Circulating Biomarkers World Congress 2019 Agenda

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

Coffee Break and Networking in the Exhibit Hall

Networking Lunch in the Exhibit Hall and Poster Viewing

Cancer–Host Crosstalk Through Exosomal miRNA
Shizhen Emily Wang, Associate Professor, Department of Pathology, University of California, San Diego, United States of America

Extracellular microRNAs (miRNAs) that can be detected in the circulation are novel mediators of intercellular communication and are considered emerging biomarkers for human diseases. Using cell-secreted extracellular vesicles (e.g., exosomes) as vehicles, miRNAs secreted by cancer cells can travel to and enter various types of niche cells in primary and pre-metastatic tumor microenvironments. Upon entering niche cells, miRNAs regulate gene expression to prepare the niche for cancer progression. Our lab focuses on defining the roles of breast cancer secreted miRNAs in adapting local and distal niche cells and tissues in cancer progression and metastasis. Through de novo sequencing and PCR of circulating small RNAs in the sera of breast cancer patients we identified circulating miRNAs as biomarkers for cancer progression to metastatic disease. Subsequent mechanistic studies revealed important functions of breast cancer secreted miRNAs in various aspects of cellular behaviors. Our findings reveal (1) vascular leakiness and enhanced metastasis caused by cancer-secreted miR-105, which downregulates endothelial tight junctions; (2) miR-105-induced metabolic plasticity in cancer-associated fibroblasts to convert cancer-produced metabolic wastes (e.g., lactate and ammonia) into energy-rich metabolites to re-enter cancer bioenergetics; (3) suppressed glucose utilization by lung fibroblasts and astrocytes in favor of glucose uptake by metastasized cancer cells mediated by cancer-secreted miR-122.

A Straightforward, Automated and Standardizable Approach to Purifying and Characterizing the Biophysical Properties of EVs; Size, Concentration and Surface Charge
Shayne Harrel, Chief Scientist-Americas, Izon Science US LTD

The ever increasing discovery of the role of extracellular vesicles (EVs) in a wide range of biological roles and functions has lead them to be realized as a prime candidate for the next generation platform of medical diagnostics and therapeutics.  As such, an accurate, repeatable and standardizable approach to measuring the biophysical properties of these particles such as concentration, particle size and surface charge and correlating these biophysical properties to biological role and function is desperately required.  This presentation will focus on recent developments at Izon Science which have created an integrated methodology for the straightforward, automated purification of EVs and subsequent characterization of their biophysical properties.

Modulation of Extracellular Vesicle Release and Content in SCC Cells
My Mahoney, Professor and Vice Chair, Thomas Jefferson University, United States of America

Emerging as critical in the pathobiology of cancer are nanosized extracellular membrane vesicles (EVs) secreted by tumor cells into the blood and other bodily fluids carrying molecular constituents to modulate local and distant tumor microenvironment. EVs can be taken up by recipient cells and can modulate diverse biological processes including cell polarity and tissue morphogenesis and represent novel and valuable circulating biomarkers for tumor prognosis and diagnosis. Thus, defining factors that affect the release or modify the content of EVs is critical to our understanding of not only normal tissue development but also malignant transformation and progression. We have shown that the cadherin desmoglein 2 (Dsg2), an important regulator of growth and survival signaling pathways, drives tumorigenesis in experimental models, and clinical samples confirm that Dsg2 is indeed overexpressed in those cancers. We observed that Dsg2 is enriched in EVs derived from SCC cells and patient sera. Dsg2 modulates EV release and mitogenic content including EGFR and c-Src. Dsg2-labeled EVs are taken up by dermal fibroblasts, activating Erk1/2 and Akt signaling and promoting cell proliferation. Our work thus far defines a mechanism by which cancer cells can modulate the tumor microenvironment, a step critical for tumor progression.

Coffee Break and Networking in the Exhibit Hall

Nano-Flow Cytometry: Next-Generation Platform for Comprehensive EV Analysis
Dimitri Aubert, Managing Director, NanoFCM Co., Ltd

Though of great importance, sizing and molecular profiling of individual extracellular vesicles (EVs) are technically challenging due to their nanoscale particle size, minute quantity of analytes, and overall heterogeneity. NanoFCM has developed Nano-Flow Cytometry (nFCM), a technology that allows light scattering and fluorescence detection of single EVs down to 40 nm. nFCM-based approach for quantitative multiparameter analysis of EVs, which is highly desirable to decipher their biological functions and promote the development of EV-based liquid biopsy and therapeutics.

