Exosomes & Liquid Biopsies Europe 2018 Agenda

Conference Registration, Materials Pick-Up, Morning Coffee and Breakfast Pastries

Morning Break and Networking in the Exhibit Hall

Networking Lunch in the Exhibit Hall -- Meet the Exhibitors and View Posters

mRNA Profiles of Circulating Tumor Cells and Extracellular Vesicles in Matched Samples From Metastatic Breast Cancer Patients
Corinna Keup, Researcher, Universitätsklinikum Essen, Klinik für Frauenheilkunde und Geburtshilfe

The analysis of RNA enclosed in circulating tumor cells (CTCs) or extracellular vesicles (EVs) may be sensitive enough to detect disease progression earlier than contemporary visual staging methods. A prediction of the ideal therapy strategy via characterization of CTCs or EVs would be even more beneficial. We, for the first time, compared RNA profiles of CTCs and EVs in metastatic breast cancer (MBC) patients to get insights about their feasibility for therapy stratification.

CTCs were isolated by positive immunomagnetic selection targeting EpCAM, EGFR and HER2 with the AdnaTest EMT-2/StemCell SelectTM, QIAGEN GmbH and EVs were isolated by affinity-based binding to a spin column with the exoRNeasy Serum/Plasma protocol, QIAGEN GmbH. mRNA was purified, reverse transcribed and pre-amplified cDNA was analysed for 18 genes by multimarker qPCR assays (AdnaTest TNBC Panel prototype, QIAGEN GmbH).

Liquid Biopsy: Screening for Single Nucleotide Variants and Indels From Cell-Free Tumoral DNA
Yves Rozenholc, Professor, University of Paris Descartes, France

Analyzing circulating free DNA from blood sample of patient known to have a cancer may help to build a precise picture of the tumoral mutations driving the carcinogenesis process, which can be used as input of a personalized treatment and offers the opportunity to realize a precise follow-up of patient under treatment. However, detecting single-nucleotide variations and insertions/deletions in circulating tumor DNA is challenging because of their low allele frequency. Based on quantification of error rate at each base position [position error rate (PER)], I will present a screening tool called PlasmaMutationDetector, which allows to identify mutations as the result of a statistical test comparing minor-allele frequency to measured PER at each base position. The method is based on a non-optimized commercial kit and allows actually the screening of about 20000 mutations (SNV and INDEL) potentially occurring along several oncogenes. It shows excellent sensibility and specificity and allows detection when minimal mutated allele frequencies is as low as 0.3% for single-nucleotide variations and 0.1% for insertions/deletions. During this presentation, to help adaptation of this method to other oncogenes and/or other NGS techniques, I will present the main technical steps, which allow to deal with multiplicity, hotspot and PER estimation.

Afternoon Coffee Break and Networking in the Exhibit Hall

Efficient, Automated Purification of ccfDNA From Plasma
Douglas Horejsh, Senior Research Scientist, Promega Corporation

While circulating cell-free DNA (ccfDNA) is one of the most promising sources of DNA-based information for oncology research available today, scientists continue to look for better ways to actively target the precious small fragment DNA necessary for liquid biopsy applications.

The use of ccfDNA as a sample type is not without its challenges. The low concentration and highly fragmented nature of ccfDNA, coupled with the low frequency of biomarkers of interest, present many challenges to the adoption of ccfDNA monitoring. Variations inherent to the sample type, as well as sample handling, can affect the amount of contaminating high molecular weight gDNA. Presence of high levels of contaminating gDNA may result in difficulty detecting low-level mutations present in the minor component ccfDNA

We have developed novel nucleic acid purification chemistries that improve upon current manual and automated methods for the purification of DNA from plasma and demonstrate their use in downstream assays. In addition, we seek to provide tools to assess DNA quantity to understand the composition of the nucleic acid obtained.  In those cases where samples have been contaminated by gDNA, we have created applications where we can enrich for the ccfDNA component of a sample, thereby eliminating larger material and effectively decreasing background without affecting levels of the 170bp fragment.

