Day 2 :
Keynote Forum
Biswendu B Goswami
FDA Center for Food Safety and Applied Nutrition, USA
Keynote: Microarray based identification of viral genomes
Time : 09:30 AM
Biography:
Biswendu B Goswami received his Ph.D. in Biochemistry degree from the University of Calcutta (Kolkata) in 1975, followed by a post-doctoral fellowship from CSIR, India for two years. He came to USA on a post-doctoral fellowship in the laboratory of the Late Prof. Ernest Borek. He worked also as a Research Associate at Georgetown University Medical Center at Washington D.C. He later joined The United States Food and Drug Administration, and was in charge of the Virology laboratory at the Center for Food Safety, until he retired in 2011. He has published more than 45 research articles and book chapters.
Abstract:
Procedures for virus identification are currently going through a revolution due to two recent developments. The orthodox methods for virus identification by cell culture followed by secondary confirmation where possible, by immunological techniques have been replaced by PCR based methods. But PCR methods have the added disadvantages that they are extremely sensitive to impurities in the test material, and have very narrow specificity, requiring individual primers for each virus and strain, and therefore atleast some knowledge of the sequence of the viral genetic material. Hybridization to oligonucleotide probes on solid support was developed in the 1990s. However, the true potential for identification of viruses on a massive scale based on sequence of viral genomes did not take off until the development of in situ probe synthesis and immobilization techniques. These newer photolithographic techniques allowed for the synthesis and simultaneous immobilization of hundreds of thousands of probes in a single microarray allowing re-sequencing on a large scale by hybridization to identify virus genotypes. Since then, microarrays have been developed that can incorporate over two million probes in a single array. This development opened up the possibility of identifying multiple virus species and genotypes in a single experiment. However, to realize this potential, it is necessary to have a target synthesis method that is independent of the sequence of the viruses present in the test material. The new microarray based method developed in our laboratory at Food and Drug Administration (FDA), avoids PCR amplification with virus specific primers normally used for target synthesis in microarray experiments.
- Track 3 : Biomarkers in Clinical Research and Development
Track 4 : Omics Technologies in Biomarkers Discovery and Validation
Chair
Alain Moreau
Sainte-Justine University Hospital, Canada
Co-Chair
Chee Gee See
Proteome Sciences, UK
Session Introduction
Alain Moreau
Sainte-Justine University Hospital, Canada
Title: Osteoarthritis unmasked: Identification of nuclear prohibitin as a new specific OA biomarker and therapeutic target
Biography:
Alain Moreau is Full Professor in the Faculty of Dentistry and the Faculty of Medicine at the Université de Montréal. He is the Director of Research and Chief Scientific Officer of Sainte-Justine University Hospital. He received his PhD in Microbiology and Immunology from the Université de Montréal in 1993. He did a first Postdoctoral training at the Protein Engineering Center of University of Liège, Belgium (1992-1993), followed by a second Postdoctoral fellowship at the Shriners Hospital for Children in Montrealaffiliated with McGill University (1993-1997). He is an internationally recognized expert on molecular genetics of pediatric scoliosis and osteoarthritis. His discoveries led to multiple peer-reviewed papers, international conferences as guest speaker, several awards as well as 32 patents issued.
Abstract:
Osteoarthritis (OA) is one of the most common age-related chronic disorders affecting articular cartilage, joints and bone tissues. According to datamonitor, up to 81.4 million cases in adults aged 25 and older suffer from OA in seven major markets (USA, Japan, UK, Germany, Italy, France and Spain). The current drug treatment for OA is symptom-relieving, and the need for pharmacological treatments to retard, prevent or repair cartilage destruction in OA is urgent. Target selection has been problematic, which includes the identification of selective biomarkers, establishment of appropriate preclinical animal models that reflect human OA, the limitations of the current radiographic standard for structural assessment (KL score), and the lack of stratification of patients in clinical trials by phenotype or tissue involvement. Yet, the search for disease-modifying OA drugs (DMOADs) has proven to be an exceptional challenge, largely because OA usually progresses slowly, with few patients reporting worsening symptoms throughout the clinical trial, which typically lasts months, not years. In that context, we developed a new predictive diagnostic assay intended to measure a specific biomarker, Prohibitin (PHB1) through an immunoassay on blood samples. This test is sensitive enough to detect OA at its earliest stage in asymptomatic subjects and for the first time provides a therapeutic window for intervention, to stop or reverse the mechanism of cartilage matrix degradation associated with primary OA. The integration of specific biomarkers in a companion diagnostic test has the potential to accelerate drug development and thus time to market access for the pharma industry.
Alexander M Buko
Human Metabolome Technologies, USA
Title: Developing a blood metabolite HMT biomarker for Major Depression Disorder (MDD)
Biography:
Alexander M Buko received his PhD in 1980 from the University of Virginia under Professor Donald F. Hunt. He went onto work at the Bureau of Biologics and Biophysics (Today called CBER) for four years then moved to Abbott Labs for 18 years as a distinguished research fellow. From 2002 to 2012, he was Sr. Director Translational Medicine at Biogen Idec. Currently, he is the Vice President of Business and Product Development for HMT-America (Human Metabolome Technologies).
Abstract:
Depression is the leading cause of disability worldwide, affecting about 121 million people in the United States. WHO predicts that it will be the second most common global burden of disease by the year 2020. However, the diagnosis and treatment of depression can be elusive and difficult. Many depressed patients would benefit from objective biological information to determine their condition prior to referral to a therapist. PCPs are only able to recognize about half of the patients with clinical depression while 20% of non-depressed people are falsely diagnosed as depressed. There is a large unmet need for additional diagnostic tests for patient referrals and therapeutic effectiveness. To develop a blood biomarker for MDD, HMT (Human Metabolome Technologies) profiled 538 plasma metabolites from 34 clinically depressed subjects and 38 demographically matched non-depressed subjects. Results identified a potential biomarker, ethanolamine phosphate (EAP). The study was repeated on 241 patients showing patients with MDD had specifically lower plasma concentrations of EAP, as well as, showing severity-dependent behavior. The diagnostic ability of EAP was further confirmed in a single-blinded independent validation group. HMT has transferred the discovery test to a specific blood CoDx assay to measure the level of EAP and we are studying environmental and circadian influences on EAP levels as well.