A Novel Extracellular Vesicle Isolation Method Used to Discover Urine Liver Disease Candidate Biomarkers
Shannon Pendergrast, Chief Scientific Officer, Ymir Genomics, United States of America

Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide, with increasing incidence, and is the third most common cause of cancer death. The clinical management of patients suffering from HCC, despite the fact that at-risk populations are well defined, is not satisfactory. This is mainly due to the unavailability of current diagnostics to predict and monitor the disease. OHSU and Ymir Genomics LLC have teamed up to develop a high-compliance urinary diagnostic for liver diseases, including HCC. We have developed a new method for the rapid and effective isolation of urine extracellular vesicles (EVs) and integrated that into Mass Spectrometry and miRNA array workflows. We have applied  the workflow to 3 groups of 20 urine samples each from 1) healthy controls, 2) patients with cirrhosis, and 3) and cirrhotic patients with untreated HCC. Mass Spectrometry data shows that this protocol is capable of identifying over 2500 proteins from only 2 mls of urine; far less sample than some previously reported urine EV proteomics studies. Our protocol also yields sensitive miRNA detection from the Firefly miRNA array. Preliminary data establishes proof-of-concept by identifying multiple liver-selective and known liver disease biomarkers in urine EV preps. Several candidate protein and miRNA biomarkers that are potentially capable of distinguishing disease from healthy groups have also been identified. The methods developed in this study are suitable for a larger longitudinal study, as they are already compatible with standard clinical chemistry laboratory assay requirements.  Hopefully, candidates biomarkers identified in this study will provide clinicians with powerful weapons in the fight against HCC.

EV Biomarker Detection using Particle Metrix ZetaView® Instruments and Fluorescent Nanoparticle Tracking Analysis (F-NTA)
Sven Rudolf Kreutel, Chief Executive Officer, Particle Metrix GmbH and CEO, Particle Metrix Inc., USA

Low Coverage, Genome-Wide Sequencing of Cell-Free DNA Enables the Monitoring of Response to Immunotherapy in Cancer Patients
Taylor Jensen, Director, Research and Development, Sequenom (a LabCorp Company), United States of America

Inhibitors of the PD-1/PD-L1/CTLA4 immune checkpoint pathway have revolutionized cancer treatment with a subset of patients showing durable responses; however, challenges remain in the development of biomarkers to predict or monitor response to these therapies.  The use of cell-free DNA (cfDNA) isolated from plasma, or liquid biopsy, provides a promising method for monitoring response.  In contrast to methods that use ultra-deep (>30,000X) targeted sequencing, we will describe a recently completed a proof-of-concept study using low-coverage (~0.3X), genome-wide sequencing of cfDNA to detect tumor-specific copy number alterations.  As part of this study, we have developed a novel metric, the Genome Instability Number (GIN), to monitor response to these drugs throughout treatment.  In a series of case studies, we will describe how the GIN can be used to discriminate clinical response from progression, differentiate progression from pseudoprogression and identify hyperprogressive disease.  In addition, we have utilized this metric to provide evidence for a delayed pharmacokinetic response for checkpoint inhibitors relative to targeted therapies.

Networking Reception with Beer and Wine in the Exhibit Hall

Close of Day 1 of the Conference.

Start of Exosomes/Extracellular Vesicles (EV) Training Course [Separate Registration Required to Attend this Training Course]

Morning Coffee Break and Breakfast Pastries Served in the Exhibit Hall

Silicon Chip Nanofluidics for Extracellular Vesicle Enrichment
Benjamin H. Wunsch, Research Staff Member, T.J. Watson Research Center, IBM, United States of America

Silicon lithography provides a high resolution and scalable route to fabricating nanofluidic structures on a chip. We describe a silicon chip based on the nanoscale deterministic lateral displacement (nanoDLD), for enriching extracellular vesicles from biological fluids based on size. We report on our pilot efforts to use high- throughput extraction of EVs from urine and serum to aid in the identification of cancer-specific biomarkers.

