ABSTRACT ANGSTADT, ANDREA Y. Evaluation of the Genomic

ABSTRACT ANGSTADT, ANDREA Y. Evaluation of the Genomic Aberrations in Canine Osteosarcoma and Their Resemblance to the Human Counterpart. (Under the direction of Dr. Matthew Breen). In the last decade the domestic dog has emerged as an ideal biomedical model of complex genetic diseases such as cancers. Cancer in the dog occurs spontaneously and several studies have concluded that human and canine cancers have similar characteristics such as presentation of disease, rate of metastases, genetic dysregulation, and survival rates. Furthermore, in the genomic era the dog genome was found more homologous in sequence conservation to humans than mice, making it a valuable model organism for genetic study in addition to pathophysiological analysis. Osteosarcoma (OS), the most commonly diagnosed malignant bone tumor in humans and dogs, is one such cancer that would benefit from comparative genomic analysis. In humans, OS is a rare cancer diagnosed in fewer than 1,000 people per year in the USA, while in the domestic dog population the annual number of new cases is estimated to far exceed 10,000. This high rate of disease occurrence in dogs provides a unique opportunity to study the genomic imbalances in canine OS and their translational value to human OS as a means to identify important alterations involved in disease etiology. OS in humans is characterized by extremely complex karyotypes which contain both structural changes (translocations and/or rearrangements) and DNA copy number changes. Metaphase and array comparative genomic hybridization (aCGH) has assisted in uncovering the genetic imbalances that are associated with human OS phenotype. In dog OS, previous low-resolution (10-20Mb) aCGH analysis identified a wide range of recurrent copy number aberrations (CNAs), indicative of a similar level of genomic instability to human OS. To further interpret chaotic OS karyotypes a genome-wide approach was taken to identify, characterize, and directly compare genomic instability in canine and human OS. For identification of genome-wide CNAs 123 cases of canine OS were profiled by 1Mb-resolution aCGH, 23 of the 123 cases were subsequently profiled by ~27kb-resolution aCGH and 15 cases of human OS were profiled by ~100kb-resolution aCGH. Subsequent fluorescence in-situ hybridization (FISH) analysis was used to confirm aCGH data, quantify numerical imbalances, and visualize structural abnormalities in a subset of dog OS cases. Characterization of the affect that CNA has on the expression of select cancer associated genes revealed that imbalance and transcriptional dysregulation in canine OS also paralleled human OS. Specifically, changes in RUNX2, TUSC3, and PTEN expression levels correlated with genomic copy number status in dog OS. This analysis showcased RUNX2 as an ‘OS associated gene’ and TUSC3 as a tumor suppressor gene involved in canine OS. In addition, direct comparison of genomic imbalance in human and dog OS using high resolution oligonucleotide aCGH indicated that the ‘OS associated genes’ RUNX2, CDKN2A/CDKN2B, MYC, RB1, and PTEN resided in orthologous microaberration regions (<500kb) with similar CNA patterns supporting that these genes are key genetic players driving OS progression. Similarities in genome-wide CNA patterns in OS between orthologous regions of the human and dog genome were also found suggesting that characterization of genes in these regions may identify additional alterations important for OS manifestation. Ultimately, this large scale screening of genomic imbalance in canine OS reiterates the value of the dog as a biomedical model of human OS while pinpointing key genes dysregulated in the disease in dogs. Upon further investigation, the genes with parallel CNA frequencies in human and dog OS may serve as possible targets of novel genetic therapeutics that once developed and tried in dogs could be translational to human patients. Evaluation of the Genomic Aberrations in Canine Osteosarcoma and Their Resemblance to the Human Counterpart by Andrea Y. Angstadt A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Functional Genomics Raleigh, North Carolina 2010 APPROVED BY: _______________________________ Dr. Matthew Breen ______________________________ Dr. Dahlia M. Nielsen Committee Chair _______________________________ Dr. David E. Malarkey ______________________________ Dr. Marlene L. Hauck DEDICATION To my parents, Roy and Ann Young, for your love, guidance, and support To my husband, Nathan, for joining me in this journey ii BIOGRAPHY Andrea developed her love for biology at an early age through hiking trips, raising farm animals, and grade school science fair projects. After graduating high school she went to Penn State University and completed a degree in animal bioscience with a minor in biology. During her time at Penn State she worked in a laboratory studying the genetic affect of external and internal factors on cacao plant growth and response as a means to improve plant productivity. She also completed an undergraduate research project identifying potential mutations in the leptin receptor gene associated with the diabetic and obese phenotype in the Butterball strain of mice. These projects increased her interest in genetics and its’ role in veterinary and human diseases. Therefore, she decided to continue her research training in the Functional Genomics Doctorate program at North Carolina State University. iii ACKNOWLEDGMENTS I would like to thank my committee members, Dr. David Malarkey, Dr. Dahlia Nielsen, and Dr. Marlene Hauck for providing support, discussions, and aid in my research progress. Thanks to Dr. Dahlia Nielsen and Dr. Alison Motsinger-Reif for helping me learn how to manage large datasets and conduct appropriate statistical analyses and to Dr. Eric Stone for your excellent teaching of bioinformatics. Dr. Ted Emigh, it was a pleasure to TA for your genetic class and I appreciate the aid you gave in developing my teaching skills. To the Breen Lab through the years; Tessa Breen, Benoit Hedan, Shannon Becker, Rachael Thomas, Christina Williams, Eric Seiser, Pei-Chen Tsai, Kate Kelley, Katie Kennedy, and Kristen Maloney, thanks for making the work environment so much fun. I cherish all our discussions and will miss you all! To my father, my brother Hugh, and Erin Parker thanks so much for your editing skills and advice. Lastly, a special thanks to Dr. Matthew Breen for your direction and guidance through my graduate years and future endeavors. iv TABLE OF CONTENTS LIST OF TABLES................................................................................................................. viii LIST OF FIGURES ..................................................................................................................x LIST OF ABBREVIATIONS ............................................................................................... xiii Chapter I: Literature Review .................................................................................................1 Overview....................................................................................................................................2 Dog as a suitable model of human disease ................................................................................3 The pathology of osteosarcoma .................................................................................................9 Molecular and genetic dysregulation of osteosarcoma............................................................13 