Chee Gee See
Proteome Sciences, UK
Title: Proteomic strategies to overcome tumor resistance to oncology targeted medicines
Biography:
Chee Gee See is the Director of Personalised Medicine at Proteome Sciences, a company providing high end mass-spectrometry based biomarker discovery and clinical utility tools, uniquely positioned to engage with pharma partners in our quest for personalised medicines. He was previously the clinical Biomarker and Experimental Medicine Leader at Roche for 5 years. He was the BEML for the Phase III pivotal TOGA study where Herceptin was trialled for the new indication of gastric cancer. He was also a BEML for CVD, CNS and immunology so his range and awareness of disease areas and their clinical development challenges is both deep and broad. He also spent 11 years at GSK where he was a genetics expert covering 3 key therapeutic areas in preclinical research within Genetics Research Europe. Prior to industry, he spent 6 years in post-doctoral academia at the University of Birmingham and University College London, most notably in the Human Genome Mapping Project. He also has dual specialist expertise as a consultant in regulatory affairs and value-based drug pricing, reimbursement and market access. He looks forward to engaging with like-minded professionals during this conference.
Abstract:
The identification of genomic targets in cancer pathways has opened up a whole vista of personalised medicine development in recent years. The fast-tracking of such targets from discovery to drug launch has been truly astonishing, as exemplified by the 2007 initial discovery of the EML4-ALK oncogene to the 2011 FDA approval of Pfizer\'s targeted ALK+Xalkori for NSCLC. Whilst these advances are nothing short of brilliant, resistance to such targeted pathways indicate the pitfalls of single target or canonical pathway approaches. Resistance to targeted therapies has emerged as one of the greatest challenges to the personalised medicine approach. To even begin to tackle the issue of resistance, a complete picture of both the canonical and non-canonical signalling pathways in tumor biology is required. Such a complete molecular profile is now possible and this would indicate if our drug is hitting the target and also what alternative pathways the tumor might be engaging to bypass the effects of the drug. Author will present data from proteomics-based strategies to identify global signalling pathways and will demonstrate how this strategy aids decision making on the effectiveness of the targeted oncology drug and in optimizing potential combinatorial options to combat resistance. These strategies and how they are implemented clinically will be discussed. This strategy represents a very innovative and eminently actionable clinical approach to tumor resistance.
Iulia M Lazar
Virginia Tech, USA
Title: Cell cycle and the world of biomarkers: What can we learn from proteomic studies?
Biography:
Iulia M Lazar completed her PhD in Chemistry from Brigham Young University. Following two Post-Doctoral appointments at Sensar Larson-Davis and Oak Ridge National laboratory, and a Principal Research Scientist position at The Barnett Institute/Northeastern University. Presently, she is an Associate Professor with research interests focused on oncoproteomics, breast cancer cell cycle, signaling, biomarker discovery and the development of microfluidic and mass spectrometry technologies for the interrogation of biological systems. The findings of her research led to over 55 publications, book chapters, patents and numerous presentations at national and international symposia.
Abstract:
High-throughput technologies such as LC-MS/MS generate large data-sets that flood scientists with unprecedented amounts of information. The conversion of such data into knowledge has the potential to not just answer long-sought biological questions, but to also revolutionize our approach to solve complex problems that lie at the root of human disease. The goal of our research is to study the molecular mechanisms of breast cancer cell-cycle to uncover aspects that lead to uncontrolled cell proliferation and the discovery of biomarkers and drug targets. Our model system consists of ER+ (MCF7), Her2+ (SKBR3) and non-tumorigenic (MCF10A) cells. Proteomic profiling of different stages of the cell cycle resulted in the identification of ~7000 proteins, with over half of the phosphorylated proteins being involved in proliferative or apoptotic signaling processes. The data revealed protein clusters involved in various signaling pathways, providing a preliminary vista of the cause-effect relationships that confer cancer cells a proliferative advantage. In particular, the G1 stage of the cell cycle included cell cycle inhibitory and DNA damage response proteins, and, most importantly, originators of proliferation and possible drivers through the G1/S transition point. Among the differentially expressed proteins, a rich network of ~200 proteins emerged with biomarker or drug-target potential. The impact of point mutations, insertions and deletions on the functional relevance of such proteins was explored. The findings expose the inherent complexity of the biological processes that unfold within the environment of a cell, and open bold, new opportunities for addressing the problem of diseased cell states.
Maria Paola Costi
Università degli Studi di Modena e Reggio Emilia, Italia
Title: Differential mass-spectrometry proteomic and bioinformatic analysis of Leishmania donovani treated with miltefosine and analogs.
Biography:
Maria Paola Costi has completed her Ph.D Medicinal chemistry in 1989 from the University of Modena and Reggio Emilia and visiting scientist at the University of California San Francisco (UCSF). She is professor of Medicinal Chemistry at the Department of Life Science at Unimore. She has published more than 85 papers in international journals, many patents and serving as an editorial board member of a few journals in the field. Board member of translational science in oncology group of the MITO network and WG leader of drug discovery and development of EUTROC. Coordinator/scientific responsible of some FP European projects in the area of drug discovery in cancer and parasitic diseases (www.lights.eu, www.optobacteria.eu, www.nmtrypi.eu).
Abstract:
The group of infections known as neglected tropical diseases (NTDs) collectively affects one billion people worldwide and represents an enormous burden in terms of human suffering, treatment costs and loss of income. Leishmaniasis is an infection caused by obligate intracellular protozoan Leishmania parasites, which are transmitted by the bite of certain sandfly species. Multitude of Leishmania species cause disease in humans resulting in three clinical manifestations. Visceral leishmaniasis is the most severe form of leishmaniasis and it is caused by Leishmania donovani and L. infantum. Before 2002, the available agents against visceral leishmaniasis were: pentavalent antimony (SbV) compounds, liposomal amphotericin B, paromomycin, and pentamidine. All these drugs must be administered parenterally and have severe side-effects. In 2002, miltefosine, was registered as the first oral agent that does not require hospitalization and up to date is still the only oral treatment for all forms of leishmaniasis. Miltefosine belongs to the class of alkylphosphocholine drugs, which are phosphocholine esters of aliphatic long-chain alcohols. These alkylphosphocholine compounds are structurally related to the group of alkyl-lysophospholipids, which are synthetic analogues of lysophosphatidylcholines or lysolecithins, but lack their glycerol backbone. Miltefosine has demonstrated activity against Leishmania parasites and neoplastic cells. Its biological activity is exerted through (i) induction of apoptosis and (ii) disturbance of lipid-dependent cell signaling pathways; however its mechanism of action and biological target(s) are still unclear (1). The main safety concerns for miltefosine relate to its effect on the mucosa of the gastrointestinal tract and its potential teratogenicity. For these reasons many miltefosine analogues/derivatives are under study/development, to increase activity and reduce toxicity. In this work, the effects of miltefosine and a number of new miltefosine-analogues on the proteomes of L. donovani promastigote parasites have been studied. The aim is to determine molecular events triggered directly or indirectly by the agents, in order to get a better insight on the mechanism of action of this compound class and, at the same time, to identify a protein signature that may characterize their activity. The method is based on differential mass spectrometry (MS) technique of the drug treated parasite samples versus untreated controls. Preliminary studies suggest that among the different proteins affected are ribosomal proteins, heat shock proteins and translation machinery components.
