Circulating Biomarkers 2014 Agenda

Registration, Materials Pick-up, and Morning Coffee

Plasma Vesicle Proteins in Diagnosis and Prognosis of cardiovascular Disease
Dominique PV de Kleijn, Professor Experimental Vascular Surgery, Professor Netherlands Heart Institute, University Medical Center Utrecht, The Netherlands, Netherlands

Extracellular vesicles, including microvesicles, microparticles and exosomes are abundant in plasma. All cells secrete extracellular vesicles that can be easily isolated from frozen plasma or serum and contain part of its cellular content like protein, miRNA and RNA. For this, interrogating extracellular vesicles content is like looking at a sample from the (pathological) cell they originate from. Vesicle content therefore contains information on pathological status that can be used for diagnosis and prognosis of disease. Isolating these vesicles from the blood and studying their content may provide rapid information on the pathological status of tissues. We already established that plasma extracellular vesicles can be used for the diagnosis of Acute Coronary Syndrome in an emergency department cohort of 471 patient with chest pain and prediction of secondary cardiovascular events on top of existing risk factors in 1100 patients.
We now established that plasma vesicle proteins in adult cohorts are associated with adiposity, the number of total affected vascular territories and with carotid intima-media thickness in a children cohort.
This shows that plasma extracellular vesicle proteins reflects the status of cardiovascular disease and are an important source for diagnostic and prognostic markers for this rapidly growing disease that is responsible for the number 1 and 2 cause death in the world.

Circulating miRNA Biomarkers for Alzheimer's Disease
Pavan Kumar, Senior Scientist, Eisai Inc, United States of America

Recent advances and the current state of miRNAs as circulating biomarkers, especially pertaining to the pharmaceutical and diagnostic industry, will be presented. In addition, a case study describing a circulating miRNA signature for potential AD diagnostics will also be discussed.

Coffee Break and Networking with Exhibitors in the Exhibit Hall

Detection, Isolation, and Characterization of Single Circulating Tumor Cells
Lori Millner, Clinical Chemistry Fellow, University of Louisville, United States of America

Advances in single cell capture and downstream analysis will be presented. Single cell capture of viable cells using the DEPArray by Silicon Biosystems is described.  A model of heterogeneous CTCs subtypes in breast cancer describing their capture and analysis will be discussed.

Circulating RNA in Brain Injury and Neurodegenerative Disease
Kendall Van Keuren-Jensen, Professor and Deputy Director, Translational Genomics Research Institute, United States of America

We have investigated extracellular RNAs associated with Parkinson's Disease, Alzheimer's Disease, repeated head impact, and subarachnoid hemorrhage.  We will discuss similarities and differences among the types of RNA associated with brain injury and disease.

Lunch, Networking in the Exhibit Hall, and Poster Viewing

Technology Spotlight:
Precise Quantitation of Circulating Nucleic Acids with the RainDrop™ Digital PCR System
Darren Link, Co Founder & Chief Technology Officer, RainDance Technologies

A Rapid and Non-destructive Magnetic Beads-based Exosome Isolation and Enrichment Method
Kenneth Henry, Senior Research Scientist, JSR Micro, Inc., United States of America

In order to isolate exosomes from various body fluids and cell culture supernatants, we have successfully developed ExoCap, which utilizes a magnetic beads based isolation method. ExoCap consists of magnetic particles coupled with antibodies that recognize antigens on the exosome surface, an irrigation solution, and a reagent that releases the captured exosomes for analysis. The antibodies against CD9, CD63, CD81, and EpCAM were specifically selected for this kit. ExoCap can separate easily exosomes within 30 minutes, without ultracentrifuge or any special equipment. A sample amount of 0.1mL is sufficient. In addition, it is an animal free system which is superior to other methods for mass analysis. Moreover, this method enables non-destructive purification of exosomes. To confirm exosomal isolation from diverse body fluids (such as human serum, plasma, urine) and cell culture media, exosomes were examined by western blot, particle size distribution measurement, and scanning transmission electron microscopy (TEM). Exosomes isolated by ExoCap had a lipid bilayer membrane, showed a particle size distribution around 100nm, and expressed tetraspanin molecules.

