ARB Dissertation

Molecular Stratification and Characterization of Clear Cell Renal Cell Carcinoma Angela Rose Brannon A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum in Genetics and Molecular Biology. Chapel Hill 2010 Approved by: W. Kimryn Rathmell, MD, PhD Adrienne Cox, PhD William Kim, MD Charles Perou, PhD Kristy Richards, MD, PhD ©2010 Angela Rose Brannon ALL RIGHTS RESERVED ii ABSTRACT ANGELA ROSE BRANNON: Molecular Stratification and Characterization of Clear Cell Renal Cell Carcinoma (Under the direction of W. Kimryn Rathmell, MD, PhD) It is estimated that there will be 58,240 new diagnoses of kidney cancer in 2010. Most cases will be clear cell renal cell carcinoma (ccRCC) and have little information as to how their disease will progress. This diversity of disease natural history is especially noteworthy in a disease so well characterized by the inactivation of the von Hippel Lindau (VHL) tumor suppressor and resulting stabilization of Hypoxia Inducible Factors (HIF). Previous studies had suggested the presence of two or more clusters in ccRCC. Based on the nonuniformity within the disease’s natural progression and previous research, we hypothesized that distinct inherent molecular subclasses of ccRCC must exist and, therefore, sought to define and characterize them. In fact, two robust subtypes of ccRCC were identified, designated ccA and ccB. These subtypes are associated with survival by multivariate analysis, conferring a median survival of 8.6 years versus 2 years, respectively. We postulated that the underlying molecular pathways within the data would explain the survival difference. ccA tumors overexpress angiogenesis, hypoxia, and metabolism pathways, common pathways characterizing ccRCC tumors. In contrast, ccB tumors overexpress more aggressive genes related to epithelial to mesenchymal transition, cell cycle, and Wnt targets. VHL analysis and HIF immunohistochemistry suggests that neither appear to be driving subtype differences. iii To understand what is causing the differences, underlying genetic changes were analyzed. Both subtypes show deletion of chromosome 3p, location of VHL, in greater than 75% of tumors, corresponding with previous research and suggesting a common initiating tumorigenic event. Overall, copy number patterns look very similar between the subtypes; however, more ccB tumors show deletion of chromosomes 9 and 14, which previous studies have shown to correlate with decreased survival. Additionally, ccA tumors have mutations in a number of histone modification genes, suggesting that epigenetic modification may play a role in subtype differences. Finally, a biomarker panel of 120 probes was defined to distinguish ccA and ccB tumors. This panel is the basis of an assay using FFPE tissue for clinical use. This assay will classify tumors into the inherent subtypes identified by this study, with prognostic impact and potentially predictive import. iv This dissertation is dedicated to my father, Philip Alan Brannon, who pushed me to succeed and, more importantly, to thrive. v Acknowledgements Thank you to the very many people that helped me to complete this dissertation project. I couldn’t have done it without you. Special notes of thanks go out to: My advisor – Dr. W. Kimryn Rathmell, who encouraged, pushed, and shook her head, at me over the years. She also tried to teach me tact, which only partially took. Plus, she brought us cake. My committee – Drs. Adrienne Cox, William Kim, Charles Perou, and Kristy Richards, who have given me guidance and hard questions over the years. My coworkers, past and present - Alex Arreola, Dr. Shufen Chen, Dr. Lance Cowey, Michelle DeSimone, Kate Hacker, Leslie Kennedy, Dr. Caroline Martz Lee, Courtney McGuire, Neal Rasmussen, Dr. Christie Sanford, and Dr. Tricia Wright. These people provided scientific commentary and, more importantly, laughter over the years. Collaborators, especially those at Rutgers - Dr. Gyan Bhanot, Dr. Anupama Reddy, Michael Seiler, and Erhan Bilal. Without these people, this dissertation would have taken far longer. Anupama, in particular, was a great pleasure to work with and helped me better understand how to use a variety of different computer programs. People at UNC who directly helped me in my projects – Dr. Yan Shi in the Genomics and Bioinformatics core, Dr. Dominic Moore in Biostatistics, Dr. Mei Huang in the Tissue Procurement Facility, Dr. Katie Hoadley for teaching me microarray basics, Grace Silva for helping with copy number analysis, Jeremy Simon for helping with Cygwin. vi Department, faculty and staff – IBMS was my first home here on campus. Thanks to Dr. Sharon Milgram for bringing me in and continuing to guide me since then. GMB then became my official curriculum. While TIBBS is not a department, they met a lot of my needs. Dr. Pat Phelps guided me to grow professionally and personally, and she made me laugh on countless occasions. Drs. Christy Ahn and Patrick Brandt were also helpful in a variety of situations. The Diversity Education Team, especially Drs. Cookie Newsom, Donna Bickford and Terri Phoenix, provided an outlet for my desire to grow in understanding diversity and sharing that with the campus. Finally, universities cannot run without administrative assistants. Special thanks to Kathy Allen, Becky Muller, Sausyty Hermreck, Pat White, and Dean Staley. Friends and mentors at the NIH and in Bethesda – Thank you to Dr. Alison McBride for taking me into her lab while I was still extremely green and continuing to be supportive as I progress further up the ladder. Thank you also to Kerri Penrose, who taught me some tricks to finding balance and helped me remember that laughter is very important in science – important tips that have continued to serve me. Friends – I don’t even know who to begin with, especially as many have already been listed above. Dr. Tamara Moyo, who I knew before I even came here and was my first graduate mentor, also helped me through a lot of sticky situations and is an amazing cook. My IBMS class, especially Pamela Hesker, who made graduate school a supported place. My friends at home, many of whom didn’t understand, but were supportive anyway. Rachel Faber Machacha, who could not provide indoor picnics or cheese trays, but did fly down more than once when I needed her support. My church families, both at Newman and Jubilee, and my Bible study group who provided support in my faith and cheering in my studies. My soon to be wife, Jennifer D. Polley, who inadvertently almost derailed my graduate career, but then cheered me as I worked to finish it. vii Family – I would not be here (on this planet or at UNC) without my parents, Phil and Rose Brannon. Thank you for your support over all of these years. Honey and Dziadzia, my mom’s parents, also deserve credit for helping to raise me and instilling a determination to succeed. My aunt, Terry Zajda, and my cousin, Barry Wolcyk, round out my immediate family and took me out to shoot clay pigeons when I was home to blow off some of that excess stress that comes from research. While I am sure that I am forgetting to list someone here, please know that you helped me through my time here at UNC, and I greatly appreciate it. viii Table of Contents List of Tables ..................................................................................................................xiv List of Figures.................................................................................................................. xv List of Abbreviations ......................................................................................................xviii Chapter One : Introduction .............................................................................................1 Renal Cell Carcinoma...............................................................................................2 Biomarkers ...............................................................................................................5 Prognostic Nomograms ............................................................................................6 The pVHL/HIF axis ...................................................................................................7 Other means of regulating HIF ...............................................................................11 Cytogenetic Studies................................................................................................13 Gene Expression Studies .......................................................................................16 Comparisons to normal tissue ..........................................................................19 Comparisons to other histologies .....................................................................19 Analyses focused on clinical outcomes ............................................................20 Biologically driven analyses..............................................................................21 Other Technologies ................................................................................................23 Updating Nomograms.............................................................................................25 Summary ................................................................................................................27 How this body of work builds on previous findings .................................................28

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