S K Jain
Jamia Hamdard, India
Title: Implication of cancer proteomics in development of biomarkers for early detection of HCC
Biography:
S K Jain, PhD (1974) from AIIMS, New Delhi is Professor of Medical Biochemistry & Biotechnology, Hamdard University. He was Research Associate at Washington University Medical Centre, Tufts Medical School and Harvard Medical School (USA), senior scientist at National Institute of Immunology, New Delhi (1985-2000), visiting Professor, Catholic University, Rome; visiting scientist, Institute of Virology, Oxford; served as consultant to WHO and US-AID; has been Dean, Faculties of Science & Allied Health Sciences and officiating Vice-Chancellor of Hamdard University. He has published 175 papers in peer reviewed journals, number of book chapters and guided >50 PhD students. His current research interests are molecular biology/ immunology of typhoid and proteomics of lung and liver cancers.
Abstract:
Post-HGP era opened new vistas for understanding gene expression and initiated several specialized ‘omics’. Proteomics is analysis of total cellular proteins under different physiological and pathological conditions and is useful for discovery of novel biomarker(s) for diagnosis, and monitoring of progression of cancers by direct analysis of serum proteins. Hepatocellular carcinomas highly prevalent and leads to high morbidity and mortality. Presently serum α-fetoprotein levels form standard diagnosis of HCC, which is relatively low in sensitivity and specificity. In our efforts to develop biomarkers for early detection of HCC, a novel animal model to study ¬ chemically induced liver cancer was developed. The serum protein profiles at various stages of disease progression have been analyzed by 2D electrophoresis. Number of differentially expressed proteins have been detected, some of which bear correlation with disease progression. Histopathology and marker enzyme analyses confirmed and monitored tumor formation and disease progression. The proteins of interest have been characterized by MALDI-TOF and LC-MS/MS. We report the analysis of one of these proteins whose levels are elevated during very early stage of cancer initiation and remain elevated thereafter. The cloning and high level expression of this protein has been achieved. The sequencing of its gene revealed that specific point mutations take place in this protein during tumorogenesis and mutated protein shows immunogenicity. The diseased animals have circulating antibodies against. These findings are important in understanding the molecular mechanism of HCC development. Sera of clinically confirmed HCC patients have elevated levels of this protein that supports its potential as probable biomarker for diagnosis of HCC. The implications of these studies will be discussed.
Todd M Doran
Scripps Research Institute, USA
Title: A chemical approach to mine the immunoproteome for disease biomarkers
Biography:
Todd M Doran completed his PhD under the guidance of Bradley Nilsson at the University of Rochester in 2012 and his Postdoctoral studies at The Scripps Research Institute working with Thomas Kodadek. His work focuses on the use of organic chemistry to create combinatorial libraries that serve as surrogates for unknown, disease-linked antigens. His improvements to the synthesis and screening of combinatorial libraries against human blood samples have led to several manuscripts and pending patents.
Abstract:
The adaptive immune system reacts to foreign molecules or antigens through the amplification of antibodies. Therefore, antibodies represent easily accessed biomarkers for diseases that include cancer, autoimmune and neurodegenerative disease. Unfortunately, the most suitable capture agent for an antibody biomarker is the cognate antigen which in many diseases is not known due to the limitations of conventional antigen discovery methods. We are developing unbiased antigen discovery platforms that take a novel chemical approach to discover new biomarkers. Using combinatorial chemistry, we identify small molecules that are capable of engaging the antigen-binding site of disease-associated antibodies from patient serum. If these abiological “antigen surrogates” bind with sufficiently high affinity and specificity, they are used to detect and enrich the disease antibody population in order to identify the cognate auto-antigen. This strategy has allowed us to uncover novel auto-antigens that are involved during the progression of type 1 diabetes mellitus. These new methods for biomarker discovery will be discussed in the context of identifying new autoantibody biomarkers for type 1 diabetes and their diagnostic application.
Sid Katugampola
Centre for Drug Development at Cancer Research, UK
Title: Biomarkers in early phase oncology clinical development
Biography:
Sid Katugampola completed his PhD in Cambridge. He worked across multiple departments, spanning over 11 years at Pfizer Research UK. During his last 6 years at Pfizer he led projects in biomarkers and translational medicine across multiple therapeutic areas and targets. The past 3 years of his career he has been working at the Centre for Drug Development at Cancer Research UK, where he is responsible for delivering pharmacodynamic biomarkers, across multiple modalities and cancer types, in early phase oncology clinical trials, the majority of which are first in class agents.
Abstract:
The current process of R&D is not sustainable and there is a big drive for shorter timelines and reduced development costs. In oncology, the number of targeted anti-cancer therapies is on the rise and they are showing significant promise both in terms of efficacy and improved safety profile compared to conventional chemotherapy. In early phase oncology clinical development, biomarkers are increasingly being used to identify the right drug for the right patient, at the right dose & schedule in order to clearly demonstrate proof of mechanism and proof of principle. This is fundamentally important as often in early phase oncology trials, demonstrating proof of concept in terms of efficacy is very limited and later stage development is performed at risk. With evolving technologies, numerous methods and assays are being explored to demonstrate biological end points that enable successful go/no-go decision for further development and thereby helping to minimize phase II attrition. Surrogate end points, in addition to tumor markers, add more confidence to overall trial success. Circulating markers, in particular ctDNA has shown significant promise as a liquid biopsy. Following dose escalation, a growing number of trials now incorporate expansion arms with patient enrichment strategies.There are number of limitations and challenges in using biomarkers in early phase oncology trials, including making sense of large data sets. Despite these challenges, a positive and optimistic outlook prevails in the use of pharmacodynamic and enrichment markers in early phase trials for ultimate patient benefit.
