Day 1 :
Keynote Forum
Patrizia Zavattari
Cagliari University, Italy
Keynote: CpG Islands methylation alterations in cancer: Understanding the functional role of these security locks and their use as early tumour biomarkers
Time : 09:30-10:10
Biography:
Patrizia Zavattari, received the MSc. degree in Biological Sciences at Pisa University (Italy) and the PhD at Cagliari University (Italy). She is Associate Professor of Experimental Biology and Genetics at Cagliari University, teaching in the Faculties of Biology, Pharmacy and Medicine. She is the Head of a Molecular Biology, Genomics and Epigenomics Laboratory. Her research interests are mainly focused on molecular oncology and complex traits. She worked both abroad (Paris, Oxford, Cambridge, Philadelphia) and in Italy (Milan, Novara). She is co-author of about forty publications. Since 2011 she is Review Editor of Frontiers and she reviews for various scientific journals.
Abstract:
DNA methylation is an epigenetic modification consisting in the addition of a methyl group to a cytosine in a CpG context. In normal mammalian cells, CpG islands, mostly concentrated at promoter regions, are protected from DNA methylation, while intergenic and repetitive regions are normally hypermethylated. In cancer cells, a massive change in the global methylation pattern occurs. Intergenic and repetitive regions of the genome become hypomethylated leading to the reactivation of transposable elements and genomic instability. In contrast, a focal hypermethylation of CpG islands at promoter regions occurs and it is normally associated to gene expression downregulation. Thus, aberrant DNA methylation is one of the most striking features of cancer cells and several studies have demonstrated that cancer-specific methylation patterns exist. For this reason, DNA methylation represents an extremely useful biomarker for several applications, including cancer risk definition, prediction of clinical outcomes, treatment response and cancer relapse. From a functional point of view, the association between DNA methylation and gene expression, although notoriously recognized, is not yet fully known. In our laboratory we identified early DNA methylation alterations in colorectal cancer, localization-specific changes in low-grade gliomas, alterations that predict the risk of developing chronic lymphocytic leukemias years before diagnosis and correlating with the aggressiveness of the disease. A feature shared by almost all these alterations is that hypermethylation targets CpG islands associated with genes poorly expressed in the tissue where cancer occurs. We have undertaken an expression study for each gene associated with these alterations, showing further dowregulation.
Keynote Forum
Henry M. Sobell
University of Rochester, USA
Keynote: The centers of premeltons signal the beginning and ends of genes
Time : 10:10-10:40
Biography:
Henry M. Sobell completed his studies at Brooklyn Technical High School (1948-1952), Columbia College (1952-1956), and the University of Virginia School of Medicine (1956-1960). Instead of practicing clinical medicine, he then went to the Massachusetts Institute of Technology (MIT) to join Professor Alexander Rich in the Department of Biology (1960-1965), where, as a Helen Hay Whitney Postdoctoral Fellow, he learned the technique of single crystal X-ray analysis. He then joined the Chemistry Department at the University of Rochester, having been subsequently jointly appointed to both the Chemistry and Molecular Biophysics departments (the latter at the University of Rochester School of Medicine and Dentistry), becoming a full tenured Professor in both departments (1965-1993). He is now retired and living in the Adirondacks in New York, USA.
Abstract:
Premeltons are examples of emergent structures (i.e., structural solitons) that arise spontaneously in DNA due to the presence of nonlinear excitations in its structure. They are of two kinds: B-B (or A-A) premeltons form at specific DNA-regions to nucleate site-specific DNA melting. These are stationary and, being globally nontopological, undergo breather motions that allow drugs and dyes to intercalate into DNA. B-A (or A-B) premeltons, on the other hand, are mobile, and being globally topological, act as phase-boundaries transforming B- into A- DNA during the structural phase-transition. They are not expected to undergo breather-motions. A key feature of both types of premeltons is the presence of an intermediate structural-form in their central regions (proposed as being a transition-state intermediate in DNA-melting and in the B- to A- transition), which differs from either A- or B- DNA. Called beta-DNA, this is both metastable and hyperflexible – and contains an alternating sugar-puckering pattern along the polymer-backbone combined with the partial-unstacking (in its lower energy-forms) of every other base-pair. Beta-DNA is connected to either B- or to A- DNA on either side by boundaries possessing a gradation of nonlinear structural-change, these being called the kink and the antikink regions. The presence of premeltons in DNA leads to a unifying theory to understand much of DNA physical-chemistry and molecular-biology. In particular, premeltons are predicted to define the 5’ and 3’ ends of genes in naked-DNA and DNA in active-chromatin, this having important implications for understanding physical aspects of the initiation, elongation and termination of RNA-synthesis during transcription. For these and other reasons, the model will be of broader interest to the general audience working in these areas. The model explains a wide variety of data, and carries within it a number of experimental predictions – all readily testable – as will be described in my talk.
