Prediction and diagnosis of complex disease may not always be possible with a small number of biomarkers. Modern 'omics' technologies make it possible to cheaply and quantitatively assay hundreds of ...molecules generating large amounts of data from individual samples. In this study, we describe a parenclitic network-based approach to disease classification using a synthetic data set modelled on data from the United Kingdom Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) and serological assay data from a nested set of samples from the same study. This approach allows us to integrate quantitative proteomic and categorical metadata into a single network, and then use network topologies to construct logistic regression models for disease classification. In this study of ovarian cancer, comprising of 30 controls and cases with samples taken <14 months to diagnosis (
= 30) and/or >34 months to diagnosis (
= 29), we were able to classify cases with a sensitivity of 80.3% within 14 months of diagnosis and 18.9% in samples exceeding 34 months to diagnosis at a specificity of 98%. Furthermore, we use the networks to make observations about proteins within the cohort and identify GZMH and FGFBP1 as changing in cases (in relation to controls) at time points most distal to diagnosis. We conclude that network-based approaches may offer a solution to the problem of complex disease classification that can be used in personalised medicine and to describe the underlying biology of cancer progression at a system level.
Earlier detection of pancreatic ductal adenocarcinoma (PDAC) is key to improving patient outcomes, as it is mostly detected at advanced stages which are associated with poor survival. Developing ...non-invasive blood tests for early detection would be an important breakthrough.
The primary objective of the work presented here is to use a dataset that is prospectively collected, to quantify a set of cancer-associated proteins and construct multi-marker models with the capacity to predict PDAC years before diagnosis. The data used is part of a nested case-control study within the UK Collaborative Trial of Ovarian Cancer Screening and is comprised of 218 samples, collected from a total of 143 post-menopausal women who were diagnosed with pancreatic cancer within 70 months after sample collection, and 249 matched non-cancer controls. We develop a stacked ensemble modelling technique to achieve robustness in predictions and, therefore, improve performance in newly collected datasets.
Here we show that with ensemble learning we can predict PDAC status with an AUC of 0.91 (95% CI 0.75-1.0), sensitivity of 92% (95% CI 0.54-1.0) at 90% specificity, up to 1 year prior to diagnosis, and at an AUC of 0.85 (95% CI 0.74-0.93) up to 2 years prior to diagnosis (sensitivity of 61%, 95% CI 0.17-0.83, at 90% specificity).
The ensemble modelling strategy explored here outperforms considerably biomarker combinations cited in the literature. Further developments in the selection of classifiers balancing performance and heterogeneity should further enhance the predictive capacity of the method.
Screening for pancreatic ductal adenocarcinoma (PDAC) in populations at high risk is recommended. Individuals with new-onset type 2 diabetes mellitus (NOD) are the largest high-risk group for PDAC. ...To facilitate screening, we sought biomarkers capable of stratifying NOD subjects into those with type 2 diabetes mellitus (T2DM) and those with the less prevalent PDAC-related diabetes (PDAC-DM), a form of type 3c DM commonly misdiagnosed as T2DM.
Using mass spectrometry- and immunoassay-based methodologies in a multi-stage analysis of independent sample sets (n=443 samples), blood levels of 264 proteins were considered using Ingenuity Pathway Analysis, literature review and targeted training and validation.
Of 30 candidate biomarkers evaluated in up to four independent patient sets, 12 showed statistically significant differences in levels between PDAC-DM and T2DM. The combination of adiponectin and interleukin-1 receptor antagonist (IL-1Ra) showed strong diagnostic potential, (AUC of 0.91; 95% CI: 0.84-0.99) for the distinction of T3cDM from T2DM.
Adiponectin and IL-1Ra warrant further consideration for use in screening for PDAC in individuals newly-diagnosed with T2DM.
North West Cancer Research, UK, Cancer Research UK, Pancreatic Cancer Action, UK.