Authors:
Kenneth Henry, Ph.D, Senior Research Scientist, Life Sciences, JSR Micro, Inc.
Tetsuji Yamaguchi, Ph.D. JSR Life Sciences

Heparin Affinity Purification of Extracellular Vesicles
Leonora Balaj, Instructor in Neurosurgery, Mass General Hospital (MGH)/Harvard Medical School, United States of America

Extracellular vesicles (EVs) are membrane vesicles released by cells. They carry active biomolecules which can be transferred to recipient cells. Isolation and purification of EVs from in vitro conditioned culture media and in vivo biofluids is still a major challenge and the most widely used isolation method still remains ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs due to co-pelleting of proteins and lipids. Affinity purification of biomolecules is an efficient way to achieve high purity without requiring expensive equipment. Previously we have shown that heparin blocks EV uptake in mammalian cells in culture, suggesting a possible direct EV/heparin interaction.  Here we show that EVs can be purified from conditioned media using heparin-coated agarose beads. We directly compared heparin-purified EVs to UC prepared and kit-isolated EVs and we show that we can efficiently isolate EVs  a higher purity than UC, kit-isolated EVs and even sucrose gradient-purified EVs. Importantly these, heparin-purified EVs retained the RNA content, morphology, and uptake dynamics of UC-isolated EVs.  In conclusion, we have discovered a simple and effective way to isolate a highly pure population of EVs using their affinity for heparin.

Coffee Break and Networking with Exhibitors in the Exhibit Hall

New Biosensor Platform for the Isolation and Analyses of Microvesicles
Hakho Lee, Associate Professor, Director of the Biomedical Engineering Program, Massachusetts General Hospital / Harvard Medical School, United States of America

This presentation will review new biosensor platforms developed in our laboratory for microvesicle analyses. Specifically, I will describe 1) microfluidic systems that can enrich microvesicles from native clinical specimen, and 2) microNMR sensors that can molecularly profile microvesicles. Clinical applications of these platforms on cancer detection will also be discussed.

A Hybrid Extracellular vesicle/virus Vector System for Gene Therapy Applications
Casey Maguire, Assistant Professor, Harvard Medical School, United States of America

Obtaining tissue-restricted transgene expression after intravenous (i.v.) injection of AAV vectors is a challenging task, especially for the brain, as the majority of vector is taken up by the liver. Although some vectors can cross the intact blood-brain barrier, improvements are still needed. Additionally, pre-existing antibodies against AAV can remove vector from the circulation. Other studies have shown that association of virus vectors with nanoparticles, such as microbubbles and cationic liposomes can alter the vector biodistribution to preferred sites.  Another nanoparticle which may offer utility to the field of viral vector gene delivery are extracellular vesicles(EVs).  EVs are small (50-200 nm in diameter) membrane limited structures naturally secreted by many cell types.  We have recently shown that EV-associated AAV vectors (EV-AAV, a.k.a vexosomes) can deliver genes more efficiently on a genome copy per cell basis than AAV vectors alone using cultured cells. In the current study we are using the EV-AAV for targeted gene delivery to the brain after i.v. injection in mice.  To investigate if EV-AAV can be targeted to the brain via over-expression of specific ligands on the EV surface we injected nude mice with EV-AAV9-Fluc or EV-AAV9-Fluc with a brain targeting peptide (RVG) fused to a trasmembrane domain.Enhanced transduction of the brain was observed with the RVG peptide compared to untargeted EV-AAV. The brain:peripheral organ transduction ratio was significantly higher for RVG-EV-AAV compared to standard AAV9.  This work has provides the first evidence for the in vivo use of EV-AAV for gene therapy.

Large Oncosomes as a Novel Source of Circulating DNA and miRNA in Cancer
Dolores Di Vizio, Professor, Cedars Sinai Medical Center, United States of America

Panel Discussion: Evolution of the Exosome Research Field
Johan Skog, Chief Scientific Officer, Exosome Diagnostics Inc, United States of America

Panelists:

Dominique de Kleijn, Professor, National University of Singapore
Xandra Breakefield, Professor, MGH
Shannon L. Stott, Professor, MGH
Pavan Kumar, Eisai

Networking Reception: Premium Beers, Wines, and Hors d'oeuvres for All Speakers, Delegates, and Exhibitors. Enjoy an Awesome Evening and Network with your Fellow Delegates. Sponsored by QIAGEN.

Close of Day 1 of the Conference.

Morning Coffee and Networking in the Exhibit Hall.