Keynote Forum
Jane Xiao
Axcel BioPartners, USA
Keynote: Biosimilar assessment: Quantitation of antibody variants and pharmacokinetic studies using peptide mapping LC-MS analysis
Time : 11:10-11:50
Biography:
Jane Xiao has obtained her PhD in Analytical Chemistry at University of Wales Swansea, UK. She is a Co-Founder and Senior Director in Biologics Characterization at Axcel BioPartners. Prior to that, she served as Director at Oncobiologics, Head of a proteomics lab at Johnson & Johnson and Senior Scientist at Merck. For the last 15 years she has worked for the pharmaceutical industry leading analytical method development for protein characterization, biomarker development in early phase clinical trials, biosimilar development for similarity assessment and comparability assessment.
Abstract:
Introduction: Peptide mapping has been widely accepted as an identity test for biotherapeutics in the QC lab. Recently, the peptide mapping based-multi attribute method (MAM) by ultrahigh performance liquid chromatography (UPLC) coupled to high resolution mass spectrometry (HRMS) have been employed for the confirmation of sequence, and the identification and quantitation of sequence variants and modifications for biosimilar development directly and simultaneously. In addition the peptide mapping technique combining multiple reaction monitoring (MRM) HRMS is gaining momentum as an alternative tool in the pharmacokinetic studies of large molecules including biosimilars. However, one common challenge for application of the technique is sample preparation due to specific structural complex and/or biological endogenous interference. The purpose of this presentation is to describe the approaches of optimization of sample preparation in peptide mapping for quantitation of antibody variants and for pharmacokinetic studies.
Case study 1: Optimization of oxidation detection for a mAb prone to be oxidized
Methods: UPLC-HRMS, tryptic digestion, stable isotope label, PinPoint software. A mAb was incubated with H218O2 overnight at room temperature first following by regular sample preparation for peptide mapping.
Results: The artificial oxidation formation during sample preparation and storage in autosampler was minimized. Intermediate precision was improved significant.
Conclusion: Platform peptide map MAM LC-MS method works for mAbs. Some mAbs are susceptible to oxidation formation at methionine residues. For accurate oxidation measurement, the use of isotope labelled peptide map LC-MS is essential.
Case study 2: Improvement of specificity in free and total PK assays
Methods: UPLC-MS/MS, tryptic digestion, complementarity-determining region, signature peptides, PinPoint software, immuno affinity.
Results: Specificity of the pharmacokinetic assay was obtained by using several levels of separation.
Conclusion: Reagent free LC-MS/MS approach allows suitable quantitation of total mAb for periclinical studies. Capture approach is especially useful for quantitation of free and partial free mAb in human serum.
Recent Publications
- Marini J C, et al. (2014) White paper: systematic verification of bioanalytical similarity between a biosimilar and a reference biotherapeutics. AAPS Journal 16(6):1149-1158.
- Islam R (2014) Bioanalytical challenges of biosimilars. Bioanalysis 6(3):349-356.
- Iwamoto N, et al. (2016) Validated LC/MS bioanalysis of Rituximab CDR peptides using nanosurface and molecular-orientation limited (nSMOL) proteolysis. Biol. Pharm. Bull. 39(7):1187-1194.
