Biomarkers identifying biological and physiological entities associated with disease are taking an increasingly important place at the tables of drug discovery and personalised medicine. Their discovery in biosamples requires the combined use of genomics, proteomics and bioinformatics platforms. Whilst for their application, robust techniques combining exquisite sensitivity and specificity must be developed. This conference is focused on technologies that are driving advances in this area and their application at gene and expression level in solid and fluid biosamples. As such this conference is targeted to provide leading edge information to researchers within the academic, biotechnology and pharmaceutical sectors.
We include a panel session in this event, so that delegates can discuss their work directly with a panel of experts.
This event has CPD accreditation
9:00 – 9:45 Registration
9:45 – 10:00 Introduction by the Chair: Dr Peter H Bach, Director: BioPharmaLogic LLC, Cambridge
10:00 – 10:30 Begin with biomarkers
Dr Peter H Bach, Director: BioPharmaLogic LLC, Cambridge.
Biomarkers are key to drug development and personalised medicine. The identification of the “right” fit-for-purpose biomarker is complex and resource intensive. Small companies have modest potentials to implement a biomarker strategy, but can still ensure that they build their development programs around potential biomarkers. A biomarkers strategy should be a core to the development of each molecule, and not a bolt on addition to clinical development. If undertaken as part of discovery-development transition there is a potential to help shape the assessment of possible biomarkers as part of nonclinical development so that they are credible when needed.
10:30 – 11:00 Identification and quantification of cancer biomarkers using liquid chromatography-mass spectrometry
Dr David J. Britton, Proteome Sciences plc, Institute of Psychiatry, London
Liquid chromatography-mass spectrometry based proteomics can identify and quantify a multitude of proteins and post translational modifications from many different sample types (cell culture, tissue, plasma, etc). We have used this technology to identify new biomarkers and developed quantitative assays to measure the abundance of known biomarkers
11:00 – 11:15 Discovery of novel liver fibrosis biomarkers using proteomics
Dr Bevin Gangadharan, Oxford Antiviral Drug Discovery Unit, Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, UK
Two-dimensional gel electrophoresis (2-DE) is often used to separate plasma or serum proteins in an attempt to identify novel biomarkers. A major problem with this approach is the presence of high abundant plasma/serum proteins which limit the detection of low abundance features. We used two proteomics approaches to identify new fibrosis biomarkers in patients with different stages of liver fibrosis. Plasma samples from healthy individuals and patients with hepatitis C virus (HCV) induced cirrhosis were analysed using 2-DE over a narrow pH 3-5.6 range, a range outside the pH of highly abundant albumin, transferrin and immunoglobulins. Novel markers identified by this approach were validated across all fibrosis stages by Western blotting. 44 candidate biomarkers were revealed of which 20 were novel. Western blot analysis with newly identified biomarkers showed a consistent change with increasing fibrosis stage and were promising when compared to the markers used in established fibrosis tests. This is the first time the pH 3−5.6 range has been used to separate plasma by 2-DE and this pH range could be useful for discovering novel biomarkers in other diseases. In addition, we used in-solution isoelectric focusing followed by SDS-PAGE to find biomarkers in HCV-induced liver cirrhosis and this approach was found to be beneficial for analyzing basic, high molecular weight proteins. The novel fibrosis markers identified by these proteomics approach may help to assess hepatic fibrosis and decrease the need for invasive liver biopsies.
11:15 – 11:40 Speakers’ photo then mid-morning break and trade show
Please try to visit all the exhibition stands during your day at this event. Not only do our sponsors enable Euroscicon to keep the registration fees competitive, but they are also here specifically to talk to you
11:40 – 12:10 Quantitative Liver Proteomics for Biomarker Discovery in Non-alcoholic Fatty Liver Disease
Dr Bernadette Moore, University of Surrey, UK
Non-alcoholic fatty liver disease (NAFLD) is now the most common liver disease worldwide. Given that NAFLD can progress from steatosis to non-alcoholic steatohepatitis (NASH), fibrosis and potentially hepatocellular carcinoma, early diagnosis and accurate disease staging are primary clinical concerns. We have applied a relative quantitative proteomic approach utilizing isobaric tags for relative and absolute quantitation labeling combined with nano-liquid chromatography and tandem mass spectrometry to identify liver protein biomarkers in a mouse model of NASH. Novel candidate biomarkers for NAFLD, identified by proteomics and independently confirmed in these experiments, have now been confirmed in human clinical biopsy samples.