Genomic Studies......................................................................................................................17 Thesis Outline..........................................................................................................................31 References................................................................................................................................32 Chapter II: Characterization of canine osteosarcoma by array comparative genomic hybridization and qRT-PCR: Signatures of genomic imbalance in canine osteosarcoma parallels the human counterpart..........................................................................................49 Abstract....................................................................................................................................50 Introduction..............................................................................................................................51 Materials and Methods.............................................................................................................53 Tissue Specimens...............................................................................................................53 Array Comparative Genomic Hybridization (aCGH)........................................................55 Fluorescence in-situ Hybridization (FISH)........................................................................56 Quantitative RT-PCR.........................................................................................................57 Statistical Analysis.............................................................................................................58 Results......................................................................................................................................59 Clinical Assessment............................................................................................................59 Abundant Genomic Instability in Canine OS .....................................................................60 FISH Validation of aCGH Data..........................................................................................65 v Breed and Morphological Subtype Specific Associated DNA Copy Number Aberrations .............................................................................................................................................68 qRT-PCR.............................................................................................................................70 Discussion................................................................................................................................71 Acknowledgements..................................................................................................................78 References................................................................................................................................90 Chapter III: A genome-wide approach to comparative oncology: High-resolution oligonucleotide aCGH of canine and human OS pinpoints communal microaberrations ................................................................................................................................................101 Abstract..................................................................................................................................102 Introduction............................................................................................................................103 Materials and Methods...........................................................................................................107 Tissue Specimens...............................................................................................................107 Array Comparative Genomic Hybridization (aCGH)........................................................109 Array Comparative Genomic Hybridization Analysis.......................................................110 Identification of Orthologous Canine and Human Regions...............................................111 Results....................................................................................................................................112 Refining Regions of Genomic Aberration in Canine OS...................................................112 Extensive Genomic Imbalance in Human OS ...................................................................117 Comparative Regions of Genomic Imbalance in Canine and Human OS ........................121 Discussion..............................................................................................................................125 References..............................................................................................................................131 Chapter IV: Conclusion ......................................................................................................141 Concluding remarks...............................................................................................................142 Future evaluation of the complexity in canine OS.................................................................147 References..............................................................................................................................151 Appendices............................................................................................................................158 vi Appendix I: Tackling the characterization of canine chromosomal breakpoints with an integrated in-situ/in-silco approach: The canine PAR and PAB.....................................159 Abstract..................................................................................................................................160 Introduction............................................................................................................................160 Materials and Methods...........................................................................................................162 Clone Selection..................................................................................................................162 Cytogenetic Evaluation......................................................................................................162 Computational Analysis of Overlapping BAC Clones......................................................164 Results....................................................................................................................................164 Cytogenetic interpretation of the canine PAR ...................................................................164 Computational Analysis of the PAB..................................................................................164 Comparison of Canine and Human PAR-Specific Genes..................................................165 Discussion..............................................................................................................................167 References..............................................................................................................................168 Appendix II: Visualization of copy number changes in OS by FISH.............................170 vii LIST OF TABLES Chapter I: Table 1: Tumor incidence rate (all sites) estimated in pet dogs by country and tumor