06:30 | Morning Coffee, Breakfast Pastries and Networking in the Exhibit Hall |
07:00 | Breakfast Briefing: From Discovery to Diagnostic: Building miRNA-based Tools for Pediatric Heart Failure Patients Pete Mariner, Founder, CoramiR Biomedical, United States of America
Circulating miRNAs are important biomarkers of disease, and several methodologies are available that identify circulating miRNAs with varied specificity and reproducibility. We have previously shown that circulating miRNAs can accurately predict recovery from heart failure in pediatric dilated cardiomyopathy patients, and to determine cardiac allograft vasculopathy. Our previous work was performed using serum-extracted RNA and Life Technologies miRNA array cards. Here we describe an improvement to the original methodology that consists of performing miRNA arrays directly from serum, without the need of RNA extraction. Importantly, although 250 µl of serum were necessary to perform RNA extraction, only 3 µl are necessary to perform serum-miRNA-arrays. Furthermore, a thorough description of the methodology used to improve release of miRNAs from microparticles is provided. Moreover, whereas circulating miRNA studies are often based on relative comparison values, an absolute quantification of circulating miRNAs is necessary of tests will be standardize across laboratories. We have successfully generated an absolute quantification platform that can be easily implemented in RT-PCR confirmatory studies. In summary, the work described here provides specific guidelines to detect and quantify circulating miRNAs. |
07:30 | Breakfast Briefing: Epigenetic Alterations to DNA Architecture in Cancer Eugen Molodysky, Clinical Associate Professor, Sydney Medical School, University of Sydney, Australia
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| Session Title: Technology Development in the Interrogation of Circulating Cell-Free DNA and Circulating RNAs |
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08:00 | Therapeutic Response Monitoring Using an NGS-based ctDNA Assay Abhijit Patel, Associate Professor, Yale University School of Medicine, United States of America
Our group has developed NGS-based technologies that utilize novel molecular and computational error suppression techniques to enable ultra-sensitive measurement of ctDNA. Data will be presented from ongoing studies to establish the clinical utility of these technologies, with a particular focus on monitoring of therapeutic response. |
08:30 | | Keynote Presentation Donor-Derived Cell-Free DNA: An Accurate, Precise, and Dynamic Biomarker for Improved Management of Solid Organ Transplant Patients John Sninsky, Chief Scientific Officer, CareDx, United States of America
Cell-free DNA (cfDNA) has been described as a biomarker for prenatal testing, cancer, and organ transplantation, each of which present different clinical and technological challenges. cfDNA circulating in the plasma of transplant recipients represents a mixture of recipient cfDNA and residual nucleosome-protected genomic regions released from dying cells of the allograft (“transgenome”). The genomes of the organ donor and allograft recipient are distinguishable by sequencing total cfDNA from the plasma. A clinical-grade cfDNA NextGen sequencing (NGS) assay was developed to monitor the levels of the “transgenome”, enabling assessment of the allograft status of transplant recipients with high confidence analytical validation. The NGS-based assay does not require testing of genetic material from the donor or recipient thereby simplifying the testing of transplants with cadaveric donors. Longitudinal samples from heart, lung and kidney transplant patients had higher dd-cfDNA levels at biopsy-confirmed rejection which were reduced following adjustments to immunosuppressive therapy in clinical validation studies. Serial assessment of dd cfDNA provides a measure of both the amount and kinetics of dying allograft cells, information clinicians may use to inform clinical utility.