Diagnostic Leukapheresis (DLA) to Improve CTC-Based Liquid Biopsies
Nikolas Stoecklein, Professor, Experimental Surgical Oncology, Heinrich Heine University Düsseldorf, Germany

The direct access to systemically spread cancer cells is the true potential of CTC-based liquid biopsies. However, the major obstacle to implement CTC-based liquid biopsies into clinical routine applications is the extreme low concentration of CTCs and the minimal amount of investigated blood in standard CTC-tests. To tackle this problem, we introduced Diagnostic Leukapheresis (DLA), which resulted in an increase in CTC detection frequency and an escalation of the median CTC numbers. We hope that DLA, or similar approaches derived thereof, will advance the clinical utility of CTC-based liquid biopsies.

Networking Reception with Beer and Wine.

Close of Day 1 of the Conference.

Morning Coffee, Breakfast Pastries and Networking

Exploiting Extracellular Nucleic Acids From Body Fluids For Precision Medicine Purposes
Jo Vandesompele, Professor, Ghent University and CSO, Biogazelle, Belgium

In contrast to general belief, a substantial part of the human transcriptome is abundantly present in the blood and other body fluids as extracellular mRNAs, lncRNAs and microRNAs, ready to exploited. I will discuss various workflows for RNA sequencing of body fluid derived RNA, including probe-based target capture as a sensitive RNA sequencing workflow to study thousands of mRNA and lncRNA genes in cell-free RNA from patients’ plasma and urine. Apart from RNA abundance profiling, this type of data can also be used to detect structural RNA variants, such as somatic mutations, fusion genes and RNA editing events, all known to play an important role in disease, including cancer. The resulting RNA profiles can be deconvoluted to enumerate the cells, tissues and organs that contribute to the extracellular RNA. Human body fluid RNA sequencing enables liquid biopsy guided precision oncology, such as therapy stratification, treatment response monitoring and early detection of relapse. I will also discuss the pre-analytical jungle of RNA targeted liquid biopsies and need for standardization, as part of the ongoing exRNAQC study.

Accuracy, Reproducibility and Consequences of Bias in Quantitative Small RNA-seq For Circulating Cell-free RNA Profiling
Maria Giraldez, Researcher, University of Michigan, United States of America

Small RNA-seq is increasingly being used for profiling of small RNAs, including cell-free RNA such as microRNAs present in plasma and other biofluids. In practice, small RNAseq can involve many different methods for library preparation, with distinct protocols and technical variations that could cause substantial differences in results, yet these have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters with randomized end-nucleotides for ligation. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide a foundation for reproducible, cross-laboratory small RNAseq studies of cell-free, circulating RNA in a variety of clinical contexts.

MicroRNA Based Liquid Biopsy: New Extraction, Preservation and Detection Technologies to Generate miRNA Signatures for Cancer Screening
Pablo Castan, Research Director, Arcis Biotechnology

The development of minimally-invasive tests, such as liquid biopsy, for early cancer detection in its preclinical phase is crucial to improve the outcome of oncologic patients. MicroRNAs (miRNAs) are small, non-coding and tissue-specific-RNAs, which have been reported as extracellular messengers derived from the cross-talk between tumours and their surrounding microenvironment. The development of easy (de-skilled and cost-effective) extraction and preservation solutions teamed with fast and reliable detection could thus represent the ideal format for early detection of defined types of cancer. In this presentation, a new methodological workflow for the prospective validation of a circulating miRNA test using custom made microfluidic cards and microsensing technology in plasma is discussed and compared to state of the art solutions currently used for liquid biopsy.