Maria Paola Costi
University of Modena and Reggio Emilia, Italy
Title: Identification of a protein panel as pharmacodynaimic biomarker of folate directed targeted drugs in the treatment of ovarian cancer
Biography:
Maria Paola Costi has completed her Ph.D Medicinal chemistry in 1989 from the University of Modena and Reggio Emilia and visiting scientist at the University of California San Francisco (UCSF). She is professor of Medicinal Chemistry at the Department of Life Science at Unimore. She has published more than 85 papers in international journals, many patents and serving as an editorial board member of a few journals in the field. Board member of translational science in oncology group of the MITO network and WG leader of drug discovery and development of EUTROC. Coordinator/scientific responsible of some FP European projects in the area of drug discovery in cancer and parasitic diseases (www.lights.eu, www.optobacteria.eu, www.nmtrypi.eu).
Abstract:
Ovarian cancer (OC) is the fifth most common cause of death from cancer in women. The standard first-line treatment (platinum-derivatives and paclitaxel) suffers from rapid resistance development with still unclear mechanisms and poor prognosis. However, the resistance process includes the over-expression of Thymidylate Synthase (TS), a cell replication key enzyme involved in folate metabolism. Drugs directed to the folate metabolism are under investigation for the second line therapy of platin resistant OC (R-OC). Pemetrexed (AlimtaTM, PMX), a multitarget drug, is in clinical phase II for the treatment of R-OC. Our research group has recently developed a new class of peptide inhibitors of TS showing cytotoxic activity against OC/R-OC cell lines (in particular octapeptide LR and its analog [D-Gln]LR). The peptides suppress TS activity without causing its overexpression, overcoming the limits of well-known substrate-like inhibitors (e.g., 5-FU). The current challenge is to identify predictive and/or prognostic pool of biomarker; whose expression could be informative of the effect of candidate drugs in preclinical and clinical phases of the discovery process. In this work, the effects at the proteome level of folate pathway-directed drug candidates in the treatment of R-OC have been studied. The aim is to determine molecular events triggered directly or indirectly by folate-metabolism inhibiting agents, in order to better understand the mechanism of action of drugs and, at the same time, to identify a protein signature that could characterize their activity. The study was carried on the above mentioned investigational TS-inhibitors peptides, and PMX. The method, based on the use of mass spectrometry (MS) technique, was developed on OC/R-OC cell lines. Subsequently, the same method was applied on biopsies from R-OC patients to verify the prognostic ability of the selected proteins. In-vitro identified protein panel was tested in biopsies from patients enrolled in a phase 2 clinical trial of PMX in the treatment of R-OC. Experiments were carried out on three couple of biopsies collected before/after PMX treatment from patients who have responded differently to the administration of the drug. The evaluation of the protein signature expression was performed via western blot assay. Results showed that in biopsies before PMX treatment TS, HSP90, TRAP1, GART and DHFR are low in patient with a positive outcome (complete responder), instead, the expression is higher in not responder and partial responder patients. To reach a more informative protein profile of the clinical response to PMX treatment, the protein panel was then implemented by adding other 22 proteins. To allow the simultaneously quantification of 28 proteins in the same sample, an LC-MS/MS mass spectrometer operating in Multiple-Reaction Monitoring (MRM) mode was adopted.
Judita Kinkorova
Charles University, Czech Republic
Title: Algorithm for evaluation of the tumor markers for diagnostics and therapy monitoring in cancer diseases
Biography:
Judita Kinkorová is currently the Manager of International Research Cooperation and Affairs at Faculty Hospital in Pilsen and Medical Faculty Charles University in Pilsen. She is the Manager of European Project, Biobanking and BioMolecular Resources Research Infrastructure (BBMRI) in Pilsen. She studied Biology-Mathematics in Charles University in Prague and holds a PhD from Czech University of Life Sciences in Prague. She worked at Charles University, Faculty of Natural Sciences as a Teacher and Researcher. From 2007, she is a National Contact Point for organizations such as FP7, Priority Health, Horizon 2020 SC 1 Health, and Demographic Change and Wellbeing Academy of Sciences of the Czech Republic. She is a member of European Association for Preventive, Predictive and Personalized Medicine, and International Society of Oncology and BioMarkers and an author of 30 publications.
Abstract:
Background: Tumor markers are currently used in the daily clinical practice particularly for the recurrence detection during the follow-up period of the cancer diseases. It is very rarely used for the diagnostic purpose.
Aim: The aim was to propose algorithms for the optimization of the diagnostic approach of the cancer disease and furthermore for the optimal therapy choice and monitoring based on the serum levels of the tumor markers.
Methods & Materials/Patients: Results of the serum tumor markers from 2000 patients monitored in the Faculty Hospital in Pilsen have been retrospectively evaluated. The following markers have been evaluated: CEA, AFP, mucin, cytokeratin and proliferative tumor markers. Markers assessed during the primary diagnosis were correlated with clinical status prior to any therapy. The following cancers were evaluated: Lung, breast, colorectal and prostate cancer. All data related to the detailed clinical status and the disease course during the follow-up period were available in all the patients.
Results: The optimal diagnostic algorithms were proposed for diagnostics and therapy monitoring of the lung, breast, colorectal and prostate cancer. Selected case reports will demonstrate the use of them. Clinical and economical benefit of these proposed algorithms was evaluated.
Conclusions: Multidisciplinary approach based on these algorithms will enable to use the tumor markers for the routine clinical practice much more effectively.
Youhe Gao
Beijing Normal University, China
Title: Are urinary biomarkers from clinical studies biomarkers of disease or biomarkers of medicine?
Biography:
Youhe Gao is a Professor, Beijing Normal University. He received his MD from Peking Union Medical College, his PhD from University of Connecticut and Postdoctoral training from Beth Israel Deaconess Medical Center Harvard Medical School. He was the professor of Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/ Peking Union Medical College from 2001-2014. His research interests include biomarker discovery in urine proteome, protein interaction and related bioinformatics.