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09:00 | Longitudinal Whole Exome Investigation of Mutational Heterogeneity in Single CTCs in a Patient with Triple-Negative Breast Cancer Eric Kaldjian, Chief Medical Officer, RareCyte, United States of America
We performed whole exome sequencing of single CTCs from a patient with metastatic triple-negative breast cancer to investigate the evolution of genetic heterogeneity during therapy. CTCs were identified using the AccuCyte-CyteFinder system (RareCyte) and individually retrieved from microscope slides using the integrated CytePicker function. Single cell whole genome amplication was performed followed by whole exome sequencing. Computational biology tools were employed to analyze genomic DNA sequence from multiple CTCs, white blood cells and ctDNA from various time points. Genomic sequence alterations were observed to evolve over the course of therapy in individual CTCs. These alterations appear to be generally consistent within CTC at a given time point. The number of predicted deleterious and cancer driver mutations per CTC were observed to increase in frequency after effective treatments, suggesting that the number of such alterations may be associated with resistance to therapy. |
09:30 | Micro-Array Isolation, Dynamic Time Series Classification, Capture and Enumeration of Breast Cancer Cells in Blood: The Nanotube–CTC Chip Balaji Panchapakesan, Professor, Department of Mechanical Engineering, Worcester Polytechnic Institute (WPI), United States of America
We describe a new philosophy in capture and isolation of breast cancer cells in blood using carbon nanotube micro-arrays ('The Nanotube–CTC chip'). |
10:00 | Coffee Break and Networking |
10:30 | Sequencing of ctDNA in Patient Plasma Samples: Bases, Genes, Exomes, and Genomes Brian Dougherty, Executive Director, Translational Genomics, Oncology IMED, AstraZeneca R&D, United States of America
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11:00 | Monitoring the Central Nervous System Using Circulating RNAs Kendall Van Keuren-Jensen, Professor and Deputy Director, Translational Genomics Research Institute, United States of America
There are a large variety of small and long RNAs in circulation. We will discuss the prevalence and potential for several RNA biotypes as markers of injury and disease. |
11:30 | | Keynote Presentation Will the Liquid Biopsy Ever Replace Solid Tumor Biopsies? Phil Stephens, Chief Scientific Officer, Foundation Medicine, United States of America
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12:00 | | Keynote Presentation Liquid Biopsies in the Management of Solid Tumors Minetta Liu, Associate Professor and Chair, Oncology Research, Mayo Clinic, United States of America
Identification of tumor specific molecular alterations increasingly plays a part in drug selection and prognosis in cancer. The advent of technologies that allow for the detection of specific mutations in circulating tumor cells (CTCs) and cell free DNA (cfDNA) isolated from the peripheral blood has increased interest in the “liquid biopsy”. These assays offer a less invasive, potentially more cost effective tool to assess prognosis and response to treatment, and they may facilitate the early diagnosis of recurrence or primary malignancy itself. The challenges of validating these molecular biomarkers and the need to provide solutions to promote rapid translation into clinical practice will be discussed. |
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12:30 | Networking Lunch: Visit Exhibitors and Engage with Fellow Delegates |
| Session Title: Joint Session Exploring the Areas of Synergy Between the Various Circulating Biomarker Classes |
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14:00 | | Keynote Presentation Advancing Liquid Biopsies using Exosomes Johan Skog, Chief Scientific Officer, Exosome Diagnostics Inc, United States of America
The field of liquid biopsy has gained an enormous interest the last couple of years. Being able to detect tumor derived genetic profiles and track the evolution of tumor mutations over time has been a long sought after goal of personalized medicine. Utilizing cell free tumor DNA (ctDNA) for detection of mutations in plasma have shown some promise in late stage cancer patients. However, ctDNA analysis suffer from several shortcomings. The copy numbers of ctDNA carrying the mutations are often very low in plasma, limiting the sensitivity of the assay and can also not be used to monitor splice variants and other RNA specific aberrations that are clinically important. Our exosome platform addresses both of these issues. Exosomes are small vesicles that are abundantly shed from tumor cells, and carry RNA from the cell of origin. Mutations are abundantly detected on exosome RNA from plasma, and can also be efficiently used to profile splice variants and fusions. This presentation will cover some of the latest diagnostic applications of the exosome platform and how they compare to other liquid biopsy tests. |
