Molecular Characterization of Canine Lymphoma Through the Use of Genomic Profiling

Non-Hodgkin lymphomas (NHLs) are frequent malignancies affecting both dog and man, with median five-year survival rates of 65% in people and almost zero in dogs when clinical subtypes of tumors are not taken into account. In the last decade, elucidation of human and canine genome sequences has provided a means to assess the degree of genetic homology between man and dog in normal and malignant states. Additionally, the unique genetic structure of dog breeds provides a means to study complex genetic disorders in genetically homogenous background, suggesting that dogs may be important models for the study of lymphomagenesis. While understanding of canine lymphomas pathobiology has grown in the last few decades, a need remains to further elucidate the complex genetic nature of these tumors within the dog. In this dissertation I hypothesize that 1) formalin-fixed paraffin embedded (FFPE) tissue is a valid resource for genomic profiling in canine lymphoma, 2) DNA copy number imbalances in canine lymphoma are associated with transcriptional deregulation in tumor cells, 3) canine lymphoid tumor cell lines are valid in vitro models of in vivo lymphoid tumors, and 4) conserved molecular alterations exist between canine lymphoma and human Non-Hodgkin lymphoma. Previous studies in human and canine lymphoma genomes has shown that DNA copy number aberrations (CNAs) are a hallmark of these disease and that CNAs may have diagnostic, prognostic, and functional significance. To examine the utility of FFPE tumor tissue in the genomic profiling of CNAs, we employed array comparative genomic hybridization (aCGH) at a 1 Mb resolution to a panel of 99 archival FFPE canine lymphoma tissue samples. Comparison of these data with aCGH data previously generated from independent group of 150 fresh canine lymphoma tumor specimens demonstrated statistical equivalence between sample sets at all stages of data processing, supporting the hypothesis that FFPE tissue is a useful resource for genomic profiling. This also allowed for a novel combinatorial approach using both data sets to validate that the previously defined inventory of CNAs in canine lymphoma is complete. Comparative analysis between human and dog CNAs for clinical subtypes of NHL common to both species identifies a limited number of evolutionary conservation of genomic lesions. Observation of high levels of aneuploidy within the T-cell lymphomas genome suggested the need to assess the functional significance of these recurrent CNAs. Using gene expression profiling and integrative genomics, the transcriptional characteristics of 22 canine T-cell NHLs with matched with aCGH data to explore the correlation of these two genetic characteristics. A limited number of loci demonstrated correlation between genomic imbalance and transcriptional deregulation, and genes within these loci lacked known association with lymphoid tumors, suggesting that most deregulated loci within the genome employ other mechanisms of transcriptional control. Comparison of T-cell lymphoma clinical subtypes identified transcripts that may be associated with disease phenotype, and functional analysis identified deregulation of processes such as chemokine signaling common to all clinical subtypes and perturbation of pathways such as p53 signaling specific to individual subtypes. Comparative analysis of gene sets associated with human peripheral T-cell lymphoma unspecified (PTCLu) molecular subtypes suggested that these transcripts are not relevant to the functional classification of canine PTCLu. Finally, we employed genomic and transcriptional profiling techniques and conventional cytogenetic methods to five commonly utilized canine lymphoid tumor cell lines to define their relevance as in vitro models of primary disease. Immunophenotypic classification refined clinical classifications of these cell lines while genomic profiling identified diverse genomic and transcriptional characteristics that may not globally represent the primary disease. Genomic imbalance and transcriptional deregulation in genes relevant to lymphoid tumor biology, including PTEN and FLT3, suggest the utility of these lines as model for discrete biological pathways. Overall evaluation of the data from these studies suggests that human and canine lymphomas are globally different, but that evolutionarily conserved perturbations of biological processes exist and employ either common or unique molecular mechanisms to contribute to the malignant state.