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Dott. Luca Licenziato

Phd thesis

Immuno-oncology profiling in canine cancers 



Evading immune system has recently been acknowledged as a cancer hallmark.1 Indeed, based on the immunoediting hypothesis, the constant interaction between tumor cells and host immune system can lead to equilibrium, elimination of tumor cells or their escape.2 The escaping mechanisms include reduced recognition by immune system, increased resistance or proliferation and development of an immunosuppressive microenvironment.2 

Compelling evidence has demonstrated in several canine cancers the contribution to cancer growth and progression by myeloid-derived suppressor cells, regulatory T-cells and other immune cell types, including cytokines, the immune checkpoint receptor PD-1, its ligand PD-L1 and CTLA-4.3–11 Gene expression immune profiling studies have been conducted allowing to cluster canine tumors according to immuno-signatures enrichment.12–15 Further, the presence of tumor infiltrating immune cells and expression of genes related to the immune system have been associated with survival in few cancers in dog.14,16–22 

Both in human and veterinary oncology understanding the mechanisms involved in tumor escape has paved the way to design therapeutic strategies activating the immune system against cancer cells and blocking suppressive signaling.23 Within this context canine lymphoma, osteosarcoma and melanoma represent an excellent model for development of immunotherapeutic strategies in human since murine models generally lack an immunocompetent system.23 



Despite the recent findings in the veterinary immuno-oncology field little is yet known in dog about the interplay mechanisms between cancer and immune cells, and related immune profiles. The main goals of the project are two. The first is to characterize the immuno-oncology gene expression profile of the most frequent tumors in dog, in order to shed light on the strategies used by cancer cells to evade the immunosurveillance. Analysis will be conducted on the most frequent tumor histotypes treated with chemotherapy, including lymphoma (n=50), osteosarcoma (n=50) and melanoma (n=50). Second, since chromosomal aberrations and genetic mutations have a consistent role in reducing immunogenicity of tumor cells and in conferring resistance to immune destruction, a panel of target genes whose mutations are known to be involved in immunoediting will be designed and tested by Next Generation Sequencing (NGS) to identify genetic alterations and correlate with mRNA expression.24 Finally, a cluster analysis of the selected tumors will be performed including several clinicopathological features in order to design a prognostic molecular model. 


Materials and methods 

Lymphoma, osteosarcoma and melanoma will be obtained from ICCB and prospectively collected frozen, RNA-later preserved and formalin-fixed (FF) during the project.25 Sampling will be accompanied by clinical data. For each histotype a similar approach will be pursued and is described hereby: 

1. Immuno-oncology expression profiling: to define immuno-oncology profile NanoString will be employed. Briefly, this technology works well on FF tissues and allows to measure mRNA without amplification. The Canine Immuno-Oncology Panel includes 800 genes across 47 pathways involved in canine immune. The most significative genes from this experiment will be biologically and clinically validated by RT-qPCR. Further, to localize mRNA in tumor cells RNAscope will be performed and if antibodies will be available protein analysis by immunohistochemistry will be considered; 

2. Immuno-oncology genetic profiling: the same tumors described in the previous task will be considered here and analyzed by NGS. First, database mining will be performed to identify the most frequent genes involved in immunoediting both in dog and human, then a panel will be designed for sequencing and a specific bioinformatic pipeline will be elaborated to identify somatic mutations; 

3. Prognostic model designing: to understand the clinical consequences of mutations and expression aberrations a prognostic model for each tumor histotype will be designed using the most updated programs for data integration (i.e. netDx and Nexus). 


Expected results 

Profiling immuno-oncology gene expression patterns in canine lymphoma, osteosarcoma and melanoma will shed light on the immune escape mechanisms and will define a more accurate classification including immuno-signatures, mutation landscape and clinicopathological variables. Along with gene expression, localization of transcripts and coded proteins will help to classify the role of different cytotypes colonizing tumors in the immunoediting. In addition, targeted gene sequencing will permit detecting somatic mutations to potentially propose as therapeutic targets or use as oncological screening in routine clinic visit at diagnosis and follow-up. Finally, new discoveries in the context of canine immuno-oncology will be relevant insight to veterinary and human cancer immunotherapy. 



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2. Mittal, D., Gubin, M. M., Schreiber, R. D. & Smyth, M. J. New insights into cancer immunoediting and its three component phases—elimination, equilibrium and escape. Curr. Opin. Immunol. 27, 16–25 (2014). 

3. Hartley, G. et al. Immune regulation of canine tumour and macrophage PD-L1 expression: Regulation of canine PD-L1 expression. Vet. Comp. Oncol. 15, 534–549 (2017). 

4. Hartley, G., Elmslie, R., Dow, S. & Guth, A. Checkpoint molecule expression by B and T cell lymphomas in dogs. Vet. Comp. Oncol. 16, 352–360 (2018). 

5. Withers, S. S. et al. Metastatic immune infiltrates correlate with those of the primary tumour in canine osteosarcoma. Vet. Comp. Oncol. 17, 242–252 (2019). 

6. Hutchison, S. et al. Characterization of myeloid-derived suppressor cells and cytokines GM-CSF, IL-10 and MCP-1 in dogs with malignant melanoma receiving a GD3-based immunotherapy. Vet. Immunol. Immunopathol. 216, 109912 (2019). 

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10. Maeda, S. et al. Foxp3 + Regulatory T Cells Associated With CCL17/CCR4 Expression in Carcinomas of Dogs. Vet. Pathol. 57, 497–506 (2020). 

11. Pi Castro, D. et al. Expression of FOXP3 in Canine Gliomas: Immunohistochemical Study of Tumor-Infiltrating Regulatory Lymphocytes. J. Neuropathol. Exp. Neurol. 79, 184–193 (2020). 

12. Filley, A. et al. Immunologic and gene expression profiles of spontaneous canine oligodendrogliomas. J. Neurooncol. 137, 469–479 (2018). 

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15. Gardner, H. L. et al. Canine osteosarcoma genome sequencing identifies recurrent mutations in DMD and the histone methyltransferase gene SETD2. Commun. Biol. 2, 266 (2019). 

16. Biller, B. J., Guth, A., Burton, J. H. & Dow, S. W. Decreased Ratio of CD8+ T Cells to Regulatory T Cells Associated with Decreased Survival in Dogs with Osteosarcoma: CD8:Treg in Canine Osteosarcoma. J. Vet. Intern. Med. 24, 1118–1123 (2010). 

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18. Martini, V. et al. Prognostic role of non-neoplastic lymphocytes in lymph node aspirates from dogs with diffuse large B-cell lymphoma treated with chemo-immunotherapy. Res. Vet. Sci. 125, 130–135 (2019). 

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21. Ariyarathna, H., Thomson, N., Aberdein, D. & Munday, J. S. Chemokine gene expression influences metastasis and survival time of female dogs with mammary carcinoma. Vet. Immunol. Immunopathol. 227, 110075 (2020). 

22. Porcellato, I. et al. Tumourinfiltrating lymphocytes in canine melanocytic tumours: An investigation on the prognostic role of CD3 + and CD20 + lymphocytic populations. Vet. Comp. Oncol. 18, 370–380 (2020). 

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24. González, S., Volkova, N., Beer, P. & Gerstung, M. Immuno-oncology from the perspective of somatic evolution. Semin. Cancer Biol. 52, 75–85 (2018). 

25. Aresu, L. et al. The ItalianCanine Cancer Biobank: Our 10year challenge. Hematol. Oncol. 37, 314–315 (2019). 

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