Potential early clinical stage colorectal cancer diagnosis using a proteomics blood test panel

Abstract

Background: One of the most significant challenges in colorectal cancer (CRC) management is the use of compliant early stage population-based diagnostic tests as adjuncts to confirmatory colonoscopy. Despite the near curative nature of early clinical stage surgical resection, mortality remains unacceptably high-as the majority of patients diagnosed by faecal haemoglobin followed by colonoscopy occur at latter stages. Additionally, current populationbased screens reliant on fecal occult blood test (FOBT) have low compliance (~ 40%) and tests suffer low sensitivities. Therefore, blood-based diagnostic tests offer survival benefits from their higher compliance (≥ 97%), if they can at least match the sensitivity and specificity of FOBTs. However, discovery of low abundance plasma biomarkers is difficult due to occupancy of a high percentage of proteomic discovery space by many high abundance plasma proteins (e.g., human serum albumin). Methods: A combination of high abundance protein ultradepletion (e.g., MARS-14 and an in-house IgY depletion columns) strategies, extensive peptide fractionation methods (SCX, SAX, High pH and SEC) and SWATH-MS were utilized to uncover protein biomarkers from a cohort of 100 plasma samples (i.e., pools of 20 healthy and 20 stages I-IV CRC plasmas). The differentially expressed proteins were analyzed using ANOVA and pairwise t-tests (p < 0.05; fold-change > 1.5), and further examined with a neural network classification method using in silico augmented 5000 patient datasets. Results: Ultradepletion combined with peptide fractionation allowed for the identification of a total of 513 plasma proteins, 8 of which had not been previously reported in human plasma (based on PeptideAtlas database). SWATH-MS analysis revealed 37 protein biomarker candidates that exhibited differential expression across CRC stages compared to healthy controls. Of those, 7 candidates (CST3, GPX3, CFD, MRC1, COMP, PON1 and ADAMDEC1) were validated using Western blotting and/or ELISA. The neural network classification narrowed down candidate biomarkers to 5 proteins (SAA2, APCS, APOA4, F2 and AMBP) that had maintained accuracy which could discern early (I/II) from late (III/IV) stage CRC. Conclusion: MS-based proteomics in combination with ultradepletion strategies have an immense potential of identifying diagnostic protein biosignature.

Systemic Proteome Alterations Linked to Early Stage Pancreatic Cancer in Diabetic Patients

“…APOA4 is a major component of high-density lipoproteins and chylomicrons. Previous studies reported the association of aberrant APOA4 expression with colorectal cancer development in diabetic patients [25] and a reduced plasma abundance in colorectal cancer patients [26]. In our current study, the plasma level of APOA4 was significantly reduced in the PDAC-DM cases compared to the controls (CP-DM and DM).…”

Recent advances in mass spectrometry based clinical proteomics: applications to cancer research

“…The results showed 50 proteins to be abundant in the vein draining part of the cancerous regions compared to the noncancerous sectors [158]. In another study done by Ahn et al [159] one hundred plasma samples from different stages of CRC and healthy controls were pooled to create a SWATH library. Similarly, the abundant proteins were depleted and peptides were further fractionated.…”

The separation sciences, the front end to proteomics: An historical perspective

“…Another unmet clinical need is a blood‐based test for the early detraction and staging of colorectal cancer (CRC). Ahn et al (2019) used a multidimensional purification strategy comprising high abundance protein ultradepletion (MARS‐14 and an in‐house IgY depletion column [64]), strong cation exchange (SCX), strong anion exchange (SAX), high pH, and size exclusion chromatography (SEC) followed by SWATH‐MS to reveal protein biomarkers from a cohort of 100 plasma samples (20 healthy and 20 stage I–IV CRC pooled plasmas). A total of 37 potential protein biomarkers were identified that exhibited differential expression across CRC stages compared with healthy controls.…”