Coffee Break and Networking in the Exhibit Hall

Networking Lunch in the Exhibit Hall and Poster Viewing

Afternoon Session Presentations in the EV Summit Track

Industry Panel Discussion Focusing on Circulating Biomarkers & Liquid Biopsy

Close of Day 2 of the Conference Track

Morning Coffee and Breakfast Pastries Served in the Exhibit Hall

The Unique Challenges and Opportunities of Circulating Tumor DNA
Alex Lovejoy, Principal Scientist, Roche Sequencing Solutions, United States of America

Despite the promise of circulating tumor DNA (ctDNA) as a minimally invasive biomarker, the detection and quantification of small amounts of ctDNA in a plasma sample is a difficult problem.  Limited input molecules, background noise, and cost of sequencing are all challenges that hinder the implementation of assays for ctDNA detection.  This talk will describe these difficulties in detail, as well as the development of the Roche AVENIO ctDNA Analysis Kits (Research Use Only), which have been specifically designed to overcome these challenges.

exRNA Biomarkers: Challenges in Methodology
Srimeenakshi Srinivasan, Research Fellow, University of California, San Diego, United States of America

The study of exRNAs and their roles in biological functions and potential biomarkers require robust and reproducible methods for quantification of exRNAs in biofluids, a task that is made more challenging by multiple sources of technical and biological variability. A number of factors including the variety of cells/tissues from which the exRNAs are produced, the low concentrations of exRNAs in the biofluids, the presence nucleases and association with multiple different extracellular compartments impact the reproducibility of exRNA measurements, and are likely a significant reason that the field has encountered difficulties with validation of findings both within and among different research groups. Here we compare various exRNA isolation methods used on standardized samples in multiple laboratories and evaluate their performance using qRT-PCR and small RNA sequencing. The results show that different methods enrich for different subsets of exRNAs depending on their association with extracellular carriers and the reproducibility of each method varies widely. Thus selection of the RNA isolation method is a critical consideration to be taken into account when planning a study to identify exRNA biomarkers.

Identification and Characterization of Circulating Long Non-Coding RNAs and HPV16/18 Cell-Free DNA as Cancer Biomarkers from Serum of Cervical Cancer Patients
Tawin Lempridee, Researcher, National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency, Thailand

Circulating lncRNAs and HPV16/18 E7 ctDNA may be used as cancer biomarkers for developing a blood-based cervical cancer screening test.

Probing the Extracellular RNA Landscape in Neurodegenerative Disease
Elizabeth Hutchins, Computational Scientist, The Translational Genomics Research Institute (TGen), United States of America

Molecular information from biofluids can be used to monitor and understand disease progression, identify biomarkers, develop assays, and for therapeutic discovery. The minimally invasive nature for collecting extracellular information from biofluids allows for frequent sampling, with the potential to closely monitor pathophysiological mechanisms of traumatic injury and disease. We have isolated and identified many types of extracellular RNA from biofluids using high-throughput RNA sequencing, including microRNA, protein coding RNA, lncRNA, and circRNA.

Tumor-derived Extracellular Vesicles Require ß1 Integrins to Promote Anchorage-Independent Growth
Rachel DeRita, Researcher, Thomas Jefferson University, United States of America

The ß1 integrins, known to promote cancer progression, are abundant in extracellular vesicles (EVs).  We investigated whether prostate cancer (PrCa) EVs affect anchorage-independent growth and whether ß1 integrins are required for this effect.  Specifically using a cell line-based genetic rescue and an in vivo PrCa model, we show that gradient purified small EVs (sEVs) from either cancer cells or blood from tumor-bearing TRAMP mice promote anchorage-independent growth of PrCa cells.  In contrast, sEVs from cultured PrCa cells harboring a shRNA to ß1, from wild-type mice or from TRAMP mice carrying a ß1 conditional ablation in the prostatic epithelium (ß1pc-/-), do not.  We find that sEVs, from cancer cells or TRAMP blood, are functional and co-express ß1 and sEV markers; in contrast, sEVs from ß1pc-/- /TRAMP or wild-type mice lack ß1 and sEV markers.  Our results demonstrate that ß1 integrins in tumor-derived sEVs are required for stimulation of anchorage-independent growth.

Extracellular Vesicles From Prostate Cancer Cells Promote Angiogenesis Through alphavß6 Integrin
Shiv Ram Krishn, Post-doctoral Fellow, Thomas Jefferson University, United States of America

Increased angiogenesis in prostate tumors has been shown to be promoted by cancer cells, but the underlying mechanisms are not completely understood.  Prostate cancer (PrCa) cells continuously release extracellular vesicles (EV) of endosomal origin, to mediate unique crosstalk with other cells in the tumor microenvironment by direct transfer of vesicular cargo.  Our group has shown expression of alphav-beta6 integrin (alphavß6), which is an epithelial-specific integrin, in human PrCa cell-derived EV.  We hypothesized that extracellular vesicles-associated alphavß6 (EV-alphavß6) shed by PrCa cells may impact the