Interpreting Extracellular Vesicles as Snapshots of Cells
Esther Nolte-‘t Hoen, Assistant Professor, Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Netherlands

Our research focuses on the role of EV in communication between cells at steady state and during immune activation or microbial infection. Using high-resolution flow cytometric analysis of single EV, we previously showed that EV populations are highly heterogeneous. Our recent work on EV released by immune cells and virus-infected cells indicates that both spatial and temporal elements drive EV heterogeneity. This has implications for interpreting EV as snapshots of cells. Recognition and in-depth analysis of EV heterogeneity can facilitate the search for refined EV-based biomarkers. Moreover it can help to dissect differences in the formation and function of distinct EV populations. Examples will be shown on how environmental triggers can lead to changes in the type of released EV and their small non-coding RNA content. Additionally, it will be explained how advanced flow cytometry-based technologies can be applied to study EV release dynamics and to isolate EV subpopulations for downstream functional analysis.

How Clinical Biobanks Support Precision Medicine: From Standardized Preprocessing of Liquid Biopsies to Treatment Guidance
Jens Habermann, Head, Section of Translational Surgical Oncology and Biobanking & Scientific Director, Surgical Center for Translational Oncology, Department of Surgery & Institute of Human Genetics, University of Lübeck and University Hospital Schleswig-Holstein, Campus Lübeck, Germany

CTCs and Exosomes harbor an enormous potential for precision medicine. However, clinical implementation requires standardization. This is in contrast to current praxis, which is still hampered by major inter-study heterogeneity. This talk will address (i) current challenges and solutions for standardized CTC / Exosome assessment and (ii) how clinical biobanks can support CTC / Exosome research for precision medicine, e.g. in colorectal cancer.

Boosting Clinical Validation of ctDNA Diagnostics
Gerrit Meijer, Professor of Oncologic Pathology, Netherlands Cancer Institute, Netherlands

Networking Lunch in the Exhibit Hall and Poster Viewing

Molecular and Functional Characterization of CTCs in Non-Small Cell Lung Cancer (NSCLC)
Francoise Farace, Head of Rare Circulating Cells Translational Laboratory, INSERM Gustave Roussy Institute, France

The presentation will focus on the liquid biopsy potential of CTCs to analyze resistance mutations to tyrosine kinase inhibitors in ALK-rearranged and EGFR-mutant patients. Our work on NSCLC CDX (CTC-derived xenograft) will be presented and discussed.

Single-Cell Transcriptome Analysis For Circulating Tumor Cells based on Microcavity Array
Tomoko Yoshino, Professor, Tokyo University of Agriculture and Technology, Japan

Circulating tumor cells (CTCs) are one of the powerful biomarkers for cancer diagnosis and potential targets for drug discovery. Genetic analysis of CTCs could provide detailed information of cancer patient. However, the recovery of such extremely rare cells from the peripheral blood and the achievement of genetic analysis without loss of CTCs presents a technical challenge. In this presentation, I will introduce several techniques towards single-cell transcriptome analysis for CTCs. Filtration using metal microfilters (microcavity array: MCA) has been used to develop precise CTC recovery technique. Furthermore, recovered cells on the MCA is easily manipulated by encapsulation by photopolymerizable hydrogel using multiple light projection system, resulting in simply manipulate multiple CTCs at a single cell level. The cells were applied RNA-seq analysis, and we investigated single cell gene expression analysis to reveal the cancer-specific transcripts. We believe our system have a potential to apply high-throughput single-CTC analysis and contribute for development of non-invasive cancer diagnosis method.

Liquid Biopsy in Colon Cancer
Pierre Laurent, Professor, Universite Paris Descartes, France

RAS mutations are currently sought for in tumor samples, which takes a median of almost 3 weeks in western European countries. This creates problems in clinical situations that require urgent treatment and for inclusion in therapeutic trials that need RAS status for randomization. Analysis of circulating tumor DNA might help to shorten the time required to determine RAS mutational status prior to anti-EGFR antibody therapy for metastatic colorectal cancer. Here we compared plasma versus tissue RAS analysis in a large prospective multicenter cohort.

