A Novel DLA Class I Genotyping Approach to Determine the Immunogenomic Profiles of Canine Cancer
Cancer is still the second largest cause of death in humans, despite the availability of numerous effective treatment options. At the forefront of treatment innovation is cancer immunotherapy because it utilizes the body’s natural power to fight disease by boosting a patient’s immune system to fight tumor cells. A patient’s responsiveness to immunotherapy is reliant on their Major Histocompatibility Complex (MHC) and the cancer antigens it is able to present to immune system cells. Different forms of the MHC present different cancer antigens. Therefore, understanding the MHC-antigen relationship is fundamental to improving immunotherapy treatments. Despite this fundamental need, the antigens that each form of the MHC can present are currently unknown. Knowledge of the MHC-antigen relationship can be quickly provided through the use of animal models for comparative oncology. Canines are particularly beneficial for human cancer research, as canines suffer from the same naturally-occurring cancers as humans, and both have significantly similar MHC sequences. In dogs, the MHC is called the Dog Leukocyte Antigen (DLA). There are three classes of the DLA; Class I and II genes are involved in antigen presentation. Current DLA Class I genotyping methods, most pertinently Sanger Sequencing, have significant limitations including accuracy, efficiency, and comprehensiveness. We are seeking to address these issues by developing a novel method to genotype DLA Class I genes. Our approach entails DLA Class I amplification through Polymerase Chain Reaction (PCR), amplicon separation through Gel Electrophoresis, DLA fragmentation by Tagmentation, amplification using indexed primers, and next generation sequencing using the MiSeq system. All four of the DLA-Class I genes have been successfully amplified and independently verified. The next steps are to finish the tagmentation and sequencing processes. This method may be applied to germline samples matched with canine tumors undergoing exome sequencing for identification of expressed somatic mutations and neoantigens, which are antigens encoded by tumor-specific mutated genes. This could improve understanding of the immune microenvironment in canine cancer and guide development of new immunotherapies. This research can improve veterinary cancer management and further be translated to human medicine to better clinical outcomes through immunotherapy treatment.