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Global gene expression profiling of canine lymphoma

Sharp, Margaret (2017) Global gene expression profiling of canine lymphoma. PhD thesis, Murdoch University.

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Lymphoma, a cancer of lymphoid tissue, is a relatively common form of canine malignancy, typically affecting middle-aged to older dogs, with a higher prevalance among certain breeds. Treatment choices and more precise diagnosis and classification for this disease are currently limited by deficiencies in our understanding of the underlying pathogenic mechanisms and by a lack of useful biomarkers. As with other forms of cancer, canine lymphoma is likely to be a molecular disease resulting from the abnormal expression of genes involved in fundamental cell processes such as cell differentiation, proliferation and apoptosis. Thus, molecular tools such as gene expression microarrays, which permit the analysis of thousands of genes in parallel, have the potential to greatly enhance our understanding of lymphoma pathogenesis in the dog, as previously achieved for human lymphoma.

In this study, gene expression profiling (GEP) of 45 canine lymphomas (36 B cell and 9 T cell) and 10 clinically normal canine lymph nodes was carried out using the Affymetrix Canine Genome 2.0 GeneChip microarray system. This showed that canine B cell lymphoma and T cell lymphoma could easily be distinguished from each other, as well as from non-diseased lymph node tissue. Within the B lymphoma specimens, which were dominated by DLBCL cases, there was a close molecular similarity and B lymphoma samples with a histological diagnosis of Burkitt-like (BL) were generally indiscernible as a subgroup. Significantly, however, gene expression profiling could subdivide the B cell lymphomas overall into two closely related molecular subgroups (designated B1 and B2). Direct comparison of this data with previously published canine lymphoma microarray data (the Frantz cohort), upheld these B lymphoma subgroups and confirmed the existence of two previously identified T lymphoma subgroups (high grade T-LBL and low grade TZL), in which all 9 T lymphoma specimens in this study corresponded to the high grade T-LBL subgroup.

Comparison of gene expression patterns of both the B cell and T cell lymphomas with non-diseased lymph nodes, revealed a preponderance of cell cycle genes and genes encoding components of various lymphocyte signalling pathways. A similar result was seen within the T lymphomas when the high-grade (T-LBL) subgroup was compared with the low grade (TZL) specimens. This was strongly indicative of tumour cells with drastically altered proliferative and survival capabilities. The B lymphomas also showed a marked decrease in gene transcripts associated with cell adhesion. An attempt to identify a gene signature common to both B cell and T cell lymphomas identified relatively few genes. However, among these were several coding for haemoglobin molecules, suggesting that an ability to scavenge oxygen in a hypoxic tumour environment might be an important feature of lymphomas in general. The discovery of highly related, yet discernible, canine B lymphoma (mainly DLBCL) subgroups prompted additional assessment of whether the B lymphomas would segregate according to two key molecular signatures defined for human DLBCL, namely stromal-1 (mesenchymal-like with strong extracellular matrix representation) and stromal-2 (angiogenic with increased blood vessel density). While a direct correspondence between human and canine was not readily apparent, a canine B lymphoma subgroup, largely congruent with identified subgroup B2 and referred to as the stromal subgroup, was revealed which appeared somewhat analogous to human stromal-1 signature with very high expression of genes associated with the extracellular matrix together with diffuse collagen staining histologically. This signature, which included the key regulator CTGF (connective tissue growth factor) as well as genes involved in cell adhesion, may underlie those canine tumours exhibiting a fibrotic reaction.
Using a set of 25 genes (16 on the full cohort of specimens and 9 on subsets of the cohort), the microarray data were validated by use of the independent technique of quantitative reverse transcription PCR (qRT-PCR) analysis. To further extend the study, the possibility of using routine archival formalin-fixed paraffin-embedded (FFPE) lymphoma specimens for qRT-PCR was explored. Using modified techniques for extraction of suitable RNA from FFPE tissue, highly valid results were obtained, although a lower signal-to-noise ratio compared to using unfixed tissue necessitated the use of higher cDNA template concentrations and effectively precluded the analysis of genes that exhibited only a low fold-change in mRNA expression. The ability to use archival FFPE tissues for measurement of gene expression has great potential for large-scale retrospective studies to aid the discovery of disease classifiers and therapeutic targets in canine lymphoma.

In summary, this study has shown at a molecular level that canine lymphoma has some degree of similarity with its human counterpart and that it can be stratified into distinct molecular subgroups on the basis of gene expression profiling. A better understanding of the pathogenesis of this tumour type will facilitate efforts to improve classification, discover diagnostic and prognostic biomarkers, and guide treatment decision-making, with the overall aim of improving lymphoma management and clinical outcomes.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Veterinary and Life Sciences
Supervisor(s): Greene, Wayne and Nicholls, Philip
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