Measurement of uPAR and αvꞵ6 for early stage colorectal cancer diagnosis
thesisposted on 28.03.2022, 17:54 by Sachini Fonseka
Colorectal cancer (CRC), characterised by tumours of the colon, rectum and appendix, is the 3rd leading cause of cancer-related death worldwide. The survival rate is dependent on the time of diagnosis, with early-stage detection leading to curative surgical resection. Despite this, a lack of accurate, sensitive and specific tests means that only ~30% of cases are diagnosed early enough to be cured (i.e., American Joint Committee on Cancer Stages I & II). To address this unmet clinical need, this thesis addressed developing a mass spectrometry (MS)-based assay for the detection and quantification of early-stage blood-based biomarkers of CRC. Here, we have developed targeted assays for the potential cancer biomarkers urokinase plasminogen activator receptor (μPAR) and integrin αvꞵ6. A growing portfolio of evidence implicates both proteins as important regulators of the epithelial-mesenchymal transition (EMT), a mechanism known to be critical in cancer proliferation, progression, invasion and eventual metastasis. The biggest challenge in detection and quantification of these biomarkers in plasma is their relatively low abundance (low ng/mL) compared to more highly abundant homeostatic proteins (high mg/mL). To circumvent this challenge, we developed a two-step assay that captures the proteins by affinity-based enrichment prior to quantitation by targeted multiple reaction monitoring (MRM) MS. Initial studies were aimed at determining the specificities of a range of monoclonal antibodies for their intended target. Subsequent studies using a surrogate parallel reaction monitoring (PRM)-MS established optimal peptide transitions, as well as each assay’s limits of detection. Collectively, the amalgamation of these two components (with the use of labelled peptide standards) will result in highly sensitive, specific and reproducible assays to detect and quantify μPAR and αvꞵ6 in control and diseased patient plasma. Early stage diagnosis will shift the percentage of CRCs detected towards the localized tumour stages (I and II), with consequential increased patient survival.