Immune Checkpoint Molecule Expression in Canine Lymphoma and Canine Reactive Lymphoid Hyperplasia

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.