- Baru R (2017) Applications of targeted proteomics and mass spectrometry in Trastuzumab pharmacokinetics assessments. J Syst Biol Proteome Res 1(1):7-9.
- Vialaret J, et al. (2018) What sample preparation should be chosen for targeted MS monoclonal antibody quantification in human serum? Bioanalysis 10(10):723-735.
- Genomics | Molecular Biology | Next Generation Sequencing | Structural Biology | Cancer Biology
Location: Sylt 1-2
Chair
Patrizia Zavattari
Cagliari University, Italy
Session Introduction
Yanyan Zhao
Shengjing Hospital of China Medical University, China
Title: Nkx2.5 regulates Nsd1 in response to oxidative stress in cardiac muscle cells
Biography:
Yanyan Zhao is director of Department of Clinical Genetics in Shengjing Hospital of China Medical University. She was completed MD in 1984 and PhD in 1996 at China Medical University. She did postdoctoral studies in Department of Cell Biology of State University of New York. Over the past decades she has focused on molecular genetics of cardiovascular disease and prenatal diagnosis of Genetic diseased, and published more than 50 papers.
Abstract:
Nuclear receptor-binding SET domain-containing protein 1 (Nsd1) is a histone lysine methyltransferase, and the effect of NSD1 in response to oxidative stress in cardiac muscle cells remains poorly understood. Current study demonstrated that the protein and mRNA levels of NSD1 was reduced by H2O2 in H9C2 cells. We analyzed the promoter of NSD1 gene, predicted an NK2 transcription factor related locus 5 (Nkx2.5) binding element (NKE) at position -412/-406 in the NSD1 promoter, and confirmed it by an electrophoresis mobility shift assay and a chromatin immunoprecipitation assay. Luciferase activity analysis indicated H2O2 decreased the p646-luc promoter activity, and Nkx2.5-specific siRNA weakened H2O2 inhibition on the p646-luc promoter. However, H2O2 did not affected mutant NKE promoter p646-luc-mut compared with wild p646-luc promoter. Furthermore, overexpression and depletion of Nkx2.5 led to the increase and decrease of NSD1 protein and mRNA levels in H9C2 cells. In addition, Nkx2.5 was downregulated by H2O2. Therefore we hypothesized that oxidative stress reduced NSD1 expression through suppression of Nkx2.5, and we going to focus on the alteration of histone methylation of NSD in response oxidative stress in future study.
Biography:
Maria Krzakowa works as Professor Senior at Adam Mickiewicz University in Poznań, Poland. Some years ago she did one- year training as Post Doc at the University of California Davis. She learned there enzymes detection by horizontal gel electrophoresis. At first, her scientific activity was concerned about genetic variation of natural populations of Bryophytes (different species of Hepatics and Mosses) and later on forest trees: Scots pine (Pinus sylvestris), beech (Fagus sylvatica) and ash-tree (Fraxinus excelsior). In the meantime she developed investigations on grasses, for example Apera spica-venti and Alopecurus myosuroides. Her main achievement was the first description of dimeric peroxidise in reed (Phragmites australis). It was some kind of discovery, as dimeric peroxidise was known earlier only from the rice (Oryza sativa). Now, she is working on European collection of Italian and Westerwolds ryegrasses (Lolium spp.) considering biochemical and molecular polymorphism of these important forage grasses.
Abstract:
Lolium westerwoldicum is an important forage grass originated from the Netherlands. It has been selected by farmers of Westerwolde from local rapid growing Italian ryegrass types within the populations of local variety. Westerwolths ryegrass is the shortest-life form of all annual ryegrasses. Genetic examination of this interesting species is very limited. One-month seedlings of L. westerwoldicum cultivars originated from different European countries: France (Barcomet, Gipsyl, Suxyl and Melword), Germany (Duccado, Grasser and Nerissa), Belgium (Mendoza), Netherlands (Bartimo and Gepetto) and Poland (Mowester) were treated as populations and investigated in terms of the two dimeric enzyme systems: phosphoglucose isomerase (PGI; EC 5.3.1.9) and diaforase (DIA; EC 1.6.4.3). From each cultivar, minimum 30 plants were examined in 11% starch gel (SIGMA), prepared on the basis of lithium-boric buffer system. Electrophoretically detected phenotypes were created by four alleles of one locus in PGI and two alleles in one locus in DIA were used to calculate the genetic parameters like heterozygosities (He and Ho)and polymorphic index (Pg). When all populations were compared according to PGI allozymes frequency, all populations show high polymorphism from Pg=0.53 for Mowester till Pg=0.81 for Gepetto. Polymorphic indices calculated for DIA are fluctuating from PG=050 for Bartimo till Pg=065 for Ducado.