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14:30 | | Keynote Presentation Clinical Potentials for Large Oncosome and Exosome Profiling in Prostate and Breast Cancer Dolores Di Vizio, Professor, Cedars Sinai Medical Center, United States of America
Extracellular vesicles (EVs) are important mediators of intercellular mechanisms as they can shuttle from one cell to another a reservoir of functional molecules (bioactive proteins, nucleic acids and lipids). EVs are highly heterogeneous and differ by size, composition and function. As a common characteristic, they all are surrounded by a lipid bilayer and can act in the proximity of the cell or at distance. Given the abundance of cancer-derived molecules that can be found in each particle, EVs are being recognized as appealing biomarkers of diagnosis and prognosis. Because these molecules are functional, EV profiling has also the potential to identify therapeutic targets in the personalized medicine effort. Our team recently reported that highly metastatic cells undergoing mesenchymal to amoeboid transition export large (1-10 µm diameter) bioactive EVs (large oncosomes) that originate from the shedding of bulky membrane protrusions from the plasma membrane. We have demonstrated that the abundance of large oncosomes in the circulation and in tissues correlates with advanced disease in mouse models and human subjects, and that these vesicles are promising candidates for liquid biopsy approaches through large scale profiling of protein, DNA and mRNA. |
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15:00 | Cell Communication and Therapeutic Delivery via ARMMs (ARRDC1-mediated Microvesicles) Quan Lu, Associate Professor, Harvard School of Public Health, United States of America
My lab discovered a new type of extracellular vesicles known as ARMMs
(ARRDC1-mediated microvesicles), which bud directly at the plasma
membrane and are distinct from exosomes. I will discuss the molecular
mechanism and physiological function of ARMMs. I will also present most
recent data on how ARMMs may be harnessed for therapeutic delivery of
bioactive protein and RNA molecules. |
15:30 | Companion Animal Studies Advance Understanding of Disease-Relevant Circulating Exosome specific miR Signatures Andrew Hoffman, Professor, Director -- Regenerative Medicine Laboratory, Tufts University, United States of America
Companion animal disease models strongly resemble human diseases and
biofluids from these animals are readily accessible sources of exosomes
for longitudinal studies. We present an analysis of miRNomic data from
circulating exosomes derived from dogs with naturally occurring heart
diseases also found in humans (mitral valve disease, arrythmogenic right
ventricular cardiomyopathy). Target mRNA and gene networks
underpinning miR-target interactions were identified in silico for
candidate miR. The process of functional, biological, and clinical
characterization of circulating exosome specific miR in companion
animals will be discussed. |
16:00 | Circadian Rhythm Modulates the Ability of Pulmonary-derived Extracellular Vesicles to Alter Target Marrow Cell Phenotype Laura Goldberg, Assistant Professor of Medicine, Brown University/Rhode Island Hospital, United States of America
We are interested in how circadian rhythm influences extracellular
vesicle (EV)-mediated inter-cellular communication. To begin exploring
whether circadian oscillations alter EV function, we employed a
well-established in vitro system in which lung-derived EVs, when
co-cultured with murine bone marrow cells, induce the bone marrow cells
to express pulmonary epithelial cell-specific mRNA and protein. Using
this readily manipulated in vitro system, we were able to vary the
circadian time-point of both the lung harvest for EVs and the bone
marrow cell harvest for target marrow cells prior to co-culture. We
found that 1) EVs, when harvested from lung at distinct circadian
time-points, differentially altered the expression of pulmonary
epithelial specific mRNAs in target bone marrow cells in culture, and 2)
altering the circadian time-point of the target whole bone marrow
cells, and co-culturing with lung-derived EVs similarly resulted in
statistically significant differences in pulmonary epithelial mRNA
expression due to circadian oscillations of the recipient marrow cells.
These data indicate that circadian rhythm is likely an important
component of EV-mediated inter-cellular communication. Our ongoing work
is aimed at elucidating the mechanisms by which circadian rhythm
influences EV-mediated communication with bone marrow cells. We hope
such studies will provide insight into the molecular mechanisms by which
EVs alter the mRNA expression profile of target WBM and help optimize
EV manipulations for therapeutic interventions in the future. |
16:30 | Nano-Plasmonic Exosome (nPLEX) Analysis Hyungsoon Im, Associate Professor, Center for Systems Biology, Mass General Hospital (MGH)/Harvard Medical School, United States of America
This presentation will review a recent progress of nPLEX (nano-plasmonic
exosome) technology. The sensor is based on transmission surface
plasmon resonance (SPR) through periodic nanohole arrays.
