgments ...................................................................................................... iii Table of Contents ...................................................................................................... iv List of Figures and Tables ........................................................................................ viii List of Abbreviations.................................................................................................. xi Chapter 1: Introduction ............................................................................................... 1 1.1 Epidemiology of Ovarian Cancer ......................................................... 2 1.2 Tumors of the Ovary ............................................................................ 2 1.3 Clinical Presentation of Ovarian Cancer ............................................... 3 1.4 Ovarian Cancer Screening .................................................................... 4 1.5 Ovarian Cancer Risk factors ................................................................ 5 1.6 Histological Subtypes of Ovarian Cancer ............................................. 5 1.7 Current Treatment Modalities .............................................................. 6 1.8 Prognosis ............................................................................................. 7 1.9 Theories on the Etiology of Ovarian Cancer ......................................... 9 1.10 Cancer “Drivers” ................................................................................ 10 1.11 Pathways in Cancer – Complexity Increases ...................................... 11 1.12 Molecular Genetics of Ovarian Cancer ............................................... 12 1.13 Ovarian Cancer Gene Expression ....................................................... 15 1.14 Structural Variation in Ovarian Cancer ............................................... 16 1.15 Parallels with Breast Cancer ............................................................... 17 1.16 Targeted Molecular Therapies ............................................................ 18 iv 1.17 Ovarian Cancer Genome-Wide Study ................................................. 18 1.18 Study Aims and Hypotheses ............................................................... 18 Chapter 2: Experimental Design and Methods ........................................................... 20 2.1 Introduction ...................................................................................... 21 2.2 Collaboration and Sample Selection ................................................... 21 2.3 Platform Selection – Copy Number Array Technology ....................... 22 2.4 Affymetrix Genome-Wide SNP Array 6.0 .......................................... 24 2.5 DNA Requirements for the Array ..................................................... 25 2.6 Sample and Array Batches ................................................................. 26 2.7 Array Data Analysis ........................................................................... 27 2.8 Accessing Partek Genomics Suite 6.4 ................................................. 27 2.9 Allele Intensity Import and Fragment Restriction ............................... 28 2.10 Creating Copy Number from Intensities ............................................. 28 2.11 Principal Components Analysis .......................................................... 30 2.12 Copy Number Visualization ............................................................... 30 2.13 Segmentation Algorithms ................................................................... 31 2.14 Assessment of Overall Trends in Data ................................................ 34 2.15 Segments Found in Multiple Samples ................................................ 35 2.16 Gene Annotation ................................................................................ 36 2.17 Gene List ........................................................................................... 36 2.18 Removal of Redundant Genes in “Normal” Samples .......................... 37 2.19 Gene List Parameters ......................................................................... 37 2.20 Selection of Top Hits ......................................................................... 38 v 2.21 Functional Inquiry into Genes of Interest ........................................... 39 2.22 Pathway Analysis of Genes of Interest ............................................... 39 Chapter 3: Results ..................................................................................................... 40 3.1 Introduction ....................................................................................... 41 3.2 Quality Control – Principal Components Analysis .............................. 41 3.3 Quality Control – Copy Number Histogram ....................................... 46 3.4 Copy Number Heat Maps ................................................................... 49 3.5 Serous Ovarian Cancer Heat Maps ..................................................... 49 3.6 Normal Sample Heat Maps ................................................................ 65 3.7 Normal and Serous Heat Map Comparison ......................................... 80 3.8 Copy Number Variation Karyoview ................................................... 81 3.9 Serous Gene List – Copy Number Gains ............................................ 85 3.10 Serous Gene List – Copy Number Losses ........................................... 96 3.11 Normal Gene List – Copy Number Gains ......................................... 101 3.12 Normal Gene List – Copy Number Losses ....................................... 101 3.13 Functional Inquiry for Top Genes of Interest .................................... 102 3.14 Pathway Analysis ......................................................................... ....106 Chapter 4: Discussion ............................................................................................. 108 4.1 Genomic Instability Identified in Serous Ovarian Cancer ................. 109 4.2 Cell Polarity Genes and Ovarian Cancer........................................... 110 4.3 Copy Number Alteration in Non-ovarian Cancer .............................. 111 4.4 Future Directions ............................................................................. 113 vi 4.5 Clinical Implications for Future Research......................................... 115 4.6 Conclusion ....................................................................................... 115 References ........................................................................................................ 117 Appendix ........................................................................................................ 137 vii List of Figures and Tables Figure 1 Age-adjusted cancer death rates in women from 1930-2003 (USA) ...... 2 Figure 2 Kaplan-Meier disease