Abstract:
Urine proteome was changed by different diuretics, anticoagulants. Other medications may cause changes in urine too. When clinical biomarker studies were designed, sex, age, disease stages, complications were usually taken into consideration. But different medicines taken by the patients were usually not. If this difference can be balanced off later in the study, it may not be a problem at all. But what if the disease is strongly associated with a particular medication, and healthy control is strongly associated with no medication, the result of the study may reveal the difference of the medicine instead of the disease. And the effects of disease and medicine can never be separated in the study. Normally and ethically, we can never stop medicine for the patients; we can never give the healthy volunteers, medicines they do not need, just for the sake of biomarker study. So the patients-medicine, healthy-no medicine associations exist in all of clinical biomarker studies. It is devastating for the field. Clinical biomarker studies are not cheap. We now have to reevaluate the candidate biomarkers proposed from most of early biomarker studies. We have to rule out the effect of the medicine. This becomes so urgent; it becomes part of the foundation of urinary biomarker study. This is why the author cannot wait to propose the pharmuromics (pharm-uro-mics) which studies the effect of the medicine on urine. The other parts can probably be named physiouromics, pathouromics which are the effect of a physiological or pathological process on urine.
Mala Ranjan
Osmania University, India
Title: Immunochemical detection of glycated lens crystallins and their circulating auto-antibodies in human serum during aging
Biography:
Mala Ranjan has completed her Ph.D at the age of 46 years from Osmania University and M.phil in the School of Biological Sciences, University of Wales, Swansea, U.K. Her thrust area of research is application of advanced glycation end products (AGEs) as clinical Biomarkers. She is focus towards the Diabetic complications associated problems. Presently she is working on major project ( Biological monitoring of advanced glycated end products of protein in Diabetic Neuropathy: Prospective role of antiglycating dietary agents) funded by University Grants Commission, New Delhi, India. She has published 12 papers in reputed journals. She is the currently working as Professor of Biochemistry in the prestigious organization (St.Francis College for Women) of Hyderabad, India.
Abstract:
The aim of this investigation was to use lens specific glycated crystallins as an immunogen to detect (i) human glycated crystallins and (ii) circulating auto-antibodies to glycated crystallins in human serum during aging.Polyclonal antibodies were produced against human total lens proteins (40-80 years) in rabbits. The specificity of the antibodies produced were determined by antibody capture assay, using the purified human lens (HMW+ï¡, HMW+ï¡-glycated, ï¢-, ï¢-glycated ï§- and ï§-glycated) crystallins as antigens. The cross-reactivity of these lens specific antibodies against rat ï¢-, ï¢-glycated, ï§- and ï§-glycated lens crystallins was also analyzed. A non-competitive ELISA methodology was developed for the detection of circulating lens crystallins in human sera, using HMW+ï¡, HMW+ï¡-glycated, ï¢- and ï¢-glycated crystallins from human and rat ï§-, and ï§-glycated crystallins as immobilized antigens. Further, these polyclonal antibodies were able to detect both natural and in vitro glycated crystallins, their IC50 values were: human total lens protein (55 ng), HMW+ ï¡ (16.45 ng), HMW + ï¡-glycated (273 ng), ï¢- (37.82 ng), ï¢-glycated (260 ng), ï§- (105.34 ng) and ï§-glycated (313 ng). The immunochemical analysis of human serum indicated a significant change (p< 0.001) in the levels of circulating ï¢-glycated and ï§-glycated crystallins in the age group of 40-80 years with respect to their control groups. However, there was no consistence significant change in the levels of HMW + ï¡-glycated crystallins in the age group of 40-80 years with their respective controls. Notably, the levels of serum ï§-glycated crystallins was found to be 3 folds higher than that of HMW + ï¡-glycated and ï¢-glycated crystallins in the age group of 70-80 years. Circulating auto-antibodies to HMW + ï¡-, ï¢- and ï§-glycated crystallins were detected in the serum of both apparently normal and cataract patients in the age group of 40-80 years by antibody capture assay. The levels of these auto-antibodies were significantly higher (p< 0.05 & < 0.001) at every time point with their respective controls. Auto-antibodies to ï§-gly crystallin was found to be 2 and 3.2 fold higher as compared to the levels of auto-antibodies to ï¢-gly and HMW+ï¡-gly crystallins, respectively.During the course of aging, leakage of lens crystallins (HMW+ï¡, HMW+ ï¡-glycated, ï¢-, ï¢-glycated ï§- and ï§-glycated) elicit an immune response resulting in the formation of auto-antibodies in cataract patients (40-80 years) as compared to age matched controls. For the very first time these specifically designed polyclonal antibodies to lens specific glycated crystallins were able to detect the early leakage of glycated crystallins. This immunochemical method reported may find an application for the early detection of cataract.
Shashwati Basak
Biocon Bristol-Myers Squibb Research and Development Center, India
Title: Clinical biomarkers in drug development: Quantitative PCR-Based fit-for-purpose assay qualification
Biography:
Shashwati Basak obtained her PhD from Indian Institute of Science, Bangalore, India. She carried out postdoctoral research from The Salk Institute for Biological Sciences, San Diego and Stanford School of Medicine, Palo Alto. Research in these two places was focused on understanding the role of tumor suppressor p53 in Cancer Signaling Pathways. She worked as a Research Scientist in the Veterans Affairs Medical Center, San Francisco, before moving into the current role as a Lead Investigator in Early Clinical and Translational Research, Biocon Bristol-Myers Squibb Research and Development Center, Bangalore. Current research interests involve assay development and qualification for Clinical Biomarkers and its use in clinical sample analysis during drug development.
Abstract:
Biomarkers play a significant role during all phases of drug discovery and development. Clinical biomarker-based studies provide early information on target engagement, help guide rational selection of drug combinations, optimization of dose and schedule, serve as tools for stratifying patients and has the potential to predict clinical outcome. A “fit-for purpose” assay development and validation to meet the clinical requirements plays an important role in biomarker estimation. While a rigorous validation is usually not required for discovery-phase work, as a drug progresses into preclinical and early-phase clinical evaluation, more thorough method validation increasingly becomes valuable. The real-time quantitative polymerase chain reaction (qPCR) technology is accurate, sensitive and fast and has become the method of choice for clinical biomarker detection and quantification. Numerous quality issues may arise throughout the entire workflow influencing the accuracy of the qPCR results and the reliability of the data interpretation and conclusions. Development and use of qPCR technology for robust, accurate and reliable method is required for the emerging “fit-for-purpose” biomarker assay qualification. Key factors influencing assay performance such as sample matrix, sample preparation, experimental precision, reproducibility, sensitivity, specificity, dilution linearity and dynamic range and their impact on the assay outcome will be discussed. Based on these, we will put forth recommendations for consideration and optimization while qualifying a qPCR-assay for analysis of clinical samples. As biomarkers become integrated into drug development and clinical trials, assay qualification becomes important with an increasing emphasis on establishing standardized guidelines for analytical methods.