Target-specific exosome binding to the array causes SPR signal changes,
which enables sensitive and fast detection of exosomes. We applied the
first generation nPLEX system to detect exosomes collected from ovarian
cancer patients. |
17:00 | Microfluidic Isolation of Cancer Cells: Overcoming the Liquid Biopsy Status Quo Silvina Ribeiro-Samy, Researcher, International Iberian Nanotechnology Laboratory (INL), Portugal
Development of a size-based rare cell capture device for point-of-care liquid biopsy that shall maximize the clinical value of circulating biomarkers. |
17:30 | Role of Extracellular Vesicles in Fetal Lung Morphogenesis Mediated by Mechanical Signals Juan Sanchez-Esteban, Associate Professor of Pediatrics, Staff Neonatologist, Women & Infants Hospital of Rhode Island, Brown University, United States of America
Incomplete development of the lung secondary to extreme prematurity or
pulmonary hypoplasia can cause neonatal death and serious long-term
morbidities. Currently, the management of these conditions is primarily
supportive. Lung morphogenesis has significant dependence on mechanical
signals. However, the mechanisms by which mechanical forces accelerate
lung development are not fully-characterized. Extracellular vesicles
(EVs), including exosomes and microvesicles, are increasingly recognized
as a novel mode of cell-to-cell communication. Shedding vesicles are
released from many cell types and have been identified in a variety of
body fluids. Moreover, EVs were found to be important for tissue
morphogenesis in drosophila. However, the role of EVs in fetal lung
development is unexplored. Our preliminary studies show the presence of
EVs in the lumen of the fetal lung. In addition, physiologic levels of
mechanical strain stimulate the release of EVs in fetal lung epithelial
cells. Moreover, incubation of fetal epithelial cells with EVs mimics
the effect of stretch on cell differentiation. These preliminary studies
suggest that signaling mediated by EVs could be important for fetal
lung development. Currently, we are investigating the role of EVs in
fetal lung development using ex vivo and in vivo models. |
18:00 | Inter- and Intra-Tumoral microRNA Heterogeneity Agnieszka Bronisz, Instructor in Neurosurgery, Brigham and Women's Hospital/Harvard Medical School, United States of America
Despite the importance of molecular subtype classification of glioblastoma multiforme (GBM), the extent of extracellular vesicle (EV)-driven molecular and phenotypic re-programming remains poorly understood. To reveal complex subpopulation dynamics within the heterogeneous intra-tumoral ecosystem, we characterized microRNA expression and secretion in phenotypically diverse subpopulations of patient-derived GBM stem-like cells (GSC). As EVs and microRNAs convey information that drives phenotype and re-arrange the molecular landscape in a cell type-specific manner, we argue that intra-tumoral exchange of microRNA augments the heterogeneity of GSC that is reflected in highly heterogeneous profiles of microRNA expression in GBM subtypes. |
18:30 | Terminal Complement Components are Critical in the Release of Cellular RNA in Circulation Ionita Ghiran, , Harvard Medical School/Beth Israel Deaconess Medical Center, United States of America
Despite of over 10 years of intense research, the intimate mechanisms
responsible for extracellular vesicles (EVs) formation (exosomes and
microvesicles), and the release of cellular RNA species (exRNAs) in
circulation are currently known. The complement system is comprised of
over 20 soluble and membrane bound proteins with critical roles in
recognizing, binding, and removal of foreign particles as well as
initiating and regulating innate and acquired immune responses.
Activation of the complement system occurs during both, normal
(circadian variation), and pathological conditions through either
classical, alternative, or lectine pathways leading to the formation and
transient insertion of C5b-9/Mac pore complex into cellular plasma
membrane. We hypothesis that a) MAC-insertion promotes a sudden,
significant and transient water and Ca++ influx, leading to: i)
endocytosis of the affected area, followed by delivery of
C5b-9/MAC-containing plasma membrane into the multi vesicular body
(MVB), and its incorporation into exosomes, or ii) exocytosis of the C9
channle/MAC-affected plasma membrane patch followed by microvesicles
(MVs) formation. In addition, the size of the MAC/C5b-9 pore, 12 nm, is
large enough to: i) allow cytoplasmic RNA species to be transferred into
the MVB following endocytosis of C5b-9/MAC-containing plasma membrane,
and ii) RNA species located near the plasma membrane to be released in
the extracellular space upon C5b-9/MAC insertion. Our results, for the
first time implicate MAC/C5b-9 as: i) a possible channel responsible for
exosomes and microparticle biogenesis, and ii) loading of cytosolic
RNAs into the exosomes, and iii) the direct release of cytoplasmic RNA
species into the circulation (exRNAs). |
19:00 | Close of Day 2 of the Conference |