All populations were compared on the basis of alleles frequency using Unweighted Pair Groups Method with Arithmetic Mean (UPGMA) and illustrated by dendrograms constructed for both: PGI and DIA enzyme systems. Dendrogram constructed for PGI allozymes shows two groups of populations: one composed of two cultivars: Mendosa (Belgium) and Melword (France) and the second group composed of 8 varieties. Population Bartimo from Holland is visibly separated. Dendrogram constructed for DIA on the basis of alleles frequency shows three groups: two composed of three populations and one of five populations. The first is formed by Bartimo from Holland as well as Suxyl and Gipsyl from France). The second three-species group is composed of Meword (France), Mowster (Poland) and Ducado (Germany). It is visible that connections between populations do not indicate regional similarity.
Wen Qiu
Nanjing Medical University, China
Title: IL-17 induces the proliferation and migration of glioma cells through the activation of PI3K/Akt1/NF-κB-p65
Biography:
Wen Qiu is currently an associate professor at Department of Immunology of Nanjing Medical University. He is exploring roles and mechanisms of inflammatory factors in glioma cell proliferation and migration. These include signal transduction, microRNA regulation, transcriptional factor regulation. He is also exploring the effects of post-transcriptional regulation such as ubiquitination and acetylation on the activation of signaling molecules and transcription factors
Abstract:
Interleukin 17 (IL‑17), as a pro-inflammatory cytokine, is up-regulated in the sera and tumor tissues of glioma patients; however the effects of IL-17 on glioma proliferation and migration remain unclear. In this study, the roles of IL-17 in the proliferation and migration of glioma cells and their potential mechanisms were determined. The results showed that IL-17 could not only enhance the proliferation and migration of cultured glioma cells (in vitro), but also promote the tumor formation of glioma cells in BALB/c nude mice (in vivo). Mechanical exploration revealed that IL-17 stimulation could increase the phosphorylation levels of Akt1 and NF-κB-p65 in glioma cells, and knockdown or inhibition of PI3K, Akt1 and NF-κB-p65 could also reduce the IL-17-induced proliferation and migration of the glioma cells. Moreover, PI3K/Akt1 was the upstream regulator of NF-κB-p65 activation in IL-17-incubated glioma cells. Furthermore, the inhibition of PI3K, Akt1 and NF-κB-p65 markedly suppressed the tumor formation of glioma cells induced by IL-17. Together, these data indicate that IL-17 can promote the proliferation and migration of glioma cells via PI3K/Akt1/NF-κB-p65 activation, and these findings might provide a new insight into glioma pathogenesis.
Yulia Einav
Holon Institute of Technology, Israel
Title: The novel suppression of function mutation in the cardiac RyR2 channel presents an allosteric dose-dependent effect mecchanism
Biography:
Yulia Einav has completed her PhD in Tel Aviv University. She works at Holon Institute of Technology (Israel). Currently she has published about 25 papers and book chapters in reputed journals and textbooks. In addition, she serves as Dean of Students of her institution.