Circulating Tumor Cells: Informing and Facilitating Development and Clinical Decisions
Robert McCormack, Head, Technology Innovation and Strategy, Veridex LLC/Johnson & Johnson, United States of America

Circulating tumor cells (CTCs) have been detected in the blood of patients with most types of cancers.  In all instances, elevated CTC numbers implies failure of the patient’s current therapy and portends a poor prognosis.  These validated findings and others have been translated into the basis for incorporating CTCs in both drug discovery and patient management.  This presentation will follow an algorithm to use CTCs to determine therapy effectiveness, elucidating underlying mechanisms for therapy failure, and identifying therapeutic options for patients.

Extracellular RNAs as Biomarkers in Cardiovascular Diseases
Saumya Das, Assistant Professor in Medicine, Beth Israel Deaconess Medical Center and Mass General Hospital, United States of America

Detection and Monitoring of Chromosomal Alterations in the Circulation of Cancer Patients with Whole-Genome Sequencing
Mark Sausen, Vice President, Research & Development, Personal Genome Diagnostics, United States of America

Clinical management of cancer patients could be improved through the development of noninvasive approaches for the detection of incipient, residual, and recurrent tumors. We describe an approach to directly identify tumor-derived chromosomal alterations through analysis of circulating cell-free DNA from cancer patients. Whole-genome analyses of DNA from the plasma of colorectal and breast cancer patients and healthy individuals with massively parallel sequencing identified, in all patients, structural alterations that were not present in plasma DNA from healthy subjects. Detected alterations comprised chromosomal copy number changes and rearrangements, including amplification of cancer driver genes such as ERBB2, MET, and CDK6. The level of circulating tumor DNA in the cancer patients ranged from 0.3% to 47.9%. The sensitivity and specificity of this approach are dependent on the amount of sequence data obtained and are derived from the fact that most cancers harbor multiple chromosomal alterations, each of which is unlikely to be present in normal cells. Given that chromosomal abnormalities are present in nearly all human cancers, this approach represents a useful method for the noninvasive detection of human tumors that is not dependent on the availability of tumor biopsies.

Coffee Break and Networking with Exhibitors in the Exhibit Hall

Technology Spotlight:
Blood platelets of NSCLC patients take up EML4-ALK, c-Met, BRAFv600e, KRAS G12D, and EGFR T790M RNA Biomarkers
Nik Sol, CSO, VUmc

Each year an estimated 1.3 million people are diagnosed with lung cancer, compromising 12.4% of all new cancer cases. Approximately 80% of lung cancer cases are diagnosed as non-small cell lung carcinoma (NSCLC), continuing as the leading cause of cancer-related death in both men and women. Depending on the patient’s medical status and stage of disease, systemic cytotoxic chemotherapy, surgery, and/or targeted therapies are the mainstay for treatment for NSCLC patients. The discovery of various molecular alterations that underlie lung cancer has opened-up a new era in the development of specifically targeted therapies employing specific biomarker-dependent inhibitors. The identification of alterations in oncogenes associated with NSCLC can help to determine which patients are more likely to benefit from targeted therapy. In the vast majority of NSCLC patients genetic alterations occur in EGFR (20-25%), KRAS (25-30%), BRAF (3%), and ALK (3-10%), mostly in a mutual exclusive manner. In addition, c-Met amplification is emerging as a potent stratification marker and therapeutic target. Hence, patient stratification for c-Met, EGFR, KRAS, BRAF, and ALK aberrations is becoming common practice among oncologists, and more potential mutations are being identified. NSCLC patients with activating EGFR mutations are generally responsive to the EGFR-directed inhibitors erlotinib and gefitinib, whereas NSCLC patients with ALK translocations respond significantly to the ALK inhibitor crizotinib. The genetic tumor profile of NSCLC patients is considered dynamic and can change in due course of the disease and in response to therapy. For instance, treatment with EGFR inhibitors can result in the outgrowth of resistant NSCLC clones with amplified c-Met, although point mutations in EGFR, e.g. T790M, can also convey resistance to EGFR inhibitors. Resistance to crizotinib in EML4-ALK positive NSCLC patients is described to be caused by several acquired ALK mutations, including in exons 2

Lunch, Networking in the Exhibit Hall, and Poster Viewing

Technology Spotlight:
Have you Checked your RNA Diversity Lately? Effect of microRNA Isolation Method on RNA Diversity and Biomarker Discovery
Bernard Lam, Senior Research Scientist, Norgen Biotek Corporation

Exosomes and microRNAs in Liver Disease
Gyongyi Szabo, Professor & Vice Chair for Research, Department of Medicine, University of Massachusetts Medical School, United States of America