Manjiri Bakre
OncoStem Diagnostics Pvt. Ltd., India
Title: Development of a novel IHC based test for prediction of risk of recurrence for breast cancer patients
Biography:
Manjiri Bakre is a PhD holder in Cell Biology from the Institute of Science, Bangalore has immense work experience in cell signaling and multiple technologies in the USA, Singapore & India. She led a group iIndiann cancer drug discovery in a biotech company in India, multi-disciplinary research on ‘point-of-care’ diagnostics at Philips Research. She Young Scientist Award from International Union of Biochemists and MolecularBiologists and along with it also received Paper of the Week Award by Journal of Biological Chemists. She has published in peer reviewed journals. She has patents and given seminars in many international and national conferences. She founded Onco Stem Diagnostics Pvt. Ltd. and has been instrumental in every aspect of development of multiple prognostic Oncology tests.
Abstract:
Estimation of risk of distant recurrences in breast cancer patients based on biomarkers beyond current gold standard e.g. ER, PR, Her2, node status and Stage to influence treatment decision is of critical importance. At present there are a few molecular tests viz oncotype dx, mamma-print available for patients with ER+, node and HER2 negative, Stage 1 tumors for prediction of risk of recurrence. However use of these tests in India is very minimal as >70% of patients get diagnosed in node+, Stage 2 disease and due to cost constraints. We decided to develop an Immunohistochemistry based prognostic test to predict ‘risk of recurrence’ for ER+ patients in Stage 1-2. To this aim we carried out a retrospective, non-interventional, anonymized study on 420 left over breast FFPE tumor samples of Stage 1-3 with known outcomes about distant recurrence in first 5 years. We tested about 25 different biomarkers via immunohistochemistry belonging to multiple pathways: Proliferation, resistance, quiescence, adhesion etc. Statistical analysis finalized an algorithm with 6 biomarkers which predicts risk of recurrence as low or high for each patient. Early validation on ~200 cases shows 95% specificity and 54% sensitivity of prediction. Our biomarkers are targetable as they are membrane associated and thus in future high risk patients will be treated with new targeted drugs to reduce risk of recurrence. Thus, we believe we have developed a simple, inexpensive and highly specific test useful to majority of breast cancer patients across the globe with high specificity and sensitivity. We would like to describe the details of development of the test in my talk.
Mônica Valeria Marquezini
University of São Paulo Medical School, Brazil
Title: Sao Paulo workers exposed to air pollution: Evaluation of genotoxicity
Biography:
Mônica Valeria Marquezini is a Scientific Researcher at Pro-Blood Foundation of Blood Center of São Paulo since 1991. She is part of Experimental Air Pollution Laboratory - LPAE staff at the Department of Pathology, Medical School of University of São Paulo, Brazil, since 2010. She is a expert in Cellular and Molecular Biology with cell surface proteins studies and extracellular matrix research. She is a Teacher of Cell and Molecular Biology themes at different graduation careers, Post-graduation advisor, and Coordinator of specialization courses and National and International Projects. She holds a PhD in Science and from the Federal University of São Paulo-School of Medicine (1996) and graduated in Biological Sciences Medical Modality (1983).
Abstract:
The goal of this study was to evaluate the genotoxic effects of air pollution (PM2.5) on exposed individuals, from Sao Paulo City. The study involved 58 male workers of two different exposition groups: 1) 26 taxi drivers (TD) and 17 traffic controllers (TC), high exposure and 2) 15 workers from the Forest Institute (FI), low exposure. Each voluntary used a Personal Environmental Monitoring Sampler to collect PM2.5 particles mass. The sampler were operated continuously at 4-LPM over 24-hours. Particles were collected on a polycarbonate membrane filter (37 mm diameter). The frequency of Micronuclei (MN) in buccal exfoliated cells and peripheral blood lymphocytes were determined. The average the MN in lymphocytes and buccal exfoliated cells was significantly higher on group 1 (PM2.5 p < 0.001). There is a significant correlation between MN measured in xfoliated cells and in lymphocytes (correlation coefficient of 0.80; p<0.001). So the determination of MN buccal exfoliated cells is as efficient as the determination of MN in lymphocytes, a classic biomarker for the assessment of genotoxicity. Our results have shown that individuals exposed to the highest concentrations of PM2.5 have a higher level of micronucleus. However, we found in our study some individuals that even exposed to lower concentrations of pollutants, showed higher levels of micronuclei which allowed us to identify them as individuals more susceptible to PM2.5. Thus, in order to understand this susceptibility, the methylation study of genes promoters related to inflammatory response, will be carried trough in these individuals.
Fanghua Qiu
Guangzhou Hospital of Traditional Chinese Medicine, China
Title: Dermcidin is a novel biomarker of hepatic carcinoma?
Biography:
Fanghua Qiu has completed her PhD from Guangzhou Medicine University. She has published 3 papers in reputed journals. She is currently working as an Associate Professor in Guangzhou Traditional Chinese Medicine Hospital, Guangzhou, China.
Abstract:
We previously identified a group of Nck SH2 domain-binding proteins through a combination of GST-Nck1-SH2 pull down and 2DE in hepatic carcinoma (HCC) tissues, one of the proteins is Dermcidin (DCD). Western blot demonstrated DCD is overexpressed in HCC tissues and aggressive cell. To further identify potential blood-based biomarkers for the early detection of HCC cancer, we compare DCD level from HCC and healthy people, increased DCD was also detected with ELISA methods in 100 serum samples taken from HCC cancer diagnosis (p<0.001). In conclusion, these results present novel evidence that DCD levels may increase in early carcinogenesis, particularly among more aggressive forms of HCC cancer.
