Defining the tumor transcriptome and systemic immune landscape of French bulldogs with High-Grade Gliomas: insights into breed-specific survival disparities

Abstract

Canine high-grade glioma (HGG) is an aggressive primary brain tumor with few treatment options and a uniformly poor prognosis. It shares many features with glioblastoma (GB), the most common and aggressive primary brain tumor in people. In effect, clinical trials investigating novel therapies in pet dogs can benefit both patient populations. Immunotherapy trials in HGG-bearing pet dogs have provided strong preliminary data for similar clinical trials in people with GB. The suitability of the canine HGG model to predict GB treatment responsiveness hinges on continued characterization of shared features betweenthe two cancers. Relative to immunotherapy trials, exploring variables that contribute to interpatient tumor heterogeneity and classifying both the local and systemic immune landscape in HGG-bearing dogs are especially important for both canine HGG and human GB patients. In a translational clinical trial at our institution, we observed that HGG- bearing French bulldogs had a uniquely poor response to immunotherapy-based treatments. The cause of this survival disparity was unknown, but given that the observation was breed-exclusive, heritable underlying causes pertaining to the HGG and the immune system were suspected. The purpose of this research was to define the transcriptomes of French bulldog HGG and circulating peripheral immune cells as compared to breeds with favorable response to immunotherapy. First, the survival disparity was further defined by investigating survival outcomes between breeds and treatments. Pertaining to immunotherapy, French bulldogs were confirmed to have markedly worse survival outcomes than boxers and Boston terriers, two dog breeds in the same phylogenetic clade as French bulldogs and with similar HGG prevalence. Pertaining to French bulldogs treated with four different treatments, the median survival time of immunotherapy-treated dogs was not significantly different from those treated with palliative care. Both these groups had significantly worse median survival times than those treated with stereotactic radiation therapy or sonodynamic therapy. Regarding characterization of the French bulldog HGG transcriptome, two investigations were performed using RNA sequencing of formalin-fixed, paraffin- embedded HGG tissue to conduct differential gene expression analysis and geneset enrichment analysis (GSEA). First, the baseline differences in tumor transcriptomes between French bulldogs and other breeds was explored. Treatment-naïve French bulldog HGG samples were compared to treatment- naïve boxer and Boston terrier samples. Differential gene expression revealed 31 differentially expressed genes (DEGs), with many having critical roles in glioma malignancy in both GB and HGG. GSEA showed activated enrichment of 15 pathways pertaining to cell cycle progression, oncogenes, and immune pathways. Second, the evolution of HGG following immunotherapy treatment was explored. Paired analyses of post-immunotherapy HGG samples versus pre- treatment HGG were performed in French bulldogs and separately in boxers and Boston terriers to enable comparison of DEGs between the two breeds. Compared to boxers and Boston terriers, French bulldog HGG was associated with larger log2-fold change magnitude in DEGs, upregulation of known drivers ofglioma progression, and enrichment of immune-associated pathways associated with poor prognosis. Regarding characterization of the French bulldog peripheral immune landscape in the presence of HGG, RNA multiplex analysis using NanoString’s Canine Immuno-Oncology (IO) panel was used to quantify 800 genes across 47 annotated pathways in blood samples obtained from HGG-bearing and HGG-free French bulldogs and boxers and Boston terriers. Differential gene expression analysis showed that when compared to HGG-free French bulldogs, HGG-bearing French bulldogs displayed eight upregulated and 46 downregulated DEGs, many demonstrated to be similarly altered in human GB circulating immune cells. This research represents the first step in determining the molecular mechanisms that cause the survival disparity observed in HGG-bearing, immunotherapy-treated French bulldogs. This information is valuable not only for informing breed-specific treatment options for pet dogs with HGG, but also further refines the canine HGG model of human GB. Future studies to validate the observations of this research may identify key targets for novel therapy development for both dogs and people.