Droplet-Based Digital PCR Procedures and Optimized NGS to Track Circulating Tumor DNA in Liquid Biopsies: Application to Cancer Patient Follow-Up
Valérie Taly, CNRS Research Director, Professor and Group leader Translational Research and Microfluidics, Université Paris Cité, France

Tracking of circulating cell-free nucleic acids in body effluents has been greatly facilitated by recent technological developments including droplet-based digital PCR and optimized NGS. In particular, droplet based digital PCR has allowed to reach unprecedented sensitivity and accuracy for rare sequences detection. Complementarity of these technologies will be presented through several retrospective and prospective studies. Moreover, we will present their application for the detection of circulating tumor DNA and its potential use for cancer patient treatment management.

Afternoon Coffee Break

Approaches For Validation of Liquid Biopsy Testing of Circulating Tumour DNA
Alison Devonshire, Science Leader, LGC Limited, United Kingdom

The typically low concentration of ctDNA in cancer patients’ samples makes validation of analytical sensitivity a critical element in the assessment of a diagnostic test. The potential for real-time monitoring of tumor dynamics through liquid biopsy testing also necessitates a systematic evaluation of quantitative accuracy.  This presentation will discuss approaches to validate the performance of targeted methods for cancer mutations, including pre-analytical controls, and the development of reference materials and reference measurement procedures, which also support longer-term comparability and standardization of patient testing.

Successful Implantation: How Small Molecule Dynamics Play a Big Role in Cell-Cell Communication
Yael Heifetz, Principal Investigator, The Hebrew University of Jerusalem, Israel

Embryo-maternal communication is crucial for achieving successful pregnancy and for the future health of the progeny. A relay of molecular signals between mother and gametes is critical as soon as after copulation, during gamete transport in the female reproductive tract, gamete maturation, and fertilization. The next step, the process of embryo implantation, is a major obstacle for successful maintenance of pregnancy to term. For implantation to succeed, a synchronized network of molecular interactions between mother and embryo must occur. Although key molecules have been identified, we still lack comprehensive understanding of the early dynamics of implantation which set-up the stage for a successful pregnancy. To examine the early sequence of events that initiate the embryo-maternal cross-talk we used in-vitro (human endometrial and trophoblast cell lines) and in-vivo (domestic pig Susscrofa domestica) models, and various molecular and imaging approaches to monitor the spatial and temporal dynamics of intercellular interactions. This talk will focus on extracellular vesicles (EVs), which have been suggested to act as essential mediators of embryo-maternal communication. Studying the dynamics of the cross-talk, and specifically signals that promote successful implantation at an early stage, will shed more light on the implantation process and possibly guide new strategies in diagnosing and preventing implantation failure.

Panel Discussion: Emerging Themes and Trends in Liquid Biopsy Development

Close of Day 2 of the Conference.

Morning Coffee, Breakfast Pastries and Networking

Improving the Detection of Circulating Tumour DNA in Plasma by Selecting Short DNA Fragments
Florent Mouliere, Researcher, Nitzan Rosenfeld Group, University of Cambridge, United Kingdom

The sensitivity for detecting the presence of genomic changes in circulating tumour DNA (ctDNA) is limited by its low concentration in plasma. Here I will present new approaches tailoring sequencing to the biological properties of ctDNA for improving the detection of rare mutant molecules. In particular, we studied the feasibility for enrichment of ctDNA by physical and in-silico size selection in plasma samples collected from multiple cancer types. Size selection of short DNA fragments enriches for ctDNA, unlocking un-targeted genome-wide sequencing for liquid biopsy.

EV-Associated Biomarker Discovery in Liquid Biopsies
An Hendrix, Professor, Ghent University, Belgium

An Hendrix will discuss the impact of pre-analytical factors and isolation methods on the identification of extracellular vesicle-associated biomarkers in liquid biopsies. She will present the development and implementation of reference materials for extracellular vesicles and how this can help to compare and normalize results in research and clinical setting.

Technology Spotlight Presentation: Imaging Flow Cytometry Enhances the Detection and Quantification of Rare Events Such as Circulating Tumor Cells and Minimal Residual Disease
Peter Rhein, Instrument Application Scientist, Merck KGaA, Germany

Using the ImageStream^®, we can combine flow cytometric and microscopic assays such as immunophenotyping and fluorescence in situ hybridization (FISH), which contributes to significantly improve the detection and functional analysis biological rare events.

