Bruce Tabor

Prognostic and diagnostic significance of DNA methylation patterns in high grade serous ovarian cancer

OBJECTIVE Altered DNA methylation patterns hold promise as cancer biomarkers. In this study we selected a panel of genes which are commonly methylated in a variety of cancers to evaluate their potential application as biomarkers for prognosis and diagnosis in high grade serous ovarian carcinoma (HGSOC); the most common and lethal subtype of ovarian cancer. METHODS The methylation patterns of 10 genes (BRCA1, EN1, DLEC1, HOXA9, RASSF1A, GATA4, GATA5, HSULF1, CDH1, SFN) were examined and compared in a cohort of 80 primary HGSOC and 12 benign ovarian surface epithelium (OSE) samples using methylation-specific headloop suppression PCR. RESULTS The genes were variably methylated in primary HGSOC, with HOXA9 methylation observed in 95% of cases. Most genes were rarely methylated in benign OSE, with the exception of SFN which was methylated in all HGSOC and benign OSE samples examined. Methylation of DLEC1 was associated with disease recurrence, independent of tumor stage and suboptimal surgical debulking (HR 3.5 (95% CI:1.10-11.07), p=0.033). A combination of the methylation status of HOXA9 and EN1 could discriminate HGSOC from benign OSE with a sensitivity of 98.8% and a specificity of 91.7%, which increased to 100% sensitivity with no loss of specificity when pre-operative CA125 levels were also incorporated. CONCLUSIONS This study provides further evidence to support the feasibility of detecting altered DNA methylation patterns as a potential diagnostic and prognostic approach for HGSOC.

Unexpected features of the dark proteome

Significance A key remaining frontier in our understanding of biological systems is the “dark proteome”—that is, the regions of proteins where molecular conformation is completely unknown. We systematically surveyed these regions, finding that nearly half of the proteome in eukaryotes is dark and that, surprisingly, most of the darkness cannot be accounted for. We also found that the dark proteome has unexpected features, including an association with secretory tissues, disulfide bonding, low evolutionary conservation, and very few known interactions with other proteins. This work will help future research shed light on the remaining dark proteome, thus revealing molecular processes of life that are currently unknown. We surveyed the “dark” proteome–that is, regions of proteins never observed by experimental structure determination and inaccessible to homology modeling. For 546,000 Swiss-Prot proteins, we found that 44–54% of the proteome in eukaryotes and viruses was dark, compared with only ∼14% in archaea and bacteria. Surprisingly, most of the dark proteome could not be accounted for by conventional explanations, such as intrinsic disorder or transmembrane regions. Nearly half of the dark proteome comprised dark proteins, in which the entire sequence lacked similarity to any known structure. Dark proteins fulfill a wide variety of functions, but a subset showed distinct and largely unexpected features, such as association with secretion, specific tissues, the endoplasmic reticulum, disulfide bonding, and proteolytic cleavage. Dark proteins also had short sequence length, low evolutionary reuse, and few known interactions with other proteins. These results suggest new research directions in structural and computational biology.

Epigenetic Deregulation Across Chromosome 2q14.2 Differentiates Normal from Prostate Cancer and Provides a Regional Panel of Novel DNA Methylation Cancer Biomarkers

Background: Previously, we showed that gene suppression commonly occurs across chromosome 2q14.2 in colorectal cancer, through a process of long-range epigenetic silencing (LRES), involving a combination of DNA methylation and repressive histone modifications. We now investigate whether LRES also occurs in prostate cancer across this 4-Mb region and whether differential DNA methylation of 2q14.2 genes could provide a regional panel of prostate cancer biomarkers. Methods: We used highly sensitive DNA methylation headloop PCR assays that can detect 10 to 25 pg of methylated DNA with a specificity of at least 1:1,000, and chromatin immunoprecipitation assays to investigate regional epigenetic remodeling across 2q14.2 in prostate cancer, in a cohort of 195 primary prostate tumors and 90 matched normal controls. Results: Prostate cancer cells exhibit concordant deacetylation and methylation of histone H3 Lysine 9 (H3K9Ac and H3K9me2, respectively), and localized DNA hypermethylation of EN1, SCTR, and INHBB and corresponding loss of H3K27me3. EN1 and SCTR were frequently methylated (65% and 53%, respectively), whereas INHBB was less frequently methylated. Conclusions: Consistent with LRES in colorectal cancer, we found regional epigenetic remodeling across 2q14.2 in prostate cancer. Concordant methylation of EN1 and SCTR was able to differentiate cancer from normal (P < 0.0001) and improved the diagnostic specificity of GSTP1 methylation for prostate cancer detection by 26%. Impact: For the first time we show that DNA methylation of EN1 and SCTR promoters provide potential novel biomarkers for prostate cancer detection and in combination with GSTP1 methylation can add increased specificity and sensitivity to improve diagnostic potential. Cancer Epidemiol Biomarkers Prev; 20(1); 148–59. ©2011 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.

