Characterization of Low Grade Serous Carcinoma of the Ovary and its Precursor Lesions by Taymaa May A Thesis Submitted in Conformity with the Requirements for Degree of Masters of Science Institute of Medical Sciences University of Toronto © Copyright by Taymaa May 2008 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 OttawaONK1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-72054-7 Our file Notre reference ISBN: 978-0-494-72054-7 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1+1 Canada ABSTRACT Characterization of Low Grade Serous Carcinoma of the Ovary and its Precursor Lesions Taymaa May, Master of Science, Institute of Medical Sciences University of Toronto, 2008 Low-Grade-Serous-Carcinoma (LGSC) is a chemoresistant ovarian neoplasm molecularly linked to the non-invasive Low-Malignant-Potential (LMP) tumors. LMP- with-Micropapillary-features (LMP-MP) have more aggressive behavior. The objectives of this study were to clarify the classification of LMP-MP tumors as borderline or malignant neoplasms and to identify candidate genes involved in low grade serous carcinogenesis. Laser-capture-microdissection was used to isolate epithelial cells from LMP (n=16), LMP-MP (n=9) and LGSC (n=ll). RNA was extracted, amplified, reverse transcribed to cDNA and hybridized to Affymetrix-U133-Plus-2-genechip-arrays. Data were analyzed by GeneSpring, Significance-Analysis-of-Microarrays (SAM), and protein-interaction-database-I2D. Unsupervised-hierarchical-clustering revealed collective clustering of LMP-MP and LGSC, separate from LMP. SAM-analysis identified differential gene expression between LMP and LMP-MP, LMP and LGSC but not between LMP-MP and LGSC. I2D-analysis highlighted differentially expressed genes in the MAPK and EGFR pathways for validation studies. LGSC appears to have a similar genetic profile to LMP-MP and different from LMP. Selected members of the MAPK and EGFR pathways may play a role in low-grade-serous carcinogenesis. Identification of novel genes associated with carcinogenesis and malignant transformation may lead to development of more effective targeted therapy for LGSC. 11 ACKNOWLEDGMENTS I would like to thank Dr. Ted Brown for his remarkable supervision and generous support. I am truly grateful to have been a part of Dr. Brown's laboratory and to have learned from such an impressive scientist. Thank you, Dr. Brown, for allowing me to independently explore interesting ideas while providing gentle guidance and advice. I wish to thank Dr. Patricia Shaw for her dedication and support of this project. Thank you, Dr. Shaw, for the countless hours we spent reviewing pathology slides and scoring TMA slides. The quality of this work would not have been the same without Dr. Shaw's world class expertise and, for that, I am deeply grateful. In addition, I appreciate Dr. Shaw's initiative in creating and maintaining the University Health Network Ovarian Tissue Bank, which is an invaluable resource for ovarian cancer research. Above all, I am grateful to all the women who generously provided the tumor specimens used in this work. Special thanks go to Dr. Joan Murphy, who has been a strong supporter of my clinical journey and her support of my young scientific journey has been equally exceptional. As a member of my research committee, Dr. Murphy has been generous with her time and ideas and provided invaluable clinical and surgical perspectives that have defined and strengthened this project. I am honored to be learning from such an amazing mentor and a true inspiration- Thank you Dr. Murphy. I am greatly appreciative of the funding support this project received from the Toronto Ovarian Cancer Research Network through funds raised by the Toronto Fashion Show; an initiative largely headed by Dr. Murphy. iii Many thanks go to Dr. Susan Done for her input as a member of my research committee. Her expertise in pathology and gene expression profiling were an asset to the development and progress of this work. In addition, I wish to thank Mr. Carl Virtanen for sharing his expertise in microarray data analysis and I look forward to working with Carl in the future. Many thanks go to members of the Brown lab for their help and support. Special thanks go to Ms. Alicia Tone for her procedural teachings