Ilka Priebe

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 (KD value of 49 and 10 pmol/L, respectively) compared with IGF-I (∼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 Å. 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. Mol Cancer Ther; 9(6); 1809–19. ©2010 AACR.

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.

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 cells 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.

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.

Delineation of the IGF-II C domain elements involved in binding and activation of the IR-A, IR-B and IGF-IR

OBJECTIVE Human insulin-like growth factor-I and -II (IGF-I and -II) ligands share a high degree of sequence and structural homology. Despite their similarities, IGF-I and IGF-II exhibit differential receptor binding and activation characteristics. The C domains of IGF-I and IGF-II are the primary determinants of binding specificity to the insulin-like growth factor I receptor (IGF-IR), insulin receptor exon 11- (IR-A) and exon 11+ (IR-B) isoforms. DESIGN Three IGF-II analogues were generated in order to delineate the C domain residues that confer the differential receptor binding affinity and activation properties of the IGFs. Chimeric IGF-II analogues IGF-IICI(N) and IGF-IICI(C) contained partial IGF-I C domain substitutions (IGF-I residues underlined) GYGSSSRRSR and SRVSRRAPQT, respectively. RESULTS The IGF-IICI(N) analogue bound the IR-A and IGF-IR with high affinity but bound the IR-B with a relatively lower affinity than IGF-II, suggesting a negative interaction between the exon-11 encoded peptide in the IR-B and the C-domain. The ability of IGF-IICI(N) to activate receptors and elicit cell viability responses was generally proportional to its relative receptor binding affinity but appeared to act as a partial agonist equivalent to IGF-I when binding and activating the IGF-IR. In contrast, IGF-IICI(C) bound IGF-IR with high affinity but elicited lower receptor activation and cell viability responses. Analogue IGF-IICI(S) contained a truncated IGF-I C domain (GSSSRRAT) and generally displayed a relatively poor ability to bind, activate and elicit viability responses via each receptor. CONCLUSIONS Together, the IGF analogues demonstrate that both flanks of the IGF-II C domain play important roles in the greater ability of IGF-II to bind and activate IR receptors than IGF-I.

Analysis of 32 Blood-Based Protein Biomarkers for their Potential to Diagnose Colorectal Cancer

Colorectal cancer (CRC) is largely viewed as a preventable disease but the prevalence is increasing worldwide. Although many faecal and blood-based biomarkers have been proposed as potential diagnostic markers, none have been successful in large cohort studies. In this study, ELISA was used to evaluate 32 candidate protein biomarkers in a single cohort of CRC patients (n=95) and age/sex matched controls (n=50). Of these, 12 markers differed statistically between cases and controls. Receiver operating characteristic analysis identified IL8, Mac2BP, TIMP1, and OPN as the best performing markers for overall CRC diagnosis. However, further analysis determined that IL6, TGFB1, TIMP2 and IGF2 were most accurate at identifying early stage disease. We also assessed the correlation between markers and determined that the strongest correlations existed between VEGFA and TGFB1 (r=0.65, p<0.0001), M30 and M65 (r=0.59, p<0.001), and between TGFB1 and TIMP1 (r=0.55, p<0.0001). This analysis provides a consistent baseline for identifying a potential panel of diagnostic protein biomarkers in blood. Our results highlight protein biomarker combinations that reflect the disease process and which may provide the sensitivity and specificity required a reliable diagnosis of CRC.

Abstract 4920: Evaluation of a diagnostic blood test for colorectal cancer screening and as a triage tool to stratify patients for colonoscopy

Worldwide, colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer death, with an annual incidence in 2012 of 1.4 million CRC cases and an annual mortality around 700,000 [1]. Fortunately the majority of colorectal cancer (CRC) cases are preventable by early detection and removal by colonoscopy or surgery. Currently in Australia, the only widely used non-invasive screening test for CRC is the immunological faecal occult blood test (iFOBT), which has been shown to reduce CRC incidence and mortality by 15-25% [2]. There are however significant issues that limit its effectiveness as a diagnostic screening tool, such as a poor positive predictive value of 5.3% for suspected cancer and low participation rate of approximately 33.5% in 2013, as seen in the Australian National Bowel Cancer Screening Program (NBCSP)[2]. Evidence indicates a robust, blood-based, diagnostic assay would increase screening participation and compliance. In our systematic analysis of 68 individual biomarkers using logistic regression strategies, we have identified a unique 3 protein biomarker panel blood test consisting of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2)[3]. This panel not only differentiates between CRC and normal patient sera with 95% specificity and 73% sensitivity in a single measurement (c.f. FOBT: 35-50 sensitivity [4]) but early stage disease. Currently, we are prospectively collecting a new cohort of over 1,000 blood samples from volunteers that have undergone colonoscopy, that include CRC patients, controls and other pathologies (IBD, other cancers) to specifically determine the performance of this blood test in asymptomatic and surveillance screening populations. We will also evaluate directly the accuracy and performance of our biomarker panel, as a diagnostic test suitable for CRC screening versus the iFOBT used in the NBCSP, as an intermediary test in the triage of individuals with positive iFOBT result for need and urgency to have a colonoscopy. 1. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray, F. GLOBOCAN 2012 v1.1, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 2. National Bowel Cancer Screening Program: monitoring report 2012-2013. AIHW & Australian Government Department of Health and Ageing, 2014. 3. Fung KYC et al 2015. Blood-based protein biomarker panel for the detection of colorectal cancer. PloS one. 2015;10(3):e0120425. 4. Morikawa, T et al., A comparison of the immunochemical fecal occult blood test and total colonoscopy in the asymptomatic population. Gastroenterology, 2005. 129(2): p. 422-8. Citation Format: Leah J. Cosgrove, Kathy Surinya, Kim YC Fung, Ilka Priebe, Mike Buckley, Tim Adams, Bruce Tabor, Leanne Purins, Trevor Lockett, Hugo Leroux, Peter Gibbs, Jiangqin Wei, Ed Nice, Tony Burgess, Michelle Thomas, James Moore, Raj Singh, Andrew Ruszkiewicz. Evaluation of a diagnostic blood test for colorectal cancer screening and as a triage tool to stratify patients for colonoscopy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4920.

Circulating Levels of the Wnt Antagonist Dkk-3 as a Diagnostic Marker for Colorectal Cancer

The Wnt antagonist Dickkopf-3 (Dkk-3) has been implicated in several stages of tumour development in a wide range of human cancers, including colorectal cancer (CRC). However, the usefulness of serum Dkk-3 levels as a diagnostic biomarker for CRC has yet to be determined. In this study we used an ELISA immunoassay to examine serum Dkk-3 protein levels in a retrospective cohort of CRC patients (n = 89) and age, gender matched controls (n = 46). The median concentration of Dkk-3 was significantly (p = 0.0003) lower in CRC patient serum samples (29.3 ng/ml, range 10.4 – 67.8 ng/ml) when compared to control serum samples (36.8 ng/ml, range 20.7 – 67.4 ng/ml). Receiver operating characteristic analysis demonstrated at 90% specificity, serum Dkk-3 levels distinguished CRC patients with 36% sensitivity (AUC = 0.69, 95% CI 0.60 – 0.78).