Mutational Heterogeneity in Cancer Akash Kumar A dissertation Submitted in partial fulfillment of requirements for the degree of Doctor of Philosophy University of Washington 2014 June 5 Reading Committee: Jay Shendure Pete Nelson Mary Claire King Program Authorized to Offer Degree: Genome Sciences 1 University of Washington ABSTRACT Mutational Heterogeneity in Cancer Akash Kumar Chair of the Supervisory Committee: Associate Professor Jay Shendure Department of Genome Sciences Somatic mutation plays a key role in the formation and progression of cancer. Differences in mutation patterns likely explain much of the heterogeneity seen in prognosis and treatment response among patients. Recent advances in massively parallel sequencing have greatly expanded our capability to investigate somatic mutation. Genomic profiling of tumor biopsies could guide the administration of targeted therapeutics on the basis of the tumor’s collection of mutations. Central to the success of this approach is the general applicability of targeted therapies to a patient’s entire tumor burden. This requires a better understanding of the genomic heterogeneity present both within individual tumors (intratumoral) and amongst tumors from the same patient (intrapatient). My dissertation is broadly organized around investigating mutational heterogeneity in cancer. Three projects are discussed in detail: analysis of (1) interpatient and (2) intrapatient heterogeneity in men with disseminated prostate cancer, and (3) investigation of regional intratumoral heterogeneity in gliomas. I conclude with a summary of my research and a discussion of future directions. 2 Table of Contents Chapter 1 – Introduction ............................................................................................... 7 Chapter 2- Molecular Landscape of Advanced Prostate Cancers ........................... 13 2.1- ABSTRACT ....................................................................................................... 14 2.2- INTRODUCTION ................................................................................................ 15 2.3- RESULTS .......................................................................................................... 16 2.4- DISCUSSION ..................................................................................................... 22 2.5- METHODS ......................................................................................................... 27 2.6-FIGURES ............................................................................................................ 32 2.7-TABLES .............................................................................................................. 34 Chapter 3- Intrapatient Heterogeneity in Advanced Prostate Cancer ...................... 40 3.1- ABSTRACT ....................................................................................................... 40 3.2- INTRODUCTION ................................................................................................ 40 3.3- RESULTS AND DISCUSSION ........................................................................... 42 3.4- METHODS ......................................................................................................... 46 3.5- FIGURES ........................................................................................................... 48 3.6- TABLES ............................................................................................................. 53 Chapter 4- Intratumoral Heterogeneity in Glioblastoma ........................................... 54 4.1- ABSTRACT ....................................................................................................... 55 4.2- INTRODUCTION ................................................................................................ 55 4.4- RESULTS .......................................................................................................... 56 4.4- DISCUSSION ..................................................................................................... 61 4.5- CONCLUSIONS ................................................................................................. 63 4.6- METHODS ......................................................................................................... 63 4.7- FIGURES ........................................................................................................... 67 3 Chapter 5- Conclusions and Future Directions ......................................................... 71 Appendix A- Supplementary Material for Chapter 2 ................................................. 74 Appendix B- Supplementary Material for Chapter 3 ................................................. 95 Appendix C- Supplementary Material for Chapter 4 ............................................... 105 Appendix D- Inherited BTNL2 Variant in Aggressive Prostate Cancer. ................. 118 D.1- ABSTRACT ..................................................................................................... 119 D.2- INTRODUCTION ............................................................................................. 120 D.3- METHODS ....................................................................................................... 120 D.4- RESULTS ........................................................................................................ 126 D.5- DISCUSSION .................................................................................................. 128 D.6- TABLES .......................................................................................................... 132 D.7- FIGURES......................................................................................................... 135 Appendix E- Genome Sequencing of Idiopathic Pulmonary Fibrosis in Conjunction with a Medical School Human Anatomy Course ..................................................... 136 E.1- ABSTRACT ..................................................................................................... 137 E.2- INTRODUCTION ............................................................................................. 138 E.3- METHODS ....................................................................................................... 139 E.4- RESULTS ........................................................................................................ 140 E.5- DISCUSSION .................................................................................................. 144 E.6- FIGURES ......................................................................................................... 149 E.7- TABLES .......................................................................................................... 151 E.8- SUPPLEMENTARY INFORMATION ............................................................... 153 REFERENCES ............................................................................................................ 154 4 Acknowledgements First, I would like to thank my mentor, Jay Shendure, who has been a fantastic role model, source of inspiration and support. He has taught me how to think like a scientist, communicate and take risks with my work. His brilliance, energy and enthusiasm have made the past four years a joy to work in his lab and I continue to be amazed by his work ethic and work-life balance. One of Jay’s many talents has been to draw a group of exceptionally bright, motivated and collaborative people together. I have been so happy to be able to spend time with this group of people who have taught me how to think bigger and more creatively about scientific problems. I owe special thanks to several members of the lab. Charlie Lee works tirelessly at the sequencer and provided company during many late nights in the lab. Sarah Ng and Emily Turner launched me on my first projects with encouragement and patience. Joe Hiatt and Stephen Salipante offered critical advice and insight at key moments on the way to become a physician scientist. Jacob Kitzman and Aaron McKenna were entirely selfless with their experience in computational and experimental approaches. Matthew Snyder and Andrew Adey shared their knack for statistics and beautifully illustrated figures, respectively. Alex Lewis, Riza Daza and Ruolan Qui provided countless hours of methodical and precise help with experimental procedures. I had the chance to work with two excellent undergraduates during my PhD: Evan Boyle who assisted with experimental methods for the hereditary prostate cancer work en route to what I am sure will be a prolific scientific career and subsequently, Jennifer Milbank for her patient help in validating results from sequencing. Finally, Rupali Patwardhan, Jerrod Schwartz, Martin Kircher and the rest of the Shendure Lab have been invaluable sources of advice friendship and camaraderie. 5 I was blessed to also collaborate with and learn from a number of excellent scientists and clinicians in the past few years including Peter Nelson, Janet Stanford, Matthew Rabinowitz, Robert Rostomily, Jim Olson, and Marshall Horwitz. Peter Nelson served as a co-advisor for nearly all of my work with prostate cancer and his dedication to improving the health of men with cancer shaped my approach to becoming a physician scientist. Liesel Fitzgerald, Ilsa Coleman, Tom White and many other members of the Nelson, Stanford and Olson labs were essential parts of the projects described here. James Maher, Yiannis Kaznessis,