12:10 – 12:40 DNA and RNA biomarker demonstration in solid tissues
Dr Anthony Warford, University of Westminster, London, UK
Tissue donated from surgical procedures represents an important resource for biomarker identification. In ideal circumstances ‘fresh’ or frozen tissue will provide high quality DNA and RNA for analysis in situ or after extraction. However, most samples are held in the vast repositories of formalin fixed paraffin wax embedded (FFPE) tissues. In these preparations nucleic acids are degraded, principally through the fixation process. Practically this means that extraction methods have to be refined, amplicons are size limited and the possibility of the generation of spurious gene signatures needs to be considered. However, in spite of these limitations FFPE preparations provide very valuable information for the development and assessment of nucleic acid based biomarkers.
12:40 – 12:55 Tracing antigenic impurities in serum controls of agglutination test kits by qs-ELISA assay and evaluation of immunological cross reactivity In-silico
Dr Surajit Debnath, Department of Medical Laboratory Technology, Women’s Polytechnic , Hapania, India
Biomarkers trace out informative hotspots on a variety of samples so that investigators can surmise a correlation. The fundamental principle mostly involves either a Watson –Crick base pairing, as in nucleic acid based technologies or Antigen-Antibody compatibility. We generally attempt to replicate the stringent specificity in-vitro as in physiological systems. We take soaring effort to avoid cross reactivity. Coupling wet lab findings with Computational algorithms can predict for us , how much stringency control will be enough for a given set of problems in our labs.
12:55 – 13:45 Lunch and trade show
13:45 – 14:30 Question and Answer Session
Delegates will be asked to submit questions to a panel of experts. Questions can be submitted before the event or on the day
14:30 -15:00 Proteomic analysis of uveal melanoma for biomarker discovery
Dr. Paula Meleady, Dublin City University, Ireland.
Uveal melanoma is the most common intraocular malignancy in adults. The survival rate of uveal melanoma patients who develop metastatic disease is very poor. We have used both two dimensional difference gel electrophoresis (2D DIGE) and quantitative label-free liquid chromatography mass spectrometry (LC-MS) approaches for biomarker discovery in uveal melanoma tissue specimens (minimum follow-up of seven years), comparing uveal melanoma tumours from patients who developed metastatic disease with those who did not. Selected differentially expressed potential protein biomarkers of uveal melanoma metastatic disease were further followed-up by immunohistochemistry and functional validation assays of cellular invasion in vitro using siRNA.
15:00 – 15:15 ERG immunohistochemistry is not predictive for PSA recurrence, local recurrence or overall survival after radical prostatectomy for prostate cancer
A Marije Hoogland, Department of Pathology, Erasmus Medical Center, Rotterdam, The Netherlands
15:15 - 15:30 Engrailed-2 (EN2): a biomarker for the detection of clinically ‘significant’ prostate cancer
Richard Morgan and Hardev Pandha, Oncology, Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK
Achieving cure for men with prostate cancer depends almost entirely on early detection, when the cancer is localized. It is also accepted that small volume prostate cancer may be carefully monitored (active surveillance) and only a small proportion of these cancers are ‘significant’ and will progress and need treatment. This spares the remaining men considerable morbidity from treatment and has obvious health economic benefits. To date the prostate specific antigen (PSA) test has been a useful biomarker for diagnosis and monitoring, but has marked limitations as its not cancer specific and cannot indicate the size and extent of the tumour. There is an urgent unmet need not only for new prostate cancer biomarkers, but more importantly those that can differentiate significant versus non-significant disease. EN2 is a homeodomain-containing transcription factor which, unusually for this type of protein, can be secreted from cells. It has a key role in early neural and limb development, but it is not expressed in adult cells. We have shown that EN2 can be released from prostatic cancer acini and ducts and detected in the urine of prostate cancer patients. We demonstrated that EN2 was not present in the urine of men with non-malignant conditions of the prostate such as prostatitis, and that EN2 was thus a potentially useful diagnostic marker for prostate