characteristics.............................................................................................................................7 Table 2: Previously defined molecular factors dysregulated in canine OS ............................14 Table 3: Summary of the cytogenetic abnormalities associated with human OS....................23 Table 4: Summary of highly recurrent genomic aberrations (≥30%) in 38 patients of canine OS indentified by low-resolution (10-20Mb) aCGH...............................................................29 Table 5: Summary of copy number imbalances for known cancer-associated genes in 38 patients of canine OS identified by low-resolution (10-20Mb) aCGH....................................29 Chapter II: Table 1: Summary of breed and morphological subtype of 123 canine OS tumor samples analyzed for DNA copy number aberrations by genome integrated 1Mb-resolution aCGH ..54 Table 2: Primer sequence of genes analyzed by qRT-PCR analysis of canine OS RNA........58 Table 3: The top twenty autosomal regions that exhibited the highest percentages of copy number gain in our canine OS cases........................................................................................64 Table 4: The top twenty autosomal regions that exhibited the highest percentages of copy number loss in our canine OS cases.........................................................................................65 Supplementary Table 1: Detailed list of clinical information concerning 123 OS tumor samples analyzed for DNA copy number aberrations by genome integrated 1Mb-resolution aCGH .......................................................................................................................................84 Supplementary Table 2: Genomic position and BAC ID for clones selected using the UCSC <a href="/goto?url=http://www.genome.ucsc.edu/) ...................................................................88" target="_blank">genome browser (http://www.genome.ucsc.edu/) ...................................................................88 </a>Supplementary Table 3: Raw Ct values of non-neoplastic bone and 11 canine OS samples for reference gene c12orf4 3.....................................................................................................89 viii Chapter III: Table 1: Clinical summary of 23 canine OS tumor samples analyzed for DNA copy number aberrations (CNAs) on Agilent’s canine180,000 feature G3 SurePrint oligonucleotide aCGH platform..................................................................................................................................108 Table 2: Clinical summary of 15 human OS tumor samples analyzed for DNA copy number aberrations on Agilent’s 60,000 feature G3 SurePrint oligonucleotide aCGH platform.......109 Table 3: Frequency of autosomal CNAs (gain and loss) identified by the ADM-2 algorithm with a threshold of 6 and log2 ratio cutoff of 0.2 in 23 cases of canine OS...........................116 Table 4: Frequency of CNAs (gain and loss) identified by the ADM-2 algorithm with a threshold of 6 and log2 ratio cutoff of 0.5 in 15 cases of human OS.....................................120 ix LIST OF FIGURES Chapter I: Figure 1: Dog cancer incidence rates for females and males estimated from tumor specimens received by the ATR from local veterinarians in Genoa, Italy across the study period (1985- 2002) for all cancers and selected cancer sites by calendar period............................................9 Figure 2: SKY analysis of human OS.....................................................................................20 Figure 3: FISH analysis of dog OS as directed by aCGH........................................................30 Chapter II: Figure 1: Frequency of copy number aberration in 123 canine OS samples...........................62 Figure 2: Bivariate fit of CNA percent loss/gain for 123 canine OS cases by the physical size of the aberrations in Mb...........................................................................................................63 Figure 3: Whole genome 1Mb aCGH profile of a 12 year old female Golden Retriever diagnosed with osteoblastic OS located in the left proximal humerus, co-hybridized with DNA derived from a blood sample from the same patient......................................................67 Figure 4: Further characterization of copy number aberration occurrences across 1,066 regions of aberration commonality in the 38 dog autosomes ..................................................69 Figure 5: Quantitative RT-PCR on 26 canine OS patients for six genes located in regions that had a high occurrence of CNAs...............................................................................................71 Supplementary Figure 1: Kaplan-Meier survival curve comparing different treatment protocols for canine OS patients..............................................................................................79 Supplementary Figure 2: Interphase multicolor FISH analysis of three zinc fixed paraffin embedded canine OS tissues using BAC clone probe sets encompassing three genes (TSC2, MYC, and TUSC3) ..................................................................................................................80 Supplementary Figure 3: Interphase multicolor FISH analysis of three zinc fixed paraffin embedded canine OS tissues using BAC clone probe sets encompassing three genes (RUNX2, RHOC, and PTEN) ..................................................................................................................83 x Supplementary Figure 4: Graphical representation of the PCA performed to evaluate global differences in genome-wide DNA copy number aberrations evident in our five breed groups ..................................................................................................................................................82 Supplementary Figure 5: Graphical representation of the PCA performed to evaluate global differences in genome-wide DNA copy number aberrations evident in our three morphological subtypes ...........................................................................................................83 Chapter III: Figure 1: Array CGH profiles of OS12, an 8 year-old male Rottweiler with osteoblastic OS ................................................................................................................................................113 Figure 2: Frequency of copy number aberrations in 23 canine OS samples .........................115 Figure 3: Box plots of frequency of autosomal CNAs (gain and loss) in 23 canine OS cases (array call resolution ~27kb) identified by the ADM-2 algorithm with a threshold of 6 and log2 ratio cutoff of 0.2............................................................................................................117 Figure 4: Array CGH profile (100kb resolution view) of HOS5, a 12-year old female with OS in the left distal femur............................................................................................................118 Figure 5: Frequency of copy number aberrations in 15 human OS samples.........................119 Figure 6: Box plots of frequencies of CNAs (gain and loss) in 15 human OS cases (array call resolution ~100kb) identified by the ADM-2 algorithm with a threshold of 6 and log2 ratio cutoff of 0.5............................................................................................................................121

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