Blood-based protein biomarker panel for the detection of colorectal cancer.

BACKGROUND: The majority of colorectal cancer (CRC) cases are preventable by early detection and removal of precancerous polyps. Even though CRC is the second most common internal cancer in Australia, only 30 per cent of the population considered to have risk factors participate in stool-based test screening programs. Evidence indicates a robust, blood-based, diagnostic assay would increase screening compliance. A number of potential diagnostic blood-based protein biomarkers for CRC have been reported, but all lack sensitivity or specificity for use as a stand-alone diagnostic. The aim of this study was to identify and validate a panel of protein-based biomarkers in independent cohorts that could be translated to a reliable, non-invasive blood-based screening test. PRINCIPAL FINDINGS: In two independent cohorts (n = 145 and n = 197), we evaluated seven single biomarkers in serum of CRC patients and age/gender matched controls that showed a significant difference between controls and CRC, but individually lack the sensitivity for diagnostic application. Using logistic regression strategies, we identified a panel of three biomarkers that discriminated between controls and CRC with 73% sensitivity at 95% specificity, when applied to either of the two cohorts. This panel comprised of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2). CONCLUSIONS: Due to the heterogeneous nature of CRC, a single biomarker is unlikely to have sufficient sensitivity or specificity for use as a stand-alone diagnostic screening test and a panel of markers may be more effective. We have identified a 3 biomarker panel that has higher sensitivity and specificity for early stage (Stage I and -II) disease than the faecal occult blood test, raising the possibility for its use as a non-invasive blood diagnostic or screening test.

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

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.

Performance of serum lipocalin 2 as a diagnostic marker for colorectal cancer.

BACKGROUND: Lipocalin 2 has been implicated in colorectal tumorigenesis but its usefulness as a diagnostic marker for the disease has previously never been determined. METHODS: We have used ELISA immunoassay to measure the level of serum lipocalin 2 in a cohort consisting of colorectal cancer patients (n=196) and age/gender matched controls (n=99). RESULTS: The median concentration of lipocalin 2 was found to be significantly higher (p< 0.0001) in the patient group (105.9 ng/mL, range 10.8-444.7 ng/mL) when compared to the control subjects (86.4 ng/mL, range 17.1-190.0 ng/mL). Additionally, no significant difference was observed between disease stage (Dukes' or T stage) in the patient cohort. Receiver operating characteristic analysis was performed to determine its performance as a diagnostic marker. The area under the curve was found to be 0.641 (95% confidence interval 0.576-0.706). Furthermore, the sensitivity of lipocalin 2 was found to be 24% at 90% specificity. CONCLUSIONS: Our study indicates that lipocalin 2 is not a suitable serum biomarker for the diagnosis of CRC.

A human monoclonal antibody against insulin-like growth factor-II blocks the growth of human hepatocellular carcinoma cell lines in vitro and in vivo.

Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR-directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1-104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (K(D) value of 49 and 10 pmol/L, respectively) compared with IGF-I (approximately 10 nmol/L) and blocked binding of IGF-II to IGF-IR, IR-A, a panel of insulin-like growth factor-binding proteins, and the mannose-6-phosphate receptor. A crystal complex of the parental Fab of DX-2647 bound to IGF-II was resolved to 2.2 A. DX-2647 inhibited IGF-II and, to a lesser extent, IGF-I-induced receptor tyrosine phosphorylation, cellular proliferation, and both anchorage-dependent and anchorage-independent colony formation in various cell lines. In addition, DX-2647 slowed tumor progression in the Hep3B xenograft model, causing decreased tumoral CD31 staining as well as reduced IGF-IIE and IGF-IR phosphorylation levels. Therefore, DX-2647 offers an alternative approach to targeting IGF-IR, blocking IGF-II signaling through both IGF-IR and IR-A.

Proteomic analysis of butyrate effects and loss of butyrate sensitivity in HT29 colorectal cancer cells.

Butyrate, a fermentation product of the large bowel microflora, is potentially protective against the development of colorectal cancer. In vitro, butyrate has been shown to induce apoptosis and inhibit proliferation in numerous cancer cell lines, including colorectal cancer. Although these tumor suppressing properties of butyrate are well-documented in experimental systems, the mechanisms underlying the induction of these effects are not fully understood. Understanding these mechanisms in cancer cells, as well as the pathways involved in a cell's ability to overcome them and progress toward malignancy, is vital to determine therapeutic approaches for disease management. We have developed a colorectal cancer cell line (HT29-BR) that is less responsive to the apoptotic effects of butyrate through sustained exposure of HT29 cells to 5 mM butyrate and have used proteomics to investigate the mechanisms involved in the development of butyrate insensitivity. Proteomic analysis identified a number of cellular processes in HT29 and HT29-BR cells influenced by butyrate including remodeling of the actin cytoskeleton, inhibition of protein biosynthesis and dysregulation of the cell stress response. We describe novel roles for butyrate in the induction of its tumor suppressing effects and outline potential cellular pathways involved in the development of butyrate insensitivity in the HT29-BR cell population.

Assessing the potential usefulness of IGF-related peptides and adiponectin for predicting disease risk.

OBJECTIVE: Insulin-like growth factors (IGF), their binding proteins and adiponectin have been investigated as potential blood-based biomarkers for a variety of diseases. Before these circulating proteins can be considered as biomarkers, their variation within and between individuals and between published studies must be critically assessed. The purpose of this study was to use the D-value to predict the potential usefulness of IGF-related peptides and adiponectin as biomarkers for the diagnosis of colorectal cancer (CRC). DESIGN: Intra- and inter-individual variation of total IGF-I and -II, IGF binding protein 1 (IGFBP-1), -2 and -3 and adiponectin, was examined in 10 healthy subjects over a 5 week period. This data was analysed in conjunction with previous publications to provide a D-value, which is a theoretical value that identifies the usefulness of the analyte individually and as a panel, as a biomarker for CRC. RESULTS: A single measurement of total IGF-I and -II, and adiponectin provided a reproducible representation of their circulating concentrations. The D-value for total IGF-II and IGFBP-3 were 0.5 and 0.47, respectively, which corresponded to area under the curve (AUC) values of 64 and 63%. Combining these analytes into a panel only slightly improved the D-value to 0.63 (AUC was 67%). CONCLUSIONS: Although serum levels of total IGF-I, total IGF-II and IGFBP-3 are stable and reproducible, the D-value calculations indicate that they have limited importance when used as biomarkers of CRC.