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers largely due to disseminated disease at the time of presentation. Here, we investigated the role and mechanism of action ...of the metastasis-associated protein anterior gradient 2 (AGR2) in the pathogenesis of pancreatic cancer. AGR2 was induced in all sporadic and familial pancreatic intraepithelial precursor lesions (PanIN), PDACs, circulating tumor cells, and metastases studied. Confocal microscopy and flow cytometric analyses indicated that AGR2 localized to the endoplasmic reticulum (ER) and the external surface of tumor cells. Furthermore, induction of AGR2 in tumor cells regulated the expression of several ER chaperones (PDI, CALU, RCN1), proteins of the ubiquitin-proteasome degradation pathway (HIP2, PSMB2, PSMA3, PSMC3, and PSMB4), and lysosomal proteases cathepsin B (CTSB) and cathepsin D (CTSD), in addition to promoting the secretion of the precursor form pro-CTSD. Importantly, the invasiveness of pancreatic cancer cells was proportional to the level of AGR2 expression. Functional downstream targets of the proinvasive activity of AGR2 included CTSB and CTSD in vitro, and AGR2, CTSB, and CTSD were essential for the dissemination of pancreatic cancer cells in vivo. Taken together, the results suggest that AGR2 promotes dissemination of pancreatic cancer and that its cell surface targeting may permit new strategies for early detection as well as therapeutic management.
Identification of serum biomarkers enabling earlier diagnosis of pancreatic ductal adenocarcinoma (PDAC) could improve outcome. Serum protein profiles in patients with preclinical disease and at ...diagnosis were investigated.
Serum from cases up to 4 years prior to PDAC diagnosis and controls (UKCTOCS,n= 174) were studied, alongside samples from patients diagnosed with PDAC, chronic pancreatitis, benign biliary disease, type 2 diabetes mellitus, and healthy subjects (n= 298). Isobaric tags for relative and absolute quantification (iTRAQ) enabled comparisons of pooled serum from a test set (n= 150). Validation was undertaken using multiple reaction monitoring (MRM) and/or Western blotting in all 472 human samples and samples from a KPC mouse model.
iTRAQ identified thrombospondin-1 (TSP-1) as reduced preclinically and in diagnosed samples. MRM confirmed significant reduction in levels of TSP-1 up to 24 months prior to diagnosis. A combination of TSP-1 and CA19-9 gave an AUC of 0.86, significantly outperforming both markers alone (0.69 and 0.77, respectively;P< 0.01). TSP-1 was also decreased in PDAC patients compared with healthy controls (P< 0.05) and patients with benign biliary obstruction (P< 0.01). Low levels of TSP-1 correlated with poorer survival, preclinically (P< 0.05) and at clinical diagnosis (P< 0.02). In PDAC patients, reduced TSP-1 levels were more frequently observed in those with confirmed diabetes mellitus (P< 0.01). Significantly lower levels were also observed in PDAC patients with diabetes compared with individuals with type 2 diabetes mellitus (P= 0.01).
Circulating TSP-1 levels decrease up to 24 months prior to diagnosis of PDAC and significantly enhance the diagnostic performance of CA19-9. The influence of diabetes mellitus on biomarker behavior should be considered in future studies.
Protein phosphorylation plays a crucial role in the signal transduction pathways that regulate gene expression, metabolism, cell adhesion, and cell survival in response to oxidative stress. In this ...study, we have used hydrogen peroxide treatment of H9C2 rat cardiomyocytes as a model of oxidative stress in heart ischemia–reperfusion injury. We show that oxidative stress induces a robust tyrosine phosphorylation of multiple proteins in this cell type. A phosphoproteomics approach using anti-phosphotyrosine affinity purification and LC–MS/MS was then used to identify the protein targets of this stress-induced phosphorylation. Twenty-three tyrosine-phosphorylated proteins were identified, with the majority known to be associated with cell–cell junctions, the actin cytoskeleton, and cell adhesion. This suggested that oxidative stress may have a profound effect on intercellular connections and the cytoskeleton to affect cell adhesion, morphology, and survival. Importantly, Src kinase was shown to be a major upstream regulator of these events. Immunofluorescence studies, fluorescence-activated cell sorting, and cell-based assays were used to demonstrate oxidative stress-induced modification of cell adhesion structures and the cytoskeleton, induced de-adhesion, and increased apoptosis, which were reversed by treatment with the Src kinase inhibitor PP1. These data demonstrate the critical role of Src kinase in oxidative stress-induced phosphorylation and cell damage in cardiomyocytes and suggest that targeting this kinase may be an effective strategy for preventing ischemia–reperfusion injury in the heart.
