Background: Although lymphoma is one of the most common malignancies in dogs, remission rates and survival times remain stagnant. Treatment with a multi-agent chemotherapy protocol induces remission for less than one year and the majority of patients relapse. Fewer than 25% of dogs live longer than two years with the currently available treatments. Targeted immunotherapy using checkpoint molecule blockade of PD-1 and PD-L1 shows promise for various types of human cancer, including relapsed/refractory lymphoma; however, little is known regarding the role of these checkpoint molecules in canine lymphoma. Objectives: To determine the patterns of expression of mRNAs encoding PD-1 and its ligands PD-L1 and PD-L2 in lymphoma and reactive lymphoid hyperplasia controls. Methods: Retrospective: formalin-fixed paraffin-embedded (FFPE) tissue from dogs with untreated lymphoma (n=10) and reactive lymphoid hyperplasia (n=10). Prospective: fine-needle aspirates (FNAs) from dogs with untreated lymphoma (n=10) and reactive lymphoid hyperplasia (n=10). Total RNA was extracted, and expression of PD-1, PD-L1, and PD-L2 was measured using qRT-PCR analysis of random-primed cDNA. Checkpoint molecule expression levels were determined using the 2^∆∆CT method. Lymphoma immunophenotype was assessed using immunohistochemical analysis of CD3 and CD79a (FFPE) and review of patient medical records (FNA). Data analysis included Wilcoxon ranksum tests, Dunn's procedure of multiple comparisons, Kruskal-Wallis tests, and regression within an ANOVA. Significance at P < 0.05. Results: PD-1, PD-L1, and PD-L2 expression (normalized internally to 18S rRNA) was lower in lymphoma compared to reactive lymphoid hyperplasia (FFPE); the difference was significant for PD-1 and PD-L2. PD-1 and PD-L2 expression was lower in lymphoma compared to reactive lymphoid hyperplasia (FNA); the difference was significant for PD-1. PD-1, PD-L1, and PD-L2 expression was lower in B cell lymphoma compared to reactive lymphoid hyperplasia (FFPE); this difference was significant for PD-1 and PD-L2. PD-1 and PD-L2 expression was lower in B cell lymphoma compared to reactive lymphoid hyperplasia (FNA); the difference was significant for PD-1. The higher relative abundance of PD-L1 vs PD-1 and PD-L2 vs PD-1 was significantly different between lymphoma and reactive lymphoid hyperplasia (FFPE and FNA). Conclusions: In this study, checkpoint molecule expression was not upregulated in canine lymphoma relative to canine reactive lymphoid hyperplasia, suggesting a limited application of PD-1 and PD-L1 blockade in canine lymphoma. The ligand:receptor relative abundance imbalances reflect the lower PD-1 expression relative to PD-L1 and PD-L2 in lymphoma. Although these results do not suggest that checkpoint inhibitors would be useful for treatment, they give insight into the mechanisms of unchecked lymphocyte proliferation in canine lymphoma. / Master of Science / Lymphoma, a cancer of the white blood cells in the body, is one of the most common malignancies in dogs. Although treatment with a multi-agent chemotherapy protocol results in high remission rates, the remission duration is usually less than one year, with the majority of patients relapsing. In an effort to improve remission rates and survival times, scientists have been working to develop therapeutic interventions that target specific points in the development and replication cycle of a cancer cell. One such strategy, targeting checkpoint molecules programed death (PD)-1 and PD-L1, has shown promise for several different types of human cancers, including lymphoma. PD-1 is a receptor on T cells, which together with its ligands, PD-L1 and PD-L2, decreases lymphocyte function when activated. This is a protective mechanism, acting to inhibit sustained harmful inflammation in a normal healthy dog. Some cancers have taken advantage of this pathway, increasing expression of PD-L1 or L-L2 in order to evade detection by the immune system. To date, little is known regarding the role and expression of these immune checkpoint molecules in dogs with lymphoma. We sought to evaluate if PD-1, PD-L1 and PD-L2 expression is significantly increased in canine lymphoma compared to reactive lymphoid hyperplasia controls. Tissue samples were collected from two sources. Cytology samples of lymphoma and reactive lymphoid hyperplasia were collected by fine needle aspiration from clinical patients. Formalin fixed paraffin embedded tissue samples of lymphoma and reactive lymphoid hyperplasia were collected from the archived tissue bank. Using a molecular analysis technique called quantitative reverse transcription PCR (qRT-PCR) we measured the amount of messenger RNA (mRNA) encoding PD-1 and its ligands PD-L1 and PD-L2 in lymphoma and in reactive lymphoid hyperplasia controls. In our results we did not observe an upregulation in the expression of checkpoint molecules in canine lymphoma relative to canine reactive lymphoid hyperplasia. This suggests there may be a limited therapeutic application for PD-1 and PD-L1/PD-L2 blockade in canine lymphoma. Although these results do not suggest that checkpoint inhibitors would be useful for treatment, they give insight into the mechanisms of unchecked lymphocyte proliferation in canine lymphoma.