cancer, with a reported sensitivity of 66% and a specificity of 88.2% (corroborated by a second independent centre) . In a recent study, we found a strong positive correlation between pre-surgical levels of urinary EN2 and cancer volume in prostatectomy specimens in a retrospective series of 125 men who had undergone prostatectomy, and that higher EN2 levels correlated with tumour stage T2 versus T3 . This has now been corroborated further in an equivalent prospective study of 57 men undergoing prostatectomy. The urinary EN2 levels strongly correlated with the tumour volume (but not total prostatic volume) in a linear regression analysis (p <0.0001, r2=0.454). EN2 levels also significantly correlated with increasing pathological T stage and margin positivity. Using any 3 different published ‘cutoff levels’ of tumour volume (0.5ml, 1.3ml and 2.5ml) used in published studies to define ‘, significant disease’, men with ‘significant disease’ had markedly higher levels of urinary EN2 (p<0.001, p<0.001 and p<0.001 respectively) . EN2 has potential utility as a biomarker to both detect prostate cancer and also indicated diseas volume, which in turn may aid the decision to treat versus observation and monitoring. EN2 is measured in first pass urine without the requirement for preceding prostatic massage, is stable at ambient temperature for up to 4 days and is detected by a simple ELISA, and is currently being developed as a lateral flow point of care test.
15:30– 16:00 Afternoon Tea
16:00 – 16:30 Quantification of HER family Dimerisation Status in Primary Invasive Breast Cancer
Mr Fabricio Barros, University of Nottingham¸UK
HER2 status is currently assessed in routine breast cancer reporting using immunohistochemistry (IHC) in addition to in situ hybridisation (ISH) in borderline cases. The ability of HER2 gene status to predict response to targeted therapy (Trastuzumab) is well documented. However, prognostic information provided by IHC expression categories and prognostic value added by using ISH in borderline cases remains unclear. Patients may acquire resistance to this drug after a period of treatment, which indicates that other molecular mechanisms might influence success of this therapy. Dimerisation between members of the HER family may contribute to resistance against treatments due to different combinations that trigger different downstream pathways. Further quantification analysis of HER family dimers using in situ Proximity Ligation Assay (PLA) may be crucial to identify subset of patients associated with distinct outcome, response to treatment and relationships with HER signalling related biomarkers
16:30 - 17:00 Chairman’s summing up
About the Chair
Peter Bach has worked in Academia, for National and International Medicine Safety Agencies, and Biotech companies on a spectrum of novel small molecules to third generation biologicals. Peter has a specialist interest in mechanistic pathology and toxicology, which allows a deeper understanding of the processes associated with disease and intervention, and identifying biomarkers that help establish efficacy and safety. This helps better match individual patients to maximised treatment.
About the Speakers
Anthony (Tony) Warford expertise is in molecular histopathology. He has set up and managed laboratories in the UK health service, academic institutions, biotechnology and Pharmaceutical companies. Technology developments he has spearheaded include the introduction of diagnostic immunohistochemical methods, validation of antibodies for use as biomarkers, production of probes and methods for in situ hybridisation and supervision and interpretation of GLP tissue based safety studies of potential therapeutic antibodies. Concurrently he has championed quality assurance programmes in histopathology and automation of immunohistochemistry coupled with image capture and analysis. He has also run laboratory safety and human bio-banking programmes. He has published in these fields and shared experience with fellow scientists by organising wet workshops, chairing symposia and lecturing in many countries.
Bernadette Moore is research scientist and a Lecturer in Molecular Nutrition within the Faculty of Health and Medical Sciences at the University of Surrey. Her research primarily focuses on the dietary, genetic and immune factors that influence non-alcoholic fatty liver disease development. Utilizing discovery-based proteomics, genomics and systems biology approaches alongside molecular cell biology techniques, her group aims both to identify disease biomarkers and characterize molecular mechanisms of NAFLD progression.