Biomarker detection using Particle Metrix ZetaView® and Nanoparticle Tracking Analysis (NTA)
Sascha Raschke, Technical and Scientific Support, Particle Metrix GmbH

By using the ZetaView® NTA instrument Extracellular Vesicles (EVs) and other biological nanoparticles in a heterogeneous sample can be individually analyzed in terms of their size, number and concentration.  Furthermore, they can be phenotypically characterized using fluorescence-labeled antibodies or intercalating membrane dyes. Two different fluorescence channels of the ZetaView® machine allow analysis of two different fluorescent labels in rapid succession with just one sample, eliminating the time and effort required to produce a second fluorescently labeled sample.

Coffee Break in the Exhibit Hall and Poster Viewing

Multilevel Analysis of Individual EVs, Direct From Sample With Minimal Purification
Andrew Malloy, VP of Sales and Marketing, NanoView Biosciences

Current analytical tools have detection limitations when used for exosome characterization due to their small size.  Specifically, exosomes have fewer surface epitopes available and low-signal relative to background levels, especially in complex samples. Current workflows require multiple measurement technologies that result in the inability to link phenotypic and physical characterization on individual EVs across a biologically relevant size range. In addition, sample purification often results in loss of sample and technique dependent biases.  

NanoView Biosciences has developed an imaging technique that allows individual EVs as small as 40nm to be characterized direct from sample with no purification in most cases. Physical characteristics such as size and EV count can be presented alongside phenotypic information at the single EV level. Low volumes of sample can be probed and a panel of up to 10 surface markers can be interrogated in parallel, in a single measurement. Lastly, through the addition of fluorescent labeling, three additional markers can be probed and colocalized across the existing panel of 10 markers.  These capabilities make the tool ideally for basic EV research, biomarker discovery and the development of EV based liquid biopsies and therapeutics.

Exosome-derived Epigenetic Biomarkers For Saliva Diagnostics
Christa Noehammer, Senior Scientist, Austrian Institute of Technology, Austria

The aim of our research activities at AIT, the Austrian Institute of Technology, is to define reliable biomarkers suitable for early and non-invasive disease diagnosis and prognosis. To this end we have been establishing and optimizing a whole range of multiplex capability technologies (e.g. microarrays, quantitative PCR, Luminex bead technology) to meet the special demands and challenges of diagnostic biomarker discovery - and validation in body fluids. Using this specific technology expertise we e.g. successfully discovered autoantibody- as well as DNA methylation -based diagnostic marker panels for the big 4 cancer entities (breast, colon, prostate, lung) in serum or plasma. Based on these success stories and the evident advantages of saliva as a diagnostic matrix our recent special interest is to go for saliva diagnostics and to evaluate saliva for its suitability for circulating biomarker-based diagnostics.  Along these lines we will report on the evaluation of different commercially available strategies for isolation of exosomes from human serum and saliva. We will further present data from genome-wide microRNA – as well as DNA-methylation profiling in salivary - and serum-derived exosomes which will also include our experiences when comparing the performance of high-density microarrays and next generation sequencing for miRNA profiling. Last but not least we will report on first salivary and plasma exosome-derived epigenetic biomarkers for type 2 diabetes diagnosis.

Lunch and Networking in the Exhibit Hall

Luncheon Tech Spotlight: Seeing is Believing - Meet the Nanoimager
Katarzyna Ginda-Mäkelä, Application Specialist, Oxford Nanoimaging (ONI)

ONI crafts desktop-compatible microscopes with miniaturized Nobel Prize winning technology capable of detecting individual molecules using fluorescence. This exquisite sensitivity is used to create images and videos of biological samples in an unprecedented fashion, opening a whole new world of discovery, leading to a better understanding of the fate and genesis of exosomes and a potential development of novel therapeutic tools. Our team is a blend of life science and microscopy experts, software and mechanical engineers as well as data scientists.