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Le lymphome est l'une des tumeurs les plus communes tant chez le chien que l’humain. Chaque année, un nombre important de chiens développe ce cancer agressif. La majorité décédant un an suivant le diagnostic. Le lymphome canin est maintenant identifié comme un excellent modèle de recherche pour la tumeur chez l'homme, particulièrement en ce qui concerne la biologie moléculaire de la maladie. En conséquence, la recherche sur le lymphome canin sera bénéfique non seulement pour les chiens mais aussi pour l’oncologie humaine. Parmi les méthodes diagnostiques de choix pour dépister de façon hâtive le lymphome se trouve la mesure de marqueurs tumoraux. Ceci a l’avantage d’être peu invasive, simple et peu dispendieuse. Ainsi, dans le but d’évaluer la protéine VCP (valosin containing protein) comme biomarqueur tumoral dans les lymphomes canins à cellules B et T, nous avons évalué la protéine VCP par immunobuvardage sur sérums et tissus tumoraux de chiens atteints et par immunohistochimie sur des tumeurs de haut grade, grade intermédiaire et bas grade. Pour mieux définir l’expression de VCP dans les cellules cancéreuses, nous avons également examiné par immunobuvardage les niveaux de VCP dans 3 lignées cellulaires: CLBL-1, CL-1, et 17-71. Il s’avère que les lymphomes à cellules B de haut grade avaient une élévation significative du taux de VCP comparé aux tumeurs de bas grade (P < 0,05). De même, une accumulation importante de VCP a également été détectée dans les lignées tumorales comparées aux cellules mononucléaires du sang périphérique (P < 0,05). D’autre part, le taux sérique de VCP est resté similaire à ceux des chiens normaux. Ces résultats suggèrent une corrélation entre le taux de VCP et le degré de malignité des lymphomes à cellules B. En conclusion, la protéine VCP doit faire l’objet d’une évaluation approfondie pour déterminer son utilité comme marqueur pronostique. / Lymphoma is one of the common malignancies in both dogs and humans. Annually, an important number of canine patients develop this aggressive cancer and a majority succumbs to the disease within one year. In recent years, canine lymphoma has been increasingly recognized as an excellent model for the disease in humans, especially with regards to the molecular biology of the disease. Consequently, research targeted at canine lymphoma benefits not only dogs but the field of human oncology as well. Among the most desirable diagnostic and screening tests for lymphoma is the measurement of cancer biomarkers. They have the advantage of being minimally invasive, simple, and inexpensive. Thus, with the aim of evaluating valosin containing protein (VCP) as a cancer biomarker in canine B and T-cell lymphomas, we first performed western blots on sera and tumor tissue of dogs with lymphoma and then immunohistochemical analysis on low, intermediate and high-grade tumors. To further determine VCP expression in cancer cells, we also examined VCP levels by immunoblotting in 3 tumor cell lines: CLBL-1, CL-1, and 17-71. High-grade B-cell lymphomas had significantly increased levels of VCP compared to low-grade tumors (P < 0.05). Additionally, we detected a corresponding accumulation of VCP in tumor cells lines compared to peripheral blood mononuclear cells (PBMCs) (P < 0.05). In contrast, VCP levels were not elevated in sera of dogs with lymphoma compared to healthy controls. These results suggest that VCP positively correlates with malignancy in canine B-cell lymphomas. We conclude that VCP merits further investigation to determine its potential as a clinically useful prognosis biomarker for canine B-cell lymphoma.