01whole.pdf (8.86 MB)
Plasma protein biomarkers of chemotherapy response in colorectal cancer
thesisposted on 2022-03-28, 22:49 authored by Sarah A. Randall
Colorectal cancer (CRC) is the third most common cause of cancer death worldwide, responsible for over 600,000 or 1.1% of total deaths. For CRC patients with metastatic disease, chemotherapy is the recommended first-line treatment. Predictive biomarkers would be useful in differentiating patients who are likely to derive better outcomes from chemotherapy from those patients that will not, thereby improving patient outcomes and potentially curbing healthcare costs. We hypothesised that a plasma protein-based test examining liver-derived plasma proteins may prove useful as a surrogate of liver function during drug metabolism. In this doctoral research, selected reaction monitoring (SRM) mass spectrometry was used in a search for potential plasma protein predictive biomarkers related to chemotherapy response in metastatic CRC patients. In Chapter 3, technical and biological variation relevant to plasma proteome analysis was explored using specimens from healthy individuals. Outcomes from targeted SRM experiments investigating quantitative levels of plasma proteins from healthy individuals over time-courses demonstrated the relative stability of the selected plasma proteome. Research in Chapter 4 detailed the discovery of candidate plasma biomarkers predictive of combination fluoropyrimidine-oxaliplatin chemotherapy response in CRC patients. iTRAQ labelled plasma was used for discovery experiments, then promising candidates were verified by SRM. One protein identified in these experiments, fibronectin, showed promise as a predictive marker. Chapter 5 followed up on preliminary data from my Honours degree research that suggested a specific tryptic peptide from vitronectin may predict chemotherapy response. Vitronectin, a highly adhesive extracellular matrix glycoprotein is known to promote cancer metastasis through interaction with integrin binding partners, similar to the actions of fibronectin within cancer development. Here, I developed new SRM assays to examine additional vitronectin peptides and acquired new data using a more advanced mass spectrometer. Immunoblotting was also conducted to support the results. Chapter 6 outlined SRM experiments utilising synthesised isotopically-labelled peptide standards to quantitate vitronectin and fibronectin peptides, in a larger CRC patient cohort. Vitronectin was confirmed to be reduced in plasma levels taken at baseline in unresponsive chemotherapy patients, compared with patients that responded to treatment. In summary, this thesis described how proteomic methodologies were used to identify and verify new candidate plasma protein biomarkers that predict for response to first-line chemotherapy in advanced CRC patients. The favourable findings for vitronectin in this cohort warrant more extensive investigation in larger patient cohorts.