Ambient Preservation of Blood-based Biomarkers: Impact on Discovery and Diagnostic Assay Development/Deployment in a Global Healthcare Market
Rolf Muller, President/CSO & Founder, Biomatrica Inc, United States of America

Biomatrica’s pioneering work over the past decade in the field of chemical biomaterial preservation at room temperature has enabled ambient alternatives to unreliable cryopreservation. This has created opportunities previously not feasible in multiple applications in the life sciences, diagnostics and therapeutics.  Biomatrica has developed a novel technology approach and discovery platform focused on the preservation of biomaterials reaching from the stabilization of high quality DNA, RNA and proteins to patient samples, diagnostic assays and live mammalian cells. We will discuss the application of ambient biopreservation for the stabilization of blood based biomarkers and its implications for research and diagnostic assay development and deployment.

Enrichment of Fetal DNA Fractions by Microfluidic Extraction of Maternal Plasma
Maiwenn Kersaudy-Kerhoas, Professor of Microfluidic Engineering, Heriot-Watt University, United Kingdom

The presence of circulating fetal nucleic acids in maternal circulation provides a unique opportunity for Non Invasive Prenatal Testing (NIPT) as an alternative to invasive techniques such as amniocentesis or chorionic villus sampling. Prompt and effective enrichment of circulating fetal DNA from blood impact the ability to perform testing. We propose a microfluidic device to rapidly enrich fetal DNA from 2-4mL maternal blood samples. We will show that in plasma extracted on-chip a three-fold average relative amount of fetal DNA (compared with total DNA) was obtained compared to plasma extracted via centrifugation (p=0.02). We therefore demonstrate that a portable microfluidic device can significantly enriches fetal fractions from maternal blood samples in one single step. The operation requires minimal training and does not need large items of specialized equipment. This device may be deployed near-patient to improve the testing accuracy and ability to test NIPT samples with low fetal fractions.

Microfluidic Isolation of Microvesicles from Serum in Glioblastoma
Shannon Stott, Assistant Professor, Massachusetts General Hospital & Harvard Medical School, United States of America

Microvesicles (MVs) released from cancer cells into the bloodstream contain genetic information about the primary tumor.  These MVs have the potential to be used to guide treatment in glioblastoma patients, but can be challenging to reliably assay due to the heterogeneous population of MVs released from normal cells.  We have taken a microfluidic approach to isolate these tumor derived MVs from human serum, using affinity based capture. Specifically, a microfluidic chaotic mixer is used to direct the MVs to the antibody coated surface of the device. This approach increases our sensitivity of detection of oncogenic mutations, while being cost effective.  Our MV capture and subsequent RNA analysis was validated using tumor MVs from a glioblastoma cell line spiked into healthy human serum. Our data demonstrates that tumor-derived MVs can be selectively captured from serum, providing a  less invasive method to obtain genetic information about the patient’s tumor.

Microfluidic Isolation and Molecular Characterization of Circulating Tumor Cells
David Miyamoto, Instructor in Radiation Oncology, Massachusetts General Hospital/Harvard Medical School, United States of America

Through a collaborative effort between bioengineers, biologists, and clinicians, our group at MGH has developed three successive generations of microfluidic devices to efficiently and gently isolate rare circulating tumor cells (CTCs) shed into the blood. Our efforts to molecularly characterize CTCs are leading to new insights into cancer biology and enabling the development of biology-driven biomarkers. I will describe our recent efforts to dissect the molecular and cell biology of CTCs, and the potential of these analyses to non-invasively monitor and predict treatment responses.

Implementation of COLD-digital-PCR for Detecting Low Level Mutations in Circulating DNA
Elena Castellanos-Rizaldos, Instructor in Radiation Oncology, Dana-Farber Cancer Institute/Harvard Medical School, United States of America

CO-amplification at Low Denaturation Temperature- PCR (PCR) amplifies preferentially rare mutations buried within an excess of wild type DNA. We have adapted COLD-PCR into emulsion-digital PCR (COLD-digital PCR) format and demonstrated highly enhanced sensitivity. We apply COLD-digital PCR to detection of T790M mutations present in tumor circulating DNA obtained from patients with lung adenocarcinoma. COLD-digital PCR improved the discrimination of T790M mutations present at a very low level, facilitating disease monitoring and selection of appropriate therapy regimens.

Poster Awards to Top 3 Posters. Presented by Chair of Poster Awards Committee Dr. Leonora Balaj, and Short Presentations by the Top 3 Poster Presenters

Close of Conference.