08:00 | Conference Registration, Materials Pick-Up, Morning Coffee and Breakfast Pastries |
| Session Title: Conference Opening Session Framing the Key Topics and Opportunities in Liquid Biopsy |
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09:00 | | Conference Chair Conference Chairman Welcome and Opening Remarks Dominique PV de Kleijn, Professor Experimental Vascular Surgery, Professor Netherlands Heart Institute, University Medical Center Utrecht, The Netherlands, Netherlands
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09:15 | | Keynote Presentation Mt-SEA Pipeline: High Throughput Flow Cytometric Analysis of Exosomes in Clinical Biofluids Jennifer Jones, NIH Stadtman Investigator, Head of Transnational Nanobiology, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, United States of America
Because Extracellular Vesicles (EVs) carry surface receptors that are characteristic of their cells of origin, EVs have tremendous potential as non-invasive biomarkers for diagnosis, risk-stratification, treatment selection, and treatment monitoring. We developed a first-in-class pipeline to characterize EV heterogeneity and provide high-sensitivity quantification of informative EVs in biofluids before, during, and after treatment. By combining multiplex assays with high-resolution, single EV flow cytometric methods together into a Mutiplex-to-Single EV Analysis (Mt-SEA) pipeline, we are able to characterize a broad range of relevant EV subsets, while also accurately measuring the concentration of specific EV populations. Detection of tumor-associated EVs and detection of EV repertoire changes during treatment paves the way to future evaluation of EVs as as biomarkers for use in personalized, adaptive therapies. |
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09:45 | | Keynote Presentation Vesicles and Cellular Communication within the Bone Environment Hans van Leeuwen, Professor and Dean, Erasmus University Medical Center, Netherlands
Bone cells are not only important in bone metabolism and building and maintaining healthy bone but are also involved in numerous other processes. This presentation will focus on two of these. One is the interplay with the cells in the bone marrow in particular the hematopoietic stem cells that give rise to the cells of our immune system. The other is the role in cancer metastasis. I will discuss the presence of extracellular vesicles in the bone environment and how these are involved in the interplay between bone cells, the osteoblasts, and hematopoietic stem cells and prostate cancer cells. In addition, I will address our studies on unraveling the content of these osteoblast as well as prostate cancer cells-derived extracellular vesicles. Hereby I will introduce extracellular vesicles in the bone environment and a role of bone cells beyond bone metabolism. The understanding of these extracellular vesicles in the either physiological or pathophysiological condition may provide novel diagnostic and therapeutic tools. |
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10:15 | Morning Break and Networking in the Exhibit Hall |
10:45 | | Keynote Presentation Liquid Biopsy: The New Diagnostic Concept in Oncology Klaus Pantel, Chairman, Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Germany
The analysis of circulating tumor cells (CTCs) in blood may provide clinically relevant information as “liquid biopsy” (Alix-Panabieres & Pantel, Nature Rev Cancer 2014) and provide new insights into tumor biology (Uhr & Pantel, PNAS, 2011, Lu et al., Cancer Cell, 2011; LeBleu et al., Nat. Cell Biol., 2014; Werner et al., Cancer Discovery, 2015). Besides CTCs the molecular analysis of ctDNA provides important complementary information as “liquid biopsy” (Pantel & Alix-Panabieres, Cancer Res., 2013; Pantel et al., Nature Med. 2013; Heitzer et al., Genome Med. 2013; Schwarzenbach et al., Nature Rev. Clin. Oncol., 2014, Joosse & Pantel, Cancer Cell 2015; Bardelli & Pantel, Cancer Cell 2017). Moreover, circulating microRNAs, extracellular vesicles and tumor-educated platelets are also interesting new members of the “Liquid Biopsy family” with potential clinical relevance in the future. In particular exosomes have gained great interest because they act as biomarkers with important functional properties for tumor progression. E.g., the integrin composition of exosomes can determine the organ site of metastases (Hoshino, Pantel, Lyden et al., Nature, 2015) and microRNAs in exosomes can impact the biology of the recipient cells (Anfossi, Bababayan, Pantel, Calin, Nature. Rev. Clin. Oncol. 