Investigating the utility of blood borne oncological biomarkers in solid tumours: glioma and melanoma

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Ita, Michael Itak
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University College Cork
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Bloodborne molecular biomarkers are increasingly emerging as significant non-invasive adjuncts to current methods of disease status evaluation in cancer patients. Investigations into the potential utility of these circulating biomarkers as analytic test measures complementing radiological imaging have been occasioned by the invasive nature of malignant tumour tissue biopsy, the need for serial evaluation of tumour burden during therapy, and the need for prognostication. In a series of five studies (four clinical studies and one pre-clinical study), this research work explored the potential utility of plasma cell-free DNA, circulating tumour DNA, cell-free messenger RNA, and bloodborne tumour related proteins as disease biomarkers in patients with glioma and metastatic melanoma. The in vivo research work employed an animal model to study micro RNA mediated epigenetic regulatory mechanisms implicated in therapeutic resistance which is prevalent in melanoma brain metastasis. Specifically, this research work sought to determine whether somatic mutations identified in the plasma samples of patients with glioma were identical or representative of the somatic mutations in synchronously obtained glioma tumour tissue samples. It further sought to determine whether significant differences exist in the plasma transcriptomic profile of glioma patients relative to differences in their tumour characteristics, and also whether any observed differences were representative of synchronously obtained glioma samples and the human cancer genome atlas (TCGA) glioma derived RNA profile. Moreover, this research work explored the relationship between plasma cell-free DNA (cfDNA), serum lactate dehydrogenase (LDH), plasma vascular endothelial growth factor (VEGF), programmed death ligand-1 (PD-L1), interferon-gamma (IFN-γ), and tumour burden in advanced melanoma patients. Furthermore, it sought to examine whether important differences exist in the plasma transcriptomic profile of advanced melanoma patients with a high disease burden compared to patients with a low disease burden or therapeutic response and whether the plasma transcriptomic profile of advanced melanoma patients was representative of TCGA melanoma tumour tissue-derived RNA profile. The methods employed in this research work include; the purification and quantification of circulating cell-free DNA and total RNA from the plasma samples of glioma and metastatic melanoma patients, somatic mutation profiling using DNA derived from FFPE glioma tumour tissue curls and plasma circulating cell-free DNA by amplification refractory mutation system (ARMS®) PCR, pathway-focused gene expression analysis using complementary DNA synthesized from the plasma circulating cell-free messenger ribonucleic acid (ccfmRNA) samples of patients with glioma and advanced melanoma, the extraction and quantification of tumour-related proteins such as LDH, VEGF, PD-L1, and IFN-γ from patients with advanced melanoma by the enzyme-linked immunosorbent assay technique (ELISA), in vivo malignant brain tumour model development, bioluminescence imaging study, immunohistochemistry and microscopy, evaluation of protein expression by flow cytometry, and genomic profiling of total cellular micro RNA using RT- PCR. This research work was able to establish that the detection of plasma circulating tumour DNA originating from the glioma tumour tissues of affected patients is feasible, albeit with a low tumour to plasma mutation concordance. It identified significant differential expression of genes involved in cancer inflammation and immunity crosstalk among patients with different glioma grades, and a positive correlation between the transcriptomic profile of these genes in plasma and tumour samples, and with TCGA glioma derived RNA. Moreover, this research work identified that the incorporation of the quantitative measures of cfDNA, LDH, VEGF and PD-L1 in a suitable multiple regression analysis model was capable of predicting changes in tumour burden in patients with advanced melanoma. Furthermore, it identified and characterized the plasma transcriptomic profile associated with therapeutic response in advanced melanoma patients during immunotherapy. This research work also characterized in a limited way, the tissue and blood markers of therapeutic response and resistance in an in vivo model of melanoma brain metastasis.
Micro RNA , Glioma , Melanoma , Blood borne biomarkers , Cell free DNA , Cell free messenger RNA
Ita, M. I. 2021. Investigating the utility of blood borne oncological biomarkers in solid tumours: glioma and melanoma. PhD Thesis, University College Cork.