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Evaluation of biomarkers for diagnosis of canine lymphoma

Al-Kinani, Layla Hashim (2021) Evaluation of biomarkers for diagnosis of canine lymphoma. PhD thesis, Murdoch University.

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PDF - Whole Thesis
Embargoed until January 2023.

Abstract

Lymphoma is a malignancy arising from lymphocytes (B cell or T cell) that are a central component of the immune system. As one of the three most common cancers encountered in the canine, lymphoma affects middle-aged to older dogs and usually stems from lymphatic tissues, such as lymph nodes, lymphoid tissue or spleen. Despite improvements in the management of canine lymphoma, better understanding concerning subtype and tumour aggressiveness is crucial for improved clinical diagnosis to differentiate malignancy from hyperplastic conditions, in addition to improve decision-making around whether to treat and what treatment type to use. This study aimed to evaluate a selection of potential novel biomarkers related to tumour stroma (collagen) and iron metabolism (embryonic haemoglobin [HBE], iron, haptoglobin [HP], ferritin [FER], hepcidin [HEP], transferrin [TF], and ceruloplasmin [CP]) that might have application in lymphoma diagnosis and monitoring response to chemotherapy. These novel biomarker candidates were identified from gene expression microarray analyses of canine lymphoma or from studies of the human disease and were used in combination with previously established markers (C-reactive protein [CRP] and lactate dehydrogenase [LDH]) in this study.

Archived samples were used in combination with prospectively collected samples from dogs with and without lymphoma. Prospectively, 36 canine patients were enrolled from two different veterinary practices, one located in Australia (Perth Veterinary Specialists) and one in Italy (Clinical Veterinary Malpensa) and blood samples collected at presentation as well as during treatment. Serum HP, HEP, FER, TF, iron, CP, CRP and LDH were measured. In addition, fine needle aspirate material (as part of the diagnostic process at presentation only) from the same patient cohort were assessed by RT-qPCR for HBE, HP, and type 1 collagen (COL1A1). Extensive archived material was available in the histopathology block bank at Murdoch University as well as frozen lymph node biopsies from 102 dogs with lymphoma. This material was used for retrospective analyses investigating the same markers by RT-qPCR, immunohistochemistry (IHC), and histochemical staining.

Collagen was confirmed to be aberrantly expressed in canine lymphoma as compared to normal lymph node tissue, which is consistent with fibrotic tumour stroma having been described in a wide range of malignancies. Aberrant collagen was particularly found in B cell lymphoma, and within these, a subgroup of tumours exhibited very high expression. Aberrant collagen expression detected by histochemical staining had high diagnostic accuracy for B cell lymphoma, with the ability to differentiate B cell lymphoma from hyperplastic and normal lymph nodes where the area under the receiver operating characteristic curve (ROC) was 0.935 (95% Cl, 0.89 to 0.98) with specificity 100% and sensitivity 78.6%. This has potential application as a relatively easy-to-perform test yielding corroborative information to aid diagnosis.

The iron-related biomarkers serum HEP and FER were found to be increased in dogs with lymphoma compared to control dogs at the time of diagnosis, while CP concentrations were decreased. Notably, the novel marker, HEP may be useful in the diagnosis of lymphoma along with the established biomarkers CRP and LDH. Not all serum iron-related biomarkers were altered with HP, TF and iron not being found to be different between dogs with lymphoma and healthy dogs at presentation. Together, these findings indicate increased iron storage and tissue iron sequestering rather than altered iron transport or haeme scavenging. No significant correlation was found between the various serum biomarkers at time of diagnosis except weak positive associations of FER with HEP and FER with iron. In addition, there were no appreciable patterns in serum iron biomarker concentrations detected over time during the monitoring of chemotherapy in the B lymphoma patients.

HBE mRNA was found to be aberrantly expressed in canine B and T lymphoma as compared to normal lymph node tissue. Specific detection of HBE protein required an immunohistochemical protocol which was successfully developed. However, using this assay aberrant HBE protein expression could not be confirmed. Due to the lack of availability to this study of canine placenta tissue as a control tissue, this IHC assay needs further validation in dogs before a final conclusion can be made about whether HBE is expressed at the protein level in canine lymphoma. Nevertheless, this assay has potential to be used in future studies to investigate ectopic expression of HBE in different diseases states and various neoplastic disorders, including lymphoma.

To identify differentially expressed genes that may have potential utility as novel biomarkers and/or provide further insight into the molecular pathogenesis of canine lymphoma RNA-Seq analysis was performed comparing B cell lymphoma and T cell lymphoma to normal lymph node tissue. This showed that canine B cell lymphoma and T cell lymphoma could readily be distinguished from each other, as well as from non-diseased lymph node tissue on the basis of gene expression. Bioinformatic analysis revealed a preponderance of cell cycle genes and genes encoding components of various signalling pathways in both the B cell and T cell lymphomas. This was strongly indicative of tumour cells with drastically altered proliferative and survival capabilities. In addition, the B lymphomas also showed a marked increase in genes associated with cilia function and formation, while the T lymphomas showed an increase in genes associated with gene transcription. As well as identifying further potential novel biomarkers for follow up studies, these findings will aid a better understanding of the pathogenesis of this malignancy.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): Medical, Molecular and Forensic Sciences
Veterinary Medicine
Supervisor(s): Greene, Wayne, Rossi, Gabriele, Sharp, Claire and Coiacetto, Flaminia
URI: http://researchrepository.murdoch.edu.au/id/eprint/61530
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