Baolin Zhang
Food and Drug Administration (FDA), USA
Title: Integrating predictive biomarkers in the development of targeted cancer therapies
Biography:
Baolin Zhang is a Senior Investigator in the Office of Biotechnology Products of the Center for Drug Evaluation and Research (CDER) at the US Food and Drug Administration (FDA). Dr. Zhang has 14 years of FDA regulatory experience in product quality review (Chemistry, Manufacturing and Control) of therapeutic protein product applications under investigational new drug applications (INDs) and biologic license applications (BLAs). In addition to performing review, inspection, policy, and training activities, Dr. Zhang also leads a research team conducting FDA mission-related research aimed at improving drug safety and efficacy. Dr. Zhang’s scientific expertise lies in the areas of protein biochemistry, cell death and cell growth regulation, cancer drug resistance and biomarkers. He has published over 70 peer-reviewed articles in reputed journals, and presented at numerous scientific and regulatory conferences on the development of therapeutic proteins. Dr. Zhang earned his Ph.D. in chemistry from Peking University in China. He is a recipient of the FDA Scientific Achievement Award, the FDA/CDER Excellence in Mentoring Award, and the FDA/CDER Excellence in Leadership Award.
Abstract:
The regulatory approval of new drugs for marketing requires a demonstration that the drug is safe and effective in the intended patient population that is specified in the drug label.In evaluating potential anti-cancer agents,there is a continued interest in using predictive biomarkers to select patients likely to respond or be resistant to a particular treatment. The ability to identify the subsets of patients with molecularly defined cancers could significantly improve patient outcome. Several clinically validated biomarkers such as HER2 and K-Ras mutation status have become an essential part of the clinical use of the HER2/EGFR targeted therapies. However, the identification of clinically useful predictive biomarkers for solid tumours has proven challenging with many initially promising biomarkers failing to translate into clinically useful applications. The problem mainly lies in the inherent heterogeneity of tumor cells as being found between tumor types, individuals with the same type of tumor, and within one tumor of a patient at any given time. The complex matter also poses challenges to the regulatory review of submissions for biomarker development and drug and biomarker co-development.This presentation will introduce the recent efforts of the US Food and Drug Administration in facilitating the development of biomarkers as well as case studies to highlight the major challenges in the discovery, qualification and regulatory review of predictive cancer biomarkers.
Hem D Shukla
University of Maryland, USA
Title: The Nrf2 mediated antioxidant defense against oxidative stress in BxPC-3 cell lines and targets for therapeutic intervention
Biography:
Hem D. Shukla is Research Scientist in Department of Pharmaceutical Sciences at University of Maryland and adjunct Professor of Genomics in Notredame of Maryland University. He has also worked as Research Faculty in Department of Biology at Johns Hopkins University. Dr. Shukla has worked on proteomic analysis of oxidative stress response in BxPC-3 pancreatic cell lines and identification of biomarkers for pancreatic cancer. He has worked on “The Nrf2 mediated antioxidant defense against oxidative stress in BxPC-3 cell lines and targets for therapeutic intervention”. He has shown elevated level of Nrf2 transcriptional factor in pancreatic cancer cell lines under oxidative stress which is phosphorylated by protein kinase C and affects phosphatidylinositol 3-kinase and MAP kinase pathways. After phosphorylation, Nrf2 translocates to the nucleus, binds AREs and transactivates detoxifying enzymes and antioxidant enzymes, such as glutathione S-transferase, and superoxide dismutase. The IPA analysis has suggested a potential role of Nrf2 under oxidative stress conditions.
Abstract:
Integrins are cell surface glycoproteins that are involved in cell-cell and cell-extracellular matrix (ECM) interactions. These interactions are the basis for a number of diverse effects that include cell migration and anchorage, cell growth and invasion. In the present investigation the proteomic analysis of oxidatively stressed BxPC3 human pancreatic cancer cells have shown the elevated level of both a6b4 integrin, caveolin-1, K-RAS, EGFR, annexin a4 and annexin a11 as compared to HPDE control. We have also identified the presence of a number of gene products involved in integrin signaling pathway and MAPK pathway. The bioinformatic analysis by Protein Center and Ingenuity Pathway Analysis have shown the altered integrin signaling pathway and MAPK pathway in Pancreatic Carcinoma Cell lines. Further, the activation of NRF2 transcriptional factor in BxPC-3 treated cells shows that It may bind to the DNA at the location of the Antioxidant Response Element (ARE) or also called hARE (Human Antioxidant Response Element) which is the master regulator of the total antioxidant system. It seems likely that upon exposure of cells to oxidative stress, Nrf2 is phosphorylated in response to the protein kinase C, phosphatidylinositol 3-kinase and MAP kinase pathways. After phosphorylation, Nrf2 translocates to the nucleus, binds AREs and transactivates detoxifying enzymes and antioxidant enzymes, such as glutathione S-transferase, and superoxide dismutase. The pathway analysis has also demonstrated that at least seven signal transduction cascades were induced by ECM interaction with integrin heterodimers, which may trigger aberrant signaling and lead to pancreatic cancer adenocarcinoma. The data have clearly shown the activation of INT-ILK-PT3K-ILKAP-AKT and Caveolin-GRB2-SOS-cRas-Raf-MEK cascade. These results may have some promise in therapeutic intervention in the treatment of pancreatic cancer adenocarcinoma.
Nalinee Sripaung
Ministry of Public Health, Thailand
Title: The usage of biomarkers in terms of biological exposure indices for health surveillance in Thailand
Biography:
Dr. Nalinee Sripaung has completed her degrees of B.Sc. and M.Sc. from universities in Thailand (Mahidol University and Chulalongkorn University, in order). She has completed her Ph.D. at the age of 34 years from Tokyo Medical and Dental University (TMDU), Japan. She has ever been the editorial board member of the repute. She has published more than 10 papers in reputed journals and government reports. Presently, She is the Director of Rayong Occupational Health and Environmental Development Center. She is serving as an Department of Disease Control of Thailand Committee on Thai Biological Indices (Thai BEIs).