2018). Detection of these miRNAs can contribute to early detection of cancer (Meng et al., Oncotarget, 2016). Liquid biopsy analyses with validated platforms provides reliable information on prognosis and may serve to identify therapeutic targets or mechanisms of resistance on metastatic cells. Metastatic cells might have unique characteristics that can differ from the bulk of cancer cells in the primary tumor currently used for stratification of patients to systemic therapy. Moreover, monitoring of blood samples before, during and after systemic therapy (e.g., chemotherapy, hormonal therapy, antibody therapy) might provide unique information for the future clinical management of the individual cancer patient and might serve as surrogate marker for response to therapy. In conclusion, liquid biopsies can be used to improve the management of individual cancer patients and contribute to the vision of personalized medicine (Alix-Panabieres & Pantel, Cancer Discovery, 2016; Bardelli & Pantel, Cancer Cell 2017). Validation of liquid biopsy assays is essential and is currently being performed by the EU/IMI consortium CANCER-ID (www.cancer-id.eu). |
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11:15 | | Keynote Presentation Circulating DNA as a Next Generation Diagnostic Tool Alain Thierry, Director of Research, Institut de Recherche en Cancérologie de Montpellier-INSERM, France
Circulating tumor DNA (ctDNA) holds promises in tumor burden monitoring or malignancies early detection. Based on crucial observation on the structure and origin of ctDNA we developed a multiparametric method for specifically analyze ctDNA. This technology enables the first clinical validation of the analysis of circulating DNA in oncology, in a prospective blinded multicentric study for the detection of KRAS and BRAF mutations from colorectal patient plasma samples. Aims of our research are now focusing on various aspects of the potential of ctDNA: (i), detecting the emergence of the mutations following targeted therapy; (ii), developing the Intplex test for the multimarker quantitative analysis of ctDNA; (iii), studying the follow up of CRC patients; (iv), the prognostic power of ctDNA and (v) the screening power of ctDNA. |
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11:45 | | Keynote Presentation Functional Analyses of Circulating Tumor Cells in Cancer Patients Catherine Alix-Panabières, Director of the Laboratory of Rare Human Circulating Tumor Cells, University Medical Centre of Montpellier, France
Circulating tumor cells (CTCs) in blood are promising new biomarkers potentially useful for prognostic prediction and monitoring of therapies in patients with solid tumors including colon cancer. Moreover, CTC research opens a new avenue for understanding the biology of metastasis in cancer patients. However, an in-depth investigation of CTCs is hampered by the very low number of these cells, especially in the blood of colorectal cancer patients. Thus, the establishment of cell cultures and permanent cell lines from CTCs has become the most challenging task over the past year. We described, for the first time, the establishment of cell cultures and a permanent cell line from CTCs of one colon cancer patient (Cayrefourcq et al. Cancer Res. 2015). The cell line designated CTC-MCC-41 is in culture for more than three years and has been characterized at the genome, transcriptome, proteome and secretome levels. This thorough analysis showed that CTC-MCC-41 cells resemble characteristics of the original tumor cells in the colon cancer patient and display a stable phenotype characterized by an intermediate epithelial/mesenchymal phenotype, stem-cell like properties and an osteomimetic signature indicating a bone marrow origin. Functional studies showed that CTC-MCC-41 cells induced rapidly in vitro endothelial cell tube formation and in vivo tumors after xenografting in immunodeficient mice. More recently, we defined the molecular portrait of these metastasis-competent CTCs (Alix-Panabières et al. Clin Chem. 2017). These results highlight that CTC-MCC-41 line display a very specific transcription program completely different than those of the primary and metastatic colon cancer cell lines. Interestingly, among the 1,624 transcripts exclusively up-regulated in CTC-MCC-41 samples, key genes related to energy metabolism, DNA repair and stemness genes were observed. Such data may supply insights for the discovery of new biomarkers to identify the most aggressive CTC sub-populations and for the development of new drugs to inhibit metastasis-initiator CTCs in colon cancer. Moreover, the development of new immunotherapeutic strategies is of utmost importance. Antibodies against proteins that block the immune response of T-cells such as PDL1 have been approved for treatment of cancer patients after showing remarkable long-term remissions in subsets of patients. It is now important to develop predictive biomarkers to identify patients with the highest benefit from these therapies. In 2015, we could show for the first time that PD-L1 is heterogeneously expressed on CTCs from metastatic breast cancer patients (Mazel et al. Mol Oncol 2015). Further functional analysis of this interesting subset of CTCs might reveal special immunosuppressive properties. |