The Molecular Control Toolkit: Controlling 3D molecular graphics via gesture and voice

Three-dimensional (3D) molecular graphic systems are widely used in the life sciences, both for research and communication. These systems need to enable a rich set of 3D operations, including three-axis rotation and translation, selection of parts of macromolecules, and the ability to redefine the center of rotation. As a result, graphical interfaces for these systems typically require users to learn complex keyboard and mouse combinations. This can be a significant barrier for new or occasional users, and even for experts, precise control of 3D molecular structures can be challenging. To help address these challenges, we developed the Molecular Control Toolkit to support multiple consumer gesture and voice recognition devices, and provide an API that allows adaption to multiple molecular graphics systems. The toolkit allows intuitive control, almost as if users are directly manipulating 3D objects in their hands. We applied the toolkit to the Kinect and Leap Motion devices, and to the Aquaria molecular graphics system. We did a pilot user study with 18 life scientists to test the resulting system in different scenarios. Overall, users gave quite favorable ratings to using the Kinect and Leap Motion gesture devices to control molecular graphics, even though these devices initially proved less efficient for common 3D control tasks, compared to the more familiar mouse/keyboard. To our knowledge, this is the first toolkit for macromolecular graphics that supports multiple devices with a set of controls sufficiently rich to be useful in the day-to-day work of a broad range of life scientists. The Molecular Control Toolkit and Aquaria can be accessed at http://aquaria.ws.

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.

An association between the PTGS2 rs5275 polymorphism and colorectal cancer risk in families with inherited non-syndromic predisposition

Recently our group completed a genome-wide linkage study investigating Australian and Spanish families with inherited risk of colorectal cancer (CRC). A minor linkage peak from that study located on chromosome 1 correlates with the location of a known CRC risk-modifying gene, prostaglandin synthase (PTGS2). PTGS2 encodes the inducible prostaglandin synthase enzyme cyclooxygenase-2 (COX-2). Prostaglandins are implicated in the initiation of carcinogenesis and progression of tumours. Sequencing of PTGS2 in a small subset of affected individuals identified a high frequency of the minor C allele of single nucleotide polymorphism rs5275. We then genotyped the rs5275 polymorphism in 183 affected and 223 unaffected individuals from our CRC predisposed families. Tests for association in the presence of linkage were made using family-based association tests. The C allele was found to be significantly associated (P<0.01) with diagnosis of hereditary non-syndromic CRC (P=0.0094, dominant model) and an earlier age of diagnosis (P=0.0089, heterozygous-advantage model). Interestingly, by stratifying the age of diagnosis data, we observed a speculative gender-discordant effect. Relative to other groups, female CC carriers were diagnosed less when young, but by 60 years of age were the most at risk group. Conversely, CT carriers of both genders showed a consistently earlier diagnosis relative to TT carriers. Our results suggest potential differential age-and gender-dependent efficacies of chemopreventative COX-2 inhibitors in the context of non-syndromic colorectal cancer.

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.

Little evidence for association between the TGFBR1*6A variant and colorectal cancer: a family- based association study on non-syndromic family members from Australia and Spain

BackgroundGenome-wide linkage studies have identified the 9q22 chromosomal region as linked with colorectal cancer (CRC) predisposition. A candidate gene in this region is transforming growth factor β receptor 1 (TGFBR1). Investigation of TGFBR1 has focused on the common genetic variant rs11466445, a short exonic deletion of nine base pairs which results in truncation of a stretch of nine alanine residues to six alanine residues in the gene product. While the six alanine (*6A) allele has been reported to be associated with increased risk of CRC in some population based study groups this association remains the subject of robust debate. To date, reports have been limited to population-based case–control association studies, or case–control studies of CRC families selecting one affected individual per family. No study has yet taken advantage of all the genetic information provided by multiplex CRC families.MethodsWe have tested for an association between rs11466445 and risk of CRC using several family-based statistical tests in a new study group comprising members of non-syndromic high risk CRC families sourced from three familial cancer centres, two in Australia and one in Spain.ResultsWe report a finding of a nominally significant result using the pedigree-based association test approach (PBAT; p = 0.028), while other family-based tests were non-significant, but with a p-value <; 0.10 in each instance. These other tests included the Generalised Disequilibrium Test (GDT; p = 0.085), parent of origin GDT Generalised Disequilibrium Test (GDT-PO; p = 0.081) and empirical Family-Based Association Test (FBAT; p = 0.096, additive model). Related-person case–control testing using the “More Powerful” Quasi-Likelihood Score Test did not provide any evidence for association (MQLS; p = 0.41).ConclusionsAfter conservatively taking into account considerations for multiple hypothesis testing, we find little evidence for an association between the TGFBR1*6A allele and CRC risk in these families. The weak support for an increase in risk in CRC predisposed families is in agreement with recent meta-analyses of case–control studies, which estimate only a modest increase in sporadic CRC risk among 6*A allele carriers.