Abstract:
Presently, the health risk assessment becomes important for health active surveillance. The significant types of chemicals residual and/or metabolites in biological samples are selected to be the biomarkers for exposure assessment and susceptibility. The results of analytical laboratory of biomarkers concentration in biological samples are compared with the health surveillance standard values. This method are used as safety measure of exposure assessment to chemicals hazard and cancer susceptibility in occupations and environmental community. Besides, the combined effects of food, drug, and other chemicals of daily life intake accompanied with the mistaken method of collecting, transferring and preserving biolological samples act as the interference factors of laboratory analyticals results. The other important interferences are the risk behaviour and the individaul differences. Thus, Department of Disease Control of Thailand has set the pilot project focused on the concentration of biomarkers, laboratory analyticals method and interfering factors of 26 chemicals (8 heavy metals, 12 volatile organic compounds, 2 pesticides, 2 gases and 2 types of radiation) for formal workers in Thailand Kingdom. This pilot project is aimed to be the tool for setting the policy of standard measure by usage of biomarkers for exposure assessment in the program of health active surveillance. This project will be involved with active surveillance and diminishment of burden diseases caused by chemicals. This tool of safety measure should be applied to prevent and control of Chemicals Diseases in any other risk area.
Maria Paola Costi
University of Modena and Reggio Emilia, Italy
Title: Identification of a protein panelas pharmacodynaimic biomarker of foalte directed targeted drugs in the treatment of ovarian cancer
Biography:
Prof.Maria Paola Costi has completed her Ph.D Medicinal chemistry at the age of 29 years from the University of Modena and Reggio Emilia and postdoctoral studies from University of California San Francisco (UCSF). She is professor of Medicinal Chemistry at the Department of Life Science at Unimore. She has published more than 85 papers in international journals, many patents and serving as an editorial board member of a few journals. Board member of translational science in oncology group of the MITO network and WG leader of drug discovery and development of EUTROC. Coordinator of a number of FP European projects in the area of drug discovery in cancer and parasitic diseases (www.nmtrypi.eu).
Abstract:
Ovarian cancer (OC) is the fifth most common cause of death from cancer in women. The standard first-line treatment (platinum-derivatives and paclitaxel) suffers from rapid resistance development with still unclear mechanisms and poor prognosis. However, the resistance process includes the over-expression of Thymidylate Synthase (TS)1, a cell replication key enzyme involved in folate metabolism. Drugs directed to the folate metabolism are under investigation for the secondline therapy of platin resistant OC (R-OC). Pemetrexed (AlimtaTM, PMX), a multitarget drug, is in clinical phase II for the treatment of R-OC. Ourresearch group has recently developed a new class of peptide inhibitors of TS showing cytotoxic activity against OC/R-OC cell lines (in particular octapeptide LR and its analog [D-Gln4]LR)2,3. The peptides suppress TS activity without causing its overexpression, overcoming the limits of well-known substrate-like inhibitors (e.g., 5-FU). The current challenge is to identify predictive and/or prognostic pool of biomarker, whoseexpression could be informative of the effect of candidate drugs in preclinical and clinical phases of the discovery process. In this work,the effects at the proteome level offolate pathway-directed drug candidates in the treatment of R-OChave been studied. The aim is to determine molecular events triggered directly or indirectly by folate-metabolism inhibiting agents, in order to better understand the mechanism of action of drugs and, at the same time, to identify a protein signature that could characterize their activity. The study was carried on the above mentioned investigationalTS-inhibitors peptides, and PMX. The method, based on the use of mass spectrometry (MS) technique, was developed on OC/R-OC cell lines. Subsequently, the same method was applied on biopsies from R-OC patients to verify the prognostic ability of the selected proteins. In-vitro identified protein panel was tested in biopsies from patients enrolled in a phase 2 clinical trial of PMX in the treatment of R-OC. Experiments were carried out on three couple of biopsies collected before/after PMX treatment from patients whose have responded differently to the administration of the drug. The evaluation of the protein signature expression was performed via western blot assay. Results showed that in biopsies before PMX treatment TS, HSP90, TRAP1, GART and DHFR are low in patient with a positive outcome (complete responder), instead, the expression is higher in not responder and partial responder patients. To reach a more informative protein profile of the clinical response to PMX treatment, the protein panel was then implemented by adding other 22 proteins. To allow the simultaneously quantification of 28 proteins in the same sample, anLC-MS/MS mass spectrometer operating in Multiple-Reaction Monitoring (MRM) mode, was adopted. 1.Cardinale D. et al. Proc. Natl. Acad. Sci. U.S.A. 2011 , 2.F.Genovese J Proteome Res. 2014,13, 5250..3. Zhang D. et al. “Establishment of pemetrexed-resistant non-small cell lung cancer cell lines.” Cancer Lett. 2011, 309, 228. This work wassupported b.y AIRC-DROC project N.10740
- Workshop - Preserving urinary proteins and nucleic acids on membrane
Session Introduction
Youhe Gao
Beijing Normal University, China
Title: Saving urinary proteins and nucleic acids on a piece of membrane simply and economically
Biography:
Youhe Gao is a Professor, Beijing Normal University. He received his MD from Peking Union Medical College, his PhD from University of Connecticut and Postdoctoral training from Beth Israel Deaconess Medical Center Harvard Medical School. He was the professor of Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/ Peking Union Medical College from 2001-2014. His research interests include biomarker discovery in urine proteome, protein interaction and related bioinformatics.
Abstract:
Urine has become more and more recognized as important biomarker source in biomarker discovery. By nature, urine accumulates all kinds of changes and changes are the most basic property of biomarker. In this sense, theoretically urine can be even better biomarker source than blood. There may be a urine biomarker era ahead of us. Even though metabolites were analyzed extensively as potential biomarker in urine, proteins and microRNAs may possess more specificity of the diseases. Saving proteins and nucleic acids were challenging because they not only took huge space but also gradually degraded even frozen in -80 degree. Here we present a simple and economical method that make storing large number of urinary proteins and nucleic acids clinical samples possible. Urine samples were filtered through the membrane, and urinary proteins were adsorbed onto the membrane. The membrane used can have high affinity to proteins or nucleic acids. Then the membrane was dried and stored in a vacuum bag, which kept the protein and microRNA faithfully preserved. The membrane may even permit storage at room temperature for weeks. This simple and inexpensive method requires minimal sample handling, uses no organic solvents, and is environmentally friendly. With this method, large number of samples will be available to biomarker discovery and validation. Biomarker research can be significantly more efficient. There has not been any new form of tissue preservation for years. This membrane, which we call urimem, may be kept with medical record of all consenting people in the future.