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Evaluation and Optimization of the Translational Potential of Array Evaluation and Optimization of the Translational Potential of Array-Based Molecular Diagnostics by Dawn Nichole Kernagis Department of Pathology Duke University Date:_______________________ Approved: ___________________________ Michael Datto, Supervisor ___________________________ Richard Moon ___________________________ Thomas Ortel ___________________________ Herman Staats ___________________________ Richard Vann Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pathology in the Graduate School of Duke University 2012 ABSTRACT Evaluation and Optimization of the Translational Potential of Array-Based Molecular Diagnostics by Dawn Nichole Kernagis Department of Pathology Duke University Date:_______________________ Approved: ___________________________ Michael Datto, Supervisor ___________________________ Richard Moon ___________________________ Thomas Ortel ___________________________ Herman Staats ___________________________ Richard Vann An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pathology in the Graduate School of Duke University 2012 Copyright by Dawn Nichole Kernagis 2012 Abstract The translational potential of diagnostic and prognostic platforms developed using microarray technology is evident given the significant number of array-based studies describing clinically-relevant molecular subclasses of disease. However, the majority of array-based diagnostics have yet to make their way into the clinical laboratory. Current approaches tend to focus on development of multi-gene classifiers of disease subtypes, but very few studies assess the translational potential of these assays with the clinical laboratory in mind. Likewise, only a handful of studies focus on development of approaches to optimize array-based tests for the ultimate goal of clinical testing utility. Prior to translation into the clinical setting, molecular diagnostic platforms should demonstrate a number of characteristics to ensure optimal and efficient testing and patient care. Assays should be accurate and precise, technically and biologically robust, and should take into account normal sources of biological variance that could ultimately affect test results. The overarching goal of the research presented in this dissertation is to develop methods for evaluating and optimizing the translational potential of molecular diagnostics developed using expression microarray technology. The first research section of this dissertation is focused on our evaluation of the impact of intratumor heterogeneity on precision in microarray-based assays in breast cancer. We conducted genome-wide expression profiling on 50 needle core biopsies iv from 18 breast cancer patients. Global profiles of expression were characterized using unsupervised clustering methods and variance components models. Array-based measures of estrogen (ER) and progesterone receptor (PR) status were compared to immunohistochemistry. The precision of genomic predictors of ER pathway status, recurrence risk, and sensitivity to chemotherapeutics were evaluated by interclass correlation. Results demonstrated that intratumor variation was substantially less than the total variation observed across the patient population. Nevertheless, a fraction of genes exhibited significant intratumor heterogeneity in expression. A high degree of reproducibility was observed in single gene predictors of ER (intraclass correlation coefficient (ICC)=0.94) and PR expression (ICC=0.90), and in a multi-gene predictor of ER pathway activation (ICC=0.98) with high concordance with immunohistochemistry. Substantial agreement was also observed for multi-gene signatures of cancer recurrence (ICC=0.71), and chemotherapeutic sensitivity (ICC=0.72 and 0.64). Together, these results demonstrated that intratumor heterogeneity, although present at the level of individual gene expression, does not preclude precise micro-array based predictions of tumor behavior or clinical outcome in breast cancer patients. Leading into the second research section, we observed that in some cancer types, certain genes behave as molecular switches and have either an "on" or "off" expression state. Specifically, we observed these molecular switch genes exist in breast cancer as v robust diagnostic and prognostic markers, including ER, PR, and HER2, and define tumor subtypes associated with different treatment and patient survival. We hypothesized that clinically relevant molecular switch (bimodal) genes exist in epithelial ovarian cancer, a type of cancer with no established molecular subgroups. To test this hypothesis, we applied a bimodal discovery algorithm to a publically available ovarian cancer expression microarray dataset (GSE9891:285 tumors; 246 malignant serous (MS), 20 endometrioid (EM), 18 low malignant potential (LMP) ovarian carcinomas). Genes with robust bimodal expression were identified across all ovarian tumor types and within selected subtypes. Of these bimodal genes, 73 demonstrated differential expression between LMP vs. MS and EM, and 22 genes distinguished MS from EM. Fourteen bimodal genes had significant association with survival among MS tumors. When these genes were combined into a single survival score, the median survival for patients with a favorable versus unfavorable score was 65 versus 29 months (p<0.0001, HR=0.4221). Two independent datasets (n=53 high grade, advanced stage serous and n=119 advanced stage ovarian tumors) validated the survival score performance. Taken together, the results of this study revealed that genes with bimodal expression patterns not only define clinically relevant molecular subtypes of ovarian carcinoma, but also provide ideal targets for translation into the clinical laboratory. vi Finally, the third research section of this dissertation focuses on development of robust blood-based molecular markers of decompression stress (DS). DS is defined as the pathophysiological response to inert gas coming out of solution in the blood and tissues when a body experiences a reduction in ambient pressure. To date, there are no established molecular markers of DS. We hypothesized that comparing gene expression before and after human decompression exposures by genome-wide expression profiling would identify candidate molecular markers of DS. Peripheral blood was collected 1hr before and 2hr after 93 hyperoxic, mixed-gas experimental dives (n=54). Control arms included samples collected 1 hour before and 2 hours after high pressure oxygen breathing (n= 9) and surface exercise (n=9), and samples collected at 7am and 5pm for time of day (n=11). Pre and post-dive expression data collected from normoxic nitrox experimental dives were utilized for independent validation. Blood samples were collected into PaxGene RNA tubes. RNA was extracted and processed for globin reduction prior to cDNA synthesis and Affymetrix U133A GeneChip hybridization. Significance analysis revealed 746 genes differentially expressed following hyperoxic, mixed-gas decompression exposures (permutation adjusted p-value cutoff 1.0E-4). After filtering for significant genes in the hyperbaric oxygen, exercise, and diurnal control groups, 726 genes remained. Pathway analysis demonstrated a significant portion of genes were associated with innate immune response (p<0.0001). A 362 multi-gene vii signature of significant, covariant genes was then applied to the independent dataset and demonstrated differentiation between pre and post-dive samples (p=0.0058). Further investigation identified four individual, robust candidate gene markers of DS associated with immune and stress response. Our results showed that expression profiling of peripheral blood following decompression exposures, while controlling for experimental and normal sources of biological variance, identifies a reproducible multi- gene signature of differentially expressed genes, primarily comprising genes associated with innate immune response and independent of venous bubble grade or dive profile. Taken together, the research and results presented in this dissertation represent considerable advances in the development of approaches to guide microarray-based diagnostics towards the ultimate goal of clinical translation. viii Dedication This dissertation is dedicated to my parents, Frank and Patricia Kernagis, for their love, support, and encouragement to venture away from the well-worn path. ix Contents Abstract ......................................................................................................................................... iv Dedication ..................................................................................................................................... ix Contents ......................................................................................................................................... x List of Tables ................................................................................................................................ xv List of Figures ............................................................................................................................. xvi List of Abbreviations ................................................................................................................. xxi Acknowledgements ................................................................................................................ xxiii 1. 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