PhD in Veterinary Sciences for Animal Health and Food Safety

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Phd thesis

Immuno-oncology profiling in canine cancers 

Cancer is a complex and dynamic disease, with outcomes shaped by the intricate dance between tumor cells and the immune system. The immunoediting hypothesis suggests that this interaction can lead to tumor cell elimination, a state of equilibrium, or immune escape. Recent insights into cancer cell biology have underlined the pivotal role of genetics in molding the immune response within the tumor.

Certain types of canine tumors are known to cause a strong immune response and are thus candidates for immunotherapy. One such example is diffuse large B-cell lymphoma (DLBCL), a particularly aggressive cancer in dogs. The marked clinical diversity in dogs with DLBCL has highlighted the need for a molecular-based prognostic classification, rather than one solely reliant on clinical features. Such a classification can aid in identifying tumors that might be responsive to immunotherapy, specifically through an autologous vaccine. Furthermore, a recent transcriptome study has emphasized the significance of the immune system in canine DLBCL (cDLBCL). This provides strong evidence that a deeper understanding of the molecular mechanisms, beyond clinical responses, in dogs undergoing treatment is needed.

The primary objective of this PhD project is to investigate the genetic and immune landscape of DLBCL in dogs to pinpoint robust molecular markers that predict responses to therapy. Subsequently, whole exome sequencing (WES) was performed on 77 cDLBCLs to identify the most common genetic abnormalities. Results identified genes already known to drive cancer in dogs (such as TRAF3, SETD2, POT1, TP53, MYC), alongside some novel findings. TP53 mutations were linked to shorter survival times, a discovery validated in an additional cohort of 56 cases. The project then delved into the immune mechanisms underlying treatment response. To achieve this, the immuno-oncology gene expression profile of cDLBCL using the NanoString nCounter platform was employed. A six-gene prognostic immune signature (IL2RB, BCL6, TXK, C2, CDKN2B, ITK) was identified and a risk score was calculated. Indeed, low-risk dogs exhibited upregulation of genes associated with complement activation, cytotoxicity, and antigen processing, as well as an abundance of natural killer and CD8+ cells when compared to high-risk dogs. Recognizing that immune checkpoints play a role in immune evasion strategies by malignant cDLBCL cells, the expression of PD-1, PD-L1 genes, and CD8A gene was investigated using RNAscope, confirming the negative prognostic impact of PD-L1.

The findings of this PhD project have brought to light the intricate genetic landscape and the complex immune interactions of cDLBCL. By unraveling these critical aspects, the research has armed with powerful tools and insights that extend beyond the laboratory and into the clinic. This knowledge can be applied to enhance the understanding and treatment of this tumor histotype in dog. The tools developed in this research can also be used for monitoring treatment progress and assessing responses to therapy. This ongoing evaluation is essential for adapting treatment plans as necessary in dogs with DLBCL.

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