Abstract
Colorectal cancer (CRC) is the third most common malignancy and fourth leading cause of cancer-related deaths worldwide. The incidence of CRC under 55 years of age, considered early-onset CRC (E-CRC), has increased in recent decades and many of these patients do not fit current criteria for CRC screening and diagnostic testing. The development of novel biomarkers for CRC could assist in prioritising patients for clinical investigation. Recent research has highlighted extracellular vesicles (EVs) and EV-encapsulated microRNA (miRNA) as promising novel blood-based biomarkers. Despite identification of potential candidate miRNA biomarkers for CRC, the expression and functional role of EV biomarkers have not been fully investigated in E-CRC, or in the context of the host-immune system and inflammatory response. This thesis explored clinical and genetic factors in E-CRC, including the expression of candidate miRNA biomarkers, and investigated the immunomodulatory role of CRC-derived EVs.
In the first chapter of this thesis, we retrospectively compared clinical and histological factors between traditional-onset colorectal cancer (T-CRC) and E-CRC patients at Wellington Hospital. We observed a longer time from symptom onset to diagnosis in E-CRC patients and found that E-CRC patients were more likely to present emergently or at a later stage of disease. E-CRC tumours were also less likely to have deficiency in mismatch repair protein expression (dMMR) compared to T-CRC tumours. These findings suggest that there may be differences in tumour characteristics between E-CRC and T-CRC patients and provide an opportunity to further identify factors that may contribute to E-CRC development and progression.
miRNA are an emergent class of biomarkers in CRC and are key molecular regulators of tumorigenesis, yet they have not been well explored in the context of E-CRC. This was addressed in our second chapter. We investigated the mutational profile of CRC tumours using a hotspot panel for DNA sequencing and identified pathogenic mutations in cancer-related genes in CRC tumour, but not normal mucosa. We found fewer APC mutations in E-CRC tumour compared to T-CRC tumour. Next, we explored the expression of potential CRC miRNA biomarkers in sporadic T-CRC and E-CRC tumour tissue and EVs. We found differential expression of miR-21-5p between E-CRC and T-CRC tumours and miR-25-3p between E-CRC and T-CRC EVs. These findings demonstrate differences in the mutational profile and genetic regulation between sporadic E-CRC and T-CRC and enhance our understanding of the pathogenesis of E-CRC.
A key role of the miRNA biomarkers that are differentially expressed between T-CRC and E-CRC is modulation of inflammatory pathways to promote aspects of tumorigenesis. This may be mediated by circulatory EVs, by interacting with immune cells within the periphery to influence the host response to tumour. Therefore, in our third chapter, we explored the interaction between CRC-derived EVs and the host-immune system. Specifically, we investigated the effects of CRC-derived EVs on peripheral blood mononuclear cell (PBMC)-derived monocytes, as a model for tumour-directed immunomodulation. While we successfully isolated EVs from CRC cell lines and identified monocyte subsets using flow cytometry, our results did not demonstrate any significant difference in the expression of phenotypic markers in response to EV application. Consequently, further studies are required to understand of the functional role of CRC-derived EVs in CRC.
Taken together, this thesis identified clinical and molecular factors that were different between E-CRC and T-CRC. These findings contribute to our understanding of the molecular mechanisms underlying E-CRC tumorigenesis and highlight the importance of developing novel biomarkers to assist in CRC diagnosis.