and for so much more. I'll miss our morning Starbucks routine. Wonderful thanks go to the division of Gynecological Oncology at Princess Margaret Hospital for their continuous help and support. Special thanks go to Dr. Barry Rosen for his insightful comments during our many discussions. I also wish to thank Mrs. Judy Brusse for coordinating many of the meetings and events related to this work and for doing it with a pleasant smile. I would like to thank Dr. Alan Bocking for his notable support and for providing my source of funding through the Bernard Ludwig Fellowship award. I am also extremely grateful to Dr. Heather Shapiro for her invaluable advice and for allowing me to go through a smooth transition from bedside to bench and back. Most of all, I would like to thank my mother, who is a true inspiration and a great role model. Thank you mom for your love, support and all the long talks. Great thanks go to my sister and her family for their unconditional love and support. Thank you sis for being my strongest supporter and best friend. iv Loving thanks go to my husband, my partner, who has been with me through every major (and minor) decision of my adult life. I am confident I wouldn't be who I am today if it weren't for his love and support. Thank you Ali. Lastly, I dedicate this work to the memory of my father, the original Dr. May, who is the inspiration and driving force behind all that I do. To you dad, with love.. v TABLE OF CONTENTS CONTENT PAGE Abstract ii Acknowledgements iii-v Table of Contents vi-ix List of Figures x-xi List of Tables xii List of Abbreviations xiii-xv CHAPTER 1: INTRODUCTION PAGE 1.1 Ovarian Carcinoma 1 1.1.1 Early Symptoms of Ovarian Carcinoma 2 1.1.2 Early Detection Strategies 3 1.2 Epithelial Ovarian Carcinoma 4 1.2.1 Embryogenesis of the Ovarian Surface Epithelium 4 1.2.2 Origin of Epithelial Ovarian Carcinoma 5 1.2.3 The Role of the Fallopian Tube Epithelium in Ovarian Carcinogenesis 5 1.3 Serous Epithelial Ovarian Carcinoma 6 1.3.1 Surgical Stage and Histological Grade in Serous Ovarian Carcinoma 6 1.3.2 Two-Tier Grading System of Ovarian Serous Carcinoma 7 VI 1.4 High Grade Serous Carcinoma 9 1.5 Low Grade Serous Carcinoma 11 1.5.1 Differences in Molecular Characteristics between LGSCandHGSC 13 1.5.2 Differences in Invasive Characteristics of LGSCandHGSC 13 1.6 Serous Low Malignant Potential Tumor 14 1.7 Serous Low Malignant Potential Tumor with Micropapillary Features 17 1.7.1 Controversies Regarding Histological Classification ofLMP-MP 19 1.8 The Relationship Between Low Malignant Potential Tumors and Low Grade Serous Carcinoma 20 1.9 Two Pathway Hypothesis of Ovarian Carcinogenesis 21 1.10 Thesis Hypothesis and Rationale 24 1.11 Objectives 25 CHAPTER 2: MATERIAL AND METHODS PAGE 2.1 Case Selection 26 2.2 Tissue Preparation and Sectioning 27 2.3 Laser Capture Microdissection 28 2.3.1 Slide Staining 28 2.3.2 Microdissection of Epithelial Cells 30 vn 2.4 RNA Extraction 2.5 RNA Quality Testing 31 2.6 RNA Amplification and Reverse Transcription to cDNA 32 2.7 cDNA Hybridization to Genechip Microarray 32 2.8 Data Analysis 33 2.9 Validation Studies 34 2.9.1 Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction 37 2.9.2 Immunohistochemistry 40 CHAPTER 3: RESULTS PAGE 3.1 Study Cases and Clinical Data 42 3.2 Unsupervised Hierarchical Clustering 48 3.3 Significant Analysis of Microarrays 51 3.4 Protein-Protein Interaction Database 63 3.5 Gene Selection 63 3.5.1 Selected Genes 65 3.6 Validation Studies 69 3.6.1 Quantitative Real-Time RT-PCR 69 3.6.2 Immunohistochemistry 69 vm CHAPTER 4: DISCUSSION PAGE 4.1 Study Design 82 4.1.1 Study Limitations 84 4.2 Clinical Association between LMP, LMP-MP and LGSC 86 4.3 LMP Tumors Have Distinct Gene Expression Profiles From LMP-MP and LGSC 87 4.4 Gene Expression Profile of LMP-MP Tumors is Similar to LGSC 88 4.5 Identification of Candidate Genes Involved in Low Grade Ovarian Carcinogenesis 90 4.5.1 Mitogen Activated Protein Kinase 91 4.5.2 Proposed Protein Cascade Significant in Low Grade Serous Carcinogenesis 93 4.6 Study Implications 94 4.7 Future Studies 96 4.7.1 Functional Assays 96 4.7.2 Differential Gene Expression between Ovarian LGSC and HGSC 97 4.8 General Conclusion and Summary 98 REFERENCES 99 IX LIST OF FIGURES FIGURE PAGE 1.