Abstract:
The cardiac ryanodine receptor RyR2 is a calcium release channel present in the sarcoplasmic reticulum, which plays a major role in regulation of Ca2+ homeostasis in the heart. We discovered a novel RyR2 missense mutation, G3118R, with recessive co-segregation, in a large family presenting with cardiac arrest and ventricular fibrillation phenotype. Interestingly, and unlike the other clinically relevant mutations in this gene, which are dominantly inherited, the clinically affected individuals in this family are homozygous for the mutation, whereas the heterozygous family members are almost unaffected. Moreover, the mechanism by which this mutation determines this unusual clinical phenotype is unknown. The aims of this study were to perform functional and structural analysis of G3118R RyR2 in order to investigate the mechanism of this mutation's pathogenicity. We inserted the mutation into HEK293 cells and performed functional studies, including measurements of changes in intracellular Ca2+ levels and the monitoring of the endoplasmic reticulum Ca2+ dynamics. G3118R causes suppression of function of the channel, which is by far less described mechanism caused by most RyR2 mutations. Next, we used a computational model to study the changes in the stability and the flexibility of RyR2 protein. We found that G3118R, a peripheral mutation located in a region, whose function is unknown, causes a major allosteric effect on the channel pore region of two out of the four RyR2 monomers, and that this effect accumulates in a dose-dependent manner.
Mariom
The University of Tokyo, Japan
Title: Expression profiles of biomineralization-related genes at different stages of pearl formation in Pinctada fucata
Biography:
Mariom is a PhD student at ‘The University of Tokyo’ and an assistant professor at ‘Bangladesh Agricultural University’. Her research interests lie in the area of molecular biology and biotechnology. Her work now focuses on molecular mechanisms underlying the formation of pearl sac and pearl because increased understanding towards producing a high quality lustrous pearl is required to further improve the effectiveness of pearl culture technique. She earned a bachelor of science in ‘Fisheries’ and a master of science in ‘Fisheries Biology and Genetics’ from ‘Bangladesh Agricultural University’.
Abstract:
In pearl farming, a piece of mantle tissues from a donor oyster is implanted into a host oyster along with an inorganic nucleus. The outer epithelial cells of the graft proliferate and form a pearl sac which secretes various shell matrix proteins (SMPs) to form a pearl surrounding the nucleus by a complex physiological process which has not been well-understood yet. Using an RNA-seq approach, here we aimed to unravel the expression of the key genes involved in pearl biomineralization at different stages. During grafting experiments for three months, we collected nine samples (donor mantle epithelial cells, donor mantle pallium, donor mantle pallium on grafting, and mantle pallium each from the host at 24 hr, 48 hr, 1 week, 2 week, 1 month, and 3 month post grafting). The pearl sac was developed by two weeks after transplantation. For the first time, we identified some stem cell marker genes differentially expressed during pearl sac formation. PCA analysis on 192 biomineralization-related genes showed clearly different expression profiles between before and after 1 week after grafting. The expression profiles of these genes demonstrated that prismatic layer forming SMPs were first up-regulated and then gradually down-regulated, indicating their involvement in the onset of pearl mineralization. Most of the nacreous layer forming SMPs were up-regulated after the formation of pearl sac with the highest expression at 1 month, suggesting the completion of nacreous layer formation. These findings provide valuable information in understanding the molecular mechanism of pearl formation in P. fucata.
- Biological Medicine | Biosimilars Pipeline
Chair
Jane Xiao
Axcel BioPartners, USA
Session Introduction
Anusha Patel
Kettering General Hospital, UK
Title: Reinvestement opportunities to enhance patient care
Biography:
Anusha Patel has completed her Graduation in 2008; Masters of Pharmacy at Kings College London and Postgraduate training in various hospitals around London. Following obtaining a Diploma in Medicines Management from the London School of Pharmacy, she has specialised in Trauma and Orthopaedics Pharmacy at UHL for a number of years. She then took on a homecare role at Kettering which has evolved into the current position. She Chairs the Regional Homecare Group for the East Midlands, and is a member of the National Homecare Medicines Committee (NHMC). She also works with the East Midlands Procurement Collaborative to assist in tenders for homecare contracts.
