Evaluation of the Genomic Aberrations in Canine Osteosarcoma and Their Resemblance to the Human Counterpart

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