Paula Meleady PhD (1997) in Cell Biology from Dublin City University. Senior Scientist at the National Institute for Cellular Biotechnology (NICB) since 2001 and currently Programme Leader of the Proteomics and Mass Spectrometry Core Facility at the NICB which is equipped with state-of-the-art proteomics and mass spectrometry equipment. Research interests include proteomic analysis of uveal melanoma, for biomarker discovery. Also have research interests in proteomics of recombinant mammalian cell lines in order to gain insights to improving productivity of biopharmaceuticals. Co-authored over 40 peer-reviewed publications, 6 book chapters and 2 patents to date in research areas related to proteomics, cancer and bioprocessing.
David J. Britton Degree; Pharmacology, University of Bristol (1998-2001). PhD; Cancer Pharmacology, Tenovus labs, Pharmacy Dept, Cardiff University (2001-2005). Post Doc; Cancer Proteomics, Benz/Gibson Lab, Buck Institute, California (2005-2008). Training Instructor; Proteomics, Thermo Fisher Scientific (2008-2010). Senior Research Scientist; Cancer Proteomics, Proteome Sciences plc, UK (2010-current).
Julian Beesley has over 22 years' experience as a research scientist in discovery research with a major pharmaceutical company and over 11 years' involvement in developing biotech businesses, operating widely throughout Europe, the USA, Asia and Japan.
Surajit Debnath, obtained PhD in the field of Animal Physiology (Tripura University, Govt. of India) after majoring with Biotechnology. As an Assistant Professor in the Department of Medical Laboratory Technology, Women’s Polytechnic Tripura, India Dr. Debnath is extensively using Biomarker tools for basic research. He received Govt of India fellowship to Hongkong (2011) & Denmark (2012) & have visited Lab of Evolutionary Biology in the J.W.Goethe University, Frankfurt, Germany. Dr. Debnath has published several research papers and is serving as editorial board member of International research journals of repute.
Fabricio Barros just concluded his Phd based on examination of expression amongst the HER family in a large series of breast cancer cases examining the clinical effect of co expression of the receptors and respective ligands. This research study is being developed using not only the standard histopathological techniques as immunohistochemistry and in situ Hybridisation but also using a novel approach (in situ Proximity Ligation Assay) to characterise HER2+ invasive breast tumours. This study is being performed at University of Nottingham within the Breast Cancer Research Group leaded by Prof. Ian Ellis and Dr. Andrew Green.
Bevin Gangadharan obtained his DPhil under the supervision of Prof. Nicole Zitzmann at the University of Oxford where he carried out the first ever gel-based proteomics study to discover novel biomarkers for liver fibrosis. He has more than a decade of experience in proteomics and biomarker discovery and first started in this field in 2000 at Smithkline Beecham looking at depletion of albumin in plasma, an important approach in biomarker discovery. He has published in several peer-reviewed journals and has two patents on novel biomarkers for liver fibrosis. He is on the editorial board for Biomarker Research.
Key words: immunohistochemistry, biomarkers, personalised health care, oncology, drug development, mass spectroscopy, proteomics, bioinformatics, DNA, RNA,Biorepositories, biomarkers, drug discovery, Histochemistry, Metabolism, Biologicals, Safety, Efficacy, predictive PharmDx, pharmacodiagnostics,Oncology, biomarker identification/quantification, quantifying biomarkers, clinical tissues, automated-analysis, ELISA, TSH, hFillotropin, hepatitis C patients; single nucleotide polymorphisms; Il-10; IL-28, MicroRNAs; Toll-like receptors;lung cancer patients, ox-LDL Ab; Insulin resistance; atherosclerosis; IMT, Adrenomedullin; SLE; nephritis, uveal melanoma, metastasis, 2D-DIGE, label-free LC-MS, biomarker, Nonalcoholic fatty liver disease; proteomics; iTRAQ; Breast Cancer, HER2, Dimerisation, PLA, PSA, prostate, ERG, : FFPET, DLBCL, microRNA, qRT-PCR, biomarkers
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