Abstract:
Homecare is a rapidly growing market with a value circa £1.5bn. Approximately 250,000 patients are receiving medicines via the homecare route. Biosimilars offer an exciting future: Opportunity to include more value added services, improved medicine administration (e.g. injectable medicines) through nurse support and patient education, improved patient compliance through offering help lines and checking product utilisation, improved product quality by enhanced supply chain, cold chain management, stock rotation, etc, improved track and trace systems, enhanced distribution models. Switching process at KGH-Currently a total of 100 patients on therapy via the homecare route. Rheumatology switched first, then dermatology, new patients commenced Benepali 21st march, existing patients to switch to biosimilar as a phased process from 1st April. Actions to take: discuss possibility of using Benepali with rheumatology team at MDT meeting, identify appropriate patients, notify patients beforehand-leaflet, clinic consultation to offer switch to existing patients, consultant/nurse to notify secretary of switch and to prepare a prescription (No registration forms needed), homecare pharmacist assistance in producing prescriptions, prescription processed by pharmacy and sent to homecare provider, homecare provider receive prescription, update patient account of switching to Benepali, homecare provider to contact patient, perform a stock check and supply “Benepali Patient Kit” on first delivery. Negotiations with the CCG-Gain share with CCG Gain share further between pharmacy and rheumatology department. Savings are to be used to recruit another nurse for department to release consultant nurse led telephone consultations for those patients who need them. Barriers to overcome were mapped out beforehand and a savings tracker was monitored to manage finances. Homecare providers are in a prime position to assist with switching as we can see in the preaparations for adalimuumab biolsimilar and trusts should work with them to ensure seamless transfer.
Biography:
Wanjie Sun is a Senior Mathematical Statistician at CDER of FDA. Prior to joining FDA, she worked at GWU as a Lead Research Scientist and a PI/co-PI for over ten years, and she also worked in pharmaceutical industry for a couple of years. She has obtained her PhD in Biostatistics at GWU. She has over 40 publications in statistical, medical, and pharmacy journals. Her research interests are in equivalence, non-inferiority, causal inference, study design, and missing data.
Abstract:
In equivalence, biosimilar, and non-inferiority studies, intent-to-treat (ITT) analysis tends to make the two treatments look similar, thereby is generally anti-conservative. The Per Protocol (PP) analysis based on completers and compliers is more able to reflect treatment differences and is usually preferred in equivalence assessment. In clinical endpoint bioequivalence studies, the current primary analysis for assessing equivalence between a generic and an innovator product is usually based on the observed per-protocol (PP) population. However, missing data and non-compliance are post-randomization intercurrent events and may introduce selection bias. Therefore, PP analysis is generally not causal. The FDA Missing Data Working Group (2016) recommended using “causal estimands of primary interest.” In this paper, we propose a principal stratification causal framework and co-primary causal estimands to test equivalence, which was one of the approaches recommended by the recently published ICH E9 (R1) addendum to address intercurrent events. We identify three conditions under which the current PP estimator is unbiased for one of the proposed co-primary causal estimands-the “Survivor Average Causal Effect” (SACE) estimand. Simulation shows that when these three conditions are not met, the PP estimator is biased, and may inflate Type 1 error and/or change power. We also propose a tipping point sensitivity analysis to evaluate the robustness of the current PP estimator in testing equivalence when the sensitivity parameters deviate from the three identified conditions, but stay within a clinically meaningful range. Our work is the first causal equivalence assessment in equivalence studies with intercurrent events and can be applied to comparative biosimilar clinical trials and non-inferiority trials.
Recent Publications
- Lou Y, Jones M P and Sun W (2018) Estimation of causal effects in clinical endpoint bioequivalence studies in the presence of intercurrent events: noncompliance and missing data. Journal of biopharmaceutical statistics 1-23.
- LaVange L M and Permutt T (2016) A regulatory perspective on missing data in the aftermath of the NRC report. Statistics in Medicine 35(17):2853-2864.
- Permutt T (2016) A taxonomy of estimands for regulatory clinical trials with discontinuations. Statistics in Medicine 35(17):2865-2875.
- Permutt T (2016) Sensitivity analysis for missing data in regulatory submissions. Statistics in Medicine 35(17):2876-2879.
- Sun W, Zhou L, Grosser S and Kim C (2016) A meta-analysis of missing data and non-compliance data in clinical endpoint bioequivalence studies. Statistics in Biopharmaceutical Research 8(3):334-344.