Colorectal cancer biomarkers: to be or not to be? Cautionary tales from a road well travelled

Abstract

Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide and places a major economic burden on the global health care system. The time frame for development from premalignant to malignant disease typically spans 10-15 years, and this latent period provides an ideal opportunity for early detection and intervention to improve patient outcomes. Currently, early diagnosis of CRC is hampered by a lack of suitable non-invasive biomarkers that are clinically or economically acceptable for population-based screening. New blood-based protein biomarkers for early detection of CRC are therefore urgently required. The success of clinical biomarker discovery and validation studies is critically dependent on understanding and adjusting for potential experimental, analytical, and biological factors that can interfere with the robust interpretation of results. In this review we outline some important considerations for research groups undertaking biomarker research with exemplars from our studies. Implementation of experimental strategies to minimise the potential effects of these problems will facilitate the identification of panels of biomarkers with the sensitivity and specificity required for the development of successful tests for the early detection and surveillance of CRC.

Keywords: Bias; Biomarker; Colorectal cancer; Diagnostic; Discovery; Validation.

Figure 1

Stability of insulin-like growth factor binding protein 2 and matrix metalloproteinase 9. A: Standard curves for insulin-like growth factor binding protein 2 (IGFBP2) and matrix metalloproteinase 9 (MMP9) over an 18 mo period indicates that the assays were stable over time; B: IGFBP2 and MMP9 stability after storage for 18 mo in liquid nitrogen and at -80 °C (n = 10 patients); C: Stability of IGFBP2 and MMP9 following three freeze/thaw cycles. All data are represented as average ± standard deviation. F/T: Freeze/thaw cycles. ^aP < 0.05 when compared to samples measured immediately.

Figure 2

Comparison of insulin-like growth factor binding protein 2 measurements after collection into serum separator tubes, plasma/EDTA tubes and plasma/citrate tubes. Data are represented as average ± SE of the mean for triplicate measurements. ^aP < 0.05; ^bP < 0.01.

Figure 3

Insulin-like growth factor binding protein 2 measured in different control cohorts and compared to the colorectal cancer patient group. A: Insulin-like growth factor binding protein 2 (IGFBP2) levels in the sera of patients from two different control cohorts and in a colorectal cancer cohort; B: IGFBP2 levels in the sera of control patients recruited from pre-admission clinics (n = 27) and the Red Cross Blood Donation Centre (n = 25). CRC: Colorectal cancer. ^aP < 0.05, ^bP < 0.01 between the median values.

Figure 4

Insulin-like growth factor binding protein 2 measured in patient sera using enzyme-linked immunosorbent assay kits sourced from two different manufacturers. A: Standard curves for insulin-like growth factor binding protein 2 (IGFBP2) enzyme-linked immunosorbent assays sourced from DSL Inc (Texas, United States) and Mediagnost (Kiel, Germany); B: Comparison of IGFBP2 levels in three colorectal cancer patients (P1-P3), five control patients (N1-N5) and a quality control sample consisting of 10 pooled samples; C: Correlation of measured values of IGFBP2 between the two different manufacturers. The correlation coefficient was 0.62 (Spearman correlation, P > 0.05). Data are represented as average ± standard deviation of three replicate measurements. ^aP < 0.05.

Figure 5

Comparison of calibration curves between commercially available enzyme-linked immunosorbent assay kits and reagents developed in-house for two protein biomarkers. Data are represented as average ± SE of the mean for two replicate measurements.