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

Simple item page
dc.contributor.authorClothier, Stacy Laurenen
dc.contributor.committeechairHuckle, William R.en
dc.contributor.committeememberLeRoith, Tanyaen
dc.contributor.committeememberKlahn, Shawna L.en
dc.contributor.committeememberBarrett, Sarah Hammonden
dc.contributor.departmentBiomedical and Veterinary Sciencesen
dc.date.accessioned2021-05-06T06:00:37Zen
dc.date.available2021-05-06T06:00:37Zen
dc.date.issued2019-11-12en
dc.description.abstractBackground: 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.en
dc.description.abstractgeneralLymphoma, 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.en
dc.description.degreeMaster of Scienceen
dc.format.mediumETDen
dc.identifier.othervt_gsexam:22603en
dc.identifier.urihttp://hdl.handle.net/10919/103200en
dc.language.isoenen
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectCanine Lymphomaen
dc.subjectImmune Checkpoint Moleculesen
dc.titleImmune Checkpoint Molecule Expression in Canine Lymphoma and Canine Reactive Lymphoid Hyperplasiaen
dc.typeThesisen
thesis.degree.disciplineBiomedical and Veterinary Sciencesen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Clothier_SL_T_2019.pdf
Size:
2.72 MB
Format:
Adobe Portable Document Format
Download

Collections

Masters Theses