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12:15 | Networking Lunch in the Exhibit Hall -- Meet the Exhibitors and View Posters |
| Session Title: Circulating Biomarkers for Cancer |
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13:30 | 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). |
14:00 | | Keynote Presentation Early Detection of Cancer by Serum Exosomes Takahiro Ochiya, Professor, Department of Molecular and Cellular Medicine, Tokyo Medical University, Japan
Recent studies have revealed a novel biomolecule, the exosome, which supports cancer metastasis and many other cancer–associated events. Exosomes are a subset of extracellular vesicles (EVs), which are lipid bilayer vesicles secreted from cells. Exosomes have a diameter of 50–150 nm, and they transfer miRNA, mRNA, DNA, and proteins to other cells, thereby mediating intercellular communications. Application of these EVs for biomarker analysis, biofunctions and cell-free regeneration medicine are actively studied, however, common methods for EVs isolation is not always accurately conducted. Especially, EVs represent an important source of potential biomarkers in many diseases that could provide a non-invasive method of early diagnosis or prognosis. Here we will discuss on our current progress on EVs detection as ExoScreen method (Yoshioka et al., Nat Commun, 2014) and usage for early detection of colorectal and pancreatic cancer with high sensitivity and specificity. |
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14:30 | 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. |
15:00 | Afternoon Coffee Break and Networking in the Exhibit Hall |
15:30 | | Keynote Presentation ISET: A New Approach for the Follow-Up and Early Diagnosis of Invasive Cancers Patrizia Paterlini-Brechot, Professor of Oncology/Molecular Biology, University Paris Descartes, France
Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients and help to detect the most aggressive invasive cancers before other diagnostic approaches like imaging. An important issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells for their diagnosis and molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on the ISET® (Isolation by SizE of Tumor/Trophoblastic Cells) open system for marker-independent isolation of tumor cells from blood. ISET® allows to reliably diagnose and count CTC with unparalleled sensitivity and specificity. Independent published data from different research teams have demonstrated the superior clinical sensitivity and specificity of ISET®. They have also demonstrated the prognostic relevance of CTC detection by ISET® in patients with melanomas, as well as lung, colorectal, liver, pancreatic, head and neck, and ovarian cancers. Furthermore, the utility of theranostic characterization of CTC by ISET® has been demonstrated for non-small-cell lung cancers, castration-resistant prostate cancers, colorectal cancers, hepatocellular carcinomas and melanomas. |
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16:00 | 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. |
16:30 | 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. |
17:00 | | Keynote Presentation The Isolation and Analysis of Rare Cells, Exosomes, and Circulating Nucleic Acids For Liquid Biopsy Daniel Chiu, A. Bruce Montgomery Professor of Chemistry, University of Washington, United States of America
This presentation will describe a rare-cell isolation instrument we call eDAR (ensemble decision aliquot ranking) for the automated enrichment and analysis of rare cells, a nanofluidic technology for the high-sensitivity and high-purity isolation of exosomes, and a digital-nucleic-acid detection platform based on our SD (self digitization) chip. I will outline the workings of these techniques, describe their performance, and discuss the clinical questions we are addressing to demonstrate the utility of liquid biopsy in precision medicine. |
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17:30 | | Keynote Presentation Circulating Tumor Cells to Guide Treatment of Cancer Patients Leon Terstappen, Chair Medical CellBiophysics, MIRA Research Institute for Biomedical Technology and Technical Medicine, University of Twente, Netherlands
The presence of Circulating Tumor Cells (CTCs) enumerated with the CellSearch system is associated with relatively poor survival and their reduction after the first weeks of therapy indicates a response to therapy. Present research focus is on the ability to extract treatment relevant information from the proteins, RNA and DNA in these CTC. |
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18:00 | Networking Reception with Beer and Wine. |
19:00 | Close of Day 1 of the Conference. |