Fetal growth restriction (FGR) is a serious obstetric condition for which there is currently no treatment. The EVERREST Prospective Study has been designed to characterise the natural history of ...pregnancies affected by severe early onset FGR and establish a well phenotyped bio-bank. The findings will provide up-to-date information for clinicians and patients and inform the design and conduct of the EVERREST Clinical Trial: a phase I/IIa trial to assess the safety and efficacy of maternal vascular endothelial growth factor (VEGF) gene therapy in severe early onset FGR. Data and samples from the EVERREST Prospective Study will be used to identify ultrasound and/or biochemical markers of prognosis in pregnancies with an estimated fetal weight (EFW) <3rd centile between 20+0 and 26+6 weeks of gestation.
This is a 6 year European multicentre prospective cohort study, recruiting women with a singleton pregnancy where the EFW is <3rd centile for gestational age and <600 g at 20+0 to 26+6 weeks of gestation. Detailed data are collected on: maternal history; antenatal, peripartum, and postnatal maternal complications; health economic impact; psychological impact; neonatal condition, progress and complications; and infant growth and neurodevelopment to 2 years of corrected age in surviving infants. Standardised longitudinal ultrasound measurements are performed, including: fetal biometry; uterine artery, umbilical artery, middle cerebral artery, and ductus venosus Doppler velocimetry; and uterine artery and umbilical vein volume blood flow. Samples of maternal blood and urine, amniotic fluid (if amniocentesis performed), placenta, umbilical cord blood, and placental bed (if caesarean delivery performed) are collected for bio-banking. An initial analysis of maternal blood samples at enrolment is planned to identify biochemical markers that are predictors for fetal or neonatal death.
The findings of the EVERREST Prospective Study will support the development of a novel therapy for severe early onset FGR by describing in detail the natural history of the disease and by identifying women whose pregnancies have the poorest outcomes, in whom a therapy might be most advantageous. The findings will also enable better counselling of couples with affected pregnancies, and provide a valuable resource for future research into the causes of FGR.
NCT02097667 registered 31
October 2013.
The technique of fluorescent two-dimensional (2D) difference gel electrophoresis for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 ...overexpression. Labeling of paired cell lysate samples with N-hydroxy succinimidyl ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive, and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrix-assisted laser-desorption ionization mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3- and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (Amersham Biosciences, Inc.) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines.
Longitudinal CA125 algorithms are the current basis of ovarian cancer screening. We report on longitudinal algorithms incorporating multiple markers. In the multimodal arm of United Kingdom ...Collaborative Trial of Ovarian Cancer Screening (UKCTOCS), 50,640 postmenopausal women underwent annual screening using a serum CA125 longitudinal algorithm. Women (cases) with invasive tubo-ovarian cancer (WHO 2014) following outcome review with stored annual serum samples donated in the 5 years preceding diagnosis were matched 1:1 to controls (no invasive tubo-ovarian cancer) in terms of the number of annual samples and age at randomisation. Blinded samples were assayed for serum human epididymis protein 4 (HE4), CA72-4 and anti-TP53 autoantibodies. Multimarker method of mean trends (MMT) longitudinal algorithms were developed using the assay results and trial CA125 values on the training set and evaluated in the blinded validation set. The study set comprised of 1363 (2-5 per woman) serial samples from 179 cases and 181 controls. In the validation set, area under the curve (AUC) and sensitivity of longitudinal CA125-MMT algorithm were 0.911 (0.871-0.952) and 90.5% (82.5-98.6%). None of the longitudinal multi-marker algorithms (CA125-HE4, CA125-HE4-CA72-4, CA125-HE4-CA72-4-anti-TP53) performed better or improved on lead-time. Our population study suggests that longitudinal HE4, CA72-4, anti-TP53 autoantibodies adds little value to longitudinal serum CA125 as a first-line test in ovarian cancer screening of postmenopausal women.
biogroup
is a human-adapted pathogen and the causative agent of Brazilian purpuric fever (BPF), an invasive disease with high mortality, that sporadically manifests in children previously suffering ...conjunctivitis. Phase variation is a rapid and reversible switching of gene expression found in many bacterial species, and typically associated with outer-membrane proteins. Phase variation of cytoplasmic DNA methyltransferases has been shown to play important roles in bacterial gene regulation and can act as epigenetic switches, regulating the expression of multiple genes as part of systems called phasevarions (phase-variable regulons). This study characterized two alleles of the ModA phasevarion present in
biogroup
, ModA13, found in non-BPF causing strains and ModA16, unique to BPF causing isolates. Phase variation of ModA13 and ModA16 led to genome-wide changes to DNA methylation resulting in altered protein expression. These changes did not affect serum resistance in
biogroup
strains.
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