New Diagnostic Biomarkers in Colorectal Cancer

Colonoscopies are currently the best method to diagnose colorectal cancer (CRC). A less invasive method, the fecal occult blood test (FOBT), can detect CRC in average-risk individuals; however, it is only 50% effective. With prognosis tied to early diagnostics and a rise in CRC worldwide, researchers such as Peltier et al. seek new diagnostic biomarkers in blood.¹  

Blood samples for this investigation came from biobanked samples collected prior to colonoscopy. Patients represented three categories: normal colonoscopy (N), polyps at colonoscopy (A) or cancer at colonoscopy (C). From these samples, the authors used an isobaric tags for relative and absolute quantitation–based (iTRAQ-based) strategy to develop a list of early CRC diagnostic biomarkers. The team used two analysis methods:

1. Reversed-phase liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF/TOF).
2. Nano electrospray ionization high energy collision dissociation–enabled mass spectrometry using an LTQ Orbitrap Velos mass spectrometer connected to an UltiMate 3000 rapid separation liquid chromatography column (both Thermo Scientific).

They identified 135 proteins using MALDI TOF/TOF and 348 proteins during the LTQ Orbitrap mass spectrometer analysis. Comparing both methods, they found the Orbitrap mass spectrometer analysis identified and quantified 60% more proteins. Meanwhile, the two methods had 30% of proteins in common. During the MALDI experiments, researchers identified 7% unique proteins, with the number of unique proteins from LTQ Orbitrap instrument totaling 63%.

Of the total proteins, researchers identified 89 distinct proteins as modulated between normal, colorectal adenoma and carcinoma patients. Analyzing expression-fold changes, they found 36 modified proteins in patients with polyps (18 upregulated and 18 downregulated) and 75 proteins modified in invasive-cancer patients (26 upregulated and 49 downregulated), compared to controls.

Next, they used non-supervised clustering to compare ratios of altered proteins. In particular, they identified proteins involved in enzyme regulator activities, specifically the serpin family (SERPINA1, SERPINA3 and SERPINC1). Correlating iTRAQ data with ELISA assays, they saw increased levels of SERPINA1 (2.0 times higher) and SERPINA3 (1.3 times higher), and decreased levels in SERPINC1 (2.0 times lower) in the CRC patients. They also saw a correlation in increasing expression in SERPINA1 with increasing cancer severity. The remaining biomarker candidates form a reserve for further evaluation of additional biomarkers for CRC diagnosis.

The authors maintain that larger studies are needed to further validate the members of the serpin family as biomarkers. Nevertheless, they are enthusiastic about the possibilities of testing that can noninvasively diagnose CRC and also provide insights into disease progression.

Reference
1. Peltier, J., et al. (2016) “Quantitative proteomic analysis exploring progression of colorectal cancer: Modulation of the serpin family,” Journal of Proteomics, 148 (pp. 139–148), doi: 10.1016/j.jprot.2016.07.031.