Early stage diagnosis of colorectal cancer using a multi-variate blood-based test

Over the past nine years, the Human Proteome Project (HPP) has made enormous progress in achieving two goals. These are: mapping the human protein repertoire under the Chromosome - centric human proteome project (C - HPP) and, understanding the pathophysiology of human biology and disease under the Biology/Disease - driven human proteome project (B/D - HPP) (Figure 1). This thesis contributes towards these two HPP goals by; ii) cataloguing human proteins, particularly the "missing proteins' (Chapter 1) and ii) diagnosing early stage colorectal cancer (CRC) through the development of a multi - variate blood - based assay (Chapters 2 - 5; with results contained across Chapters 3, 4 and 5). Chapter 1 of this thesis summarises contributions in developing an investigative approach and community - centric resource (MissingProteinPedia) to accelerate the discovery and understanding of 'missing proteins' (Publication I). Out of ~ 20,000 human protein coding genes, 2,319 proteins (at time of writing) still lack mass spectrometry - based evidence for reliable high - stringency protein existence data. These are referred to as the PE2 - 4 proteins or colloquially as the 'missing proteins' . This chapter explores the common characteristics of missing proteins, including assignment to groups of proteins, topological distribution and structural composition, and it details challenges associate with identifying these proteins. This is exemplified by investigation of an elusive class/family of GPCR proteins, known as the olfactory receptors (ORs) . The chapter provides potential approaches to facilitate identification of missing proteins in order to accelerate successful and timely completion of the HPP. Chapter 2 introduces CRC by reviewing incidence, stages, mortality rate, as well as the factors governing susceptibility and pathophysiology, staging and survival. The chapter describes existing screening tests, and CRC biomarkers in context unmet clinical needs. One major requirement is to develop a minimally invasive, blood - based markers for routine CRC screening is explored in this chapter with particular reference to technological and strategic advances across the field of proteomics. The complexities and challenges associated with plasma proteomic biomarker discovery are also discussed. Chapter 3 employs state - of - the - art proteomic techniques to provides an in - depth analysis of the CRC plasma proteome in order to develop such blood - based diagnostic test. A combination of XII patented, in - house ultradepletion methods, commercial immuno depletion approaches and multiple peptide fractionations was used to overcome the challenges of detecting low - abundance proteins in plasma. Furthermore, SWATH™ - MS was employed for specific and reliable exploration of protein biomarkers using 100 clinically staged EDTA - plasma samples (n=20 per AJCC stage for CRC (i.e . stages I, II, III and IV)). A total of 37 potential protein candidates were identified by comparing differential expression in CRC stages (I - IV) against the plasma pool of healthy controls (n =20) with stringent statistical analysis (fold change ≥ 1.5, unique peptide ≥ 1, p < 0.05). A literature search indicated some of these putative CRC biomarkers were novel while others had been previously associated with CRC. The capability of 7 of these 37 candidates to distinguish CRC early stages (I/II) from healthy controls were confirmed using Western blotting and/or ELISA - based method. In addition, a machine learning model was then utilised to confirm the potential of 5 protein candidates (from list of 37 candidates) as a putative panel to distinguish early stage (I/II) CRC from healthy controls using a 5000 synthetic patient cohort. Chapter 4 then develops and verifies a first - pass parallel reaction monitoring (PRM) assays to interrogate plasma samples for two target peptides for Complementary factor D and ADAMDEC1, both were identified as potential candidates from Chapter 3. Despite encouraging initial data, this work remained preliminary and requires further characterisation and validation studies, which are described in detail. Penultimately, Chapter 5 develops a proof - of - concept PRM assay to quantify the expression of two markers of the epithelial - to - mesenchymal transition (uPAR and αvβ6) in HCT116 (colon carcinoma) cell line and CRC plasma sample s. Both (uPAR and αvβ6) are of low abundance in plasma and have been extensively studied by our group. Increased levels of uPAR has been established as a stage II prognostic marker in CRC tissues, and equally, αvβ6 expression has shown to be elevated in ea rly stage of CRC. The objective of this chapter was to explore the increased expressions of circulating uPAR and αvβ6 as potential biomarkers in development of CRC in using plasma samples. The proof - of - concept PRM assay developed, showed a 33% decrease in uPAR expression between antisense (HCT116 AS ) and wildtype (HCT116 WT ). This study is ongoing and requires optimisation to verify and quantitate both uPAR and αvβ6 peptide from CRC plasma samples. Finally, Chapter 6 summarises the thesis in the context of the current literature providing a discussion of possible future directions and the clinical significance and limitations of the studies outlined. I describe the importance of population - based studies of the selected CRC protein biomarkers and the need for direct comparisons to other existing screening methodologies.