Challenges in the Detection, Isolation and Quantification of Extracellular Vesicles and Exosomes
Alain Brisson, Professor Emeritus, University of Bordeaux, France

The emerging field of extracellular vesicle (EV) and exosome research faces a notorious lack of reliable characterization methods. This is mainly due to the small size of EV/exosomes (mostly from 30 to 500 nm) and the heterogeneity of their suspensions. This presentation will illustrate the advantages of novel methods allowing to image, phenotype, quantify and isolate EVs in complex fluids.

Production of Functionally Bioactive EVs from an Immortalized Human Neural Stem Cell (hNSC) Line
Randolph Corteling, Head of Research, ReNeuron Ltd., United Kingdom

To ensure the scale required for clinical research and development, producer cell immortalization and clonal isolation is a practical strategy to produce consistent, functionally bioactive exosomes for use as therapeutic agents.

Afternoon Coffee Break

Circulating Microvesicles – Biomarkers and Effectors and Cardiovascular Health and Disease
Felix Jansen, Leader of the Microvesicle Research Group, Department for Cardiology, Pulmology and Angiology, University Hospital Bonn, Germany

My talk will summarize the diagnostic potential and current obstacles in using circulating MVs as biomarkers in CVD. Furthermore, I will highlight the regulatory role of MV in the progression of atherosclerosis.

Liquid Biopsy for Breast Cancer Biomarkers Discovery
Clotilde Costa, Translational Medical Oncology Group, Joint Unit Roche-Chus, Oncomet, Universitary Cinical Hospital of Santiago de Compostela, Spain

Metastasis is responsible for 90% of cancer related deaths, thus, biomarkers discovery to predict and monitor tumor progression is an urgent need in oncology. Liquid Biopsy analysis permit to obtain tumoral information in real time, in a non-invasive way and in a systemic manner. Thus, molecular characterization of circulating material of metastatic breast cancer patients in different time points of the disease (before and after treatment) had led us to identify biomarkers with interest for clinical oncology translation.

Pre-Clinical Test Requirements For EVs Drugs
Marcin Jurga, R&D Director, The Cell Factory bvba, Netherlands

Extracellular vesicles (EVs) are promising drug candidates and drug delivery tools in treatment of multiple diseases. Currently, one of the most popular source of EVs are mesenchymal stromal cells (MSCs) with a broad spectrum of anti-inflammatory and immunosuppressive activity. Translation of the EVs drug candidates into clinic requires well-established production system and effective quality control procedures. The presentation will give an overview of the clinical-grade production process of the EVs drugs and will describe the quality control and activity assays developed and validated for clinical translation of the EVs drugs.

Relevance of EV Heterogeneity For Their Application in Liquid Biopsy
Irina Nazarenko, Exosome and Tumor Biology Group Leader, University of Freiburg, Germany

The need to develop better approaches allowing comprehensive molecular characterization of a progressing tumor has led to the development of the “Liquid Biopsy” concept, which is based on the application of body fluids as a biomarker source. In view on the complexity of blood and availability of components of different origins, including circulating tumor cells, free-circulating nucleic acids and Extracellular Vesicles (EVs), a question of a specific impact of each of these different compartments on the outcome of liquid biopsy-based diagnostic may arise. We address this question comparing amounts of mutated oncogenes in different EV subtypes and in the free-circulating fractions in blood of cancer patients. Our results show that different tumor types at different stages may contain varying portions of potential biomarkers in the free circulating fraction and in different EV subtypes, indicating a potential relevance of a comparative analysis of different blood compartments prior the choice of the most appropriate source and technique for the liquid biopsy-based diagnostic.

Glioblastoma Extracellular Vesicles As a Tool To Investigate The Hijacking of Multiple Microglial Functions To Promote Tumor Growth
Niek Maas, Clinical Researcher, University Medical Center Utrecht, Netherlands

This presentation will focus on the role of extracellular vesicles (EVs) in the microenvironment of glioblastoma malignant brain tumors. We will present data on EV associated modification of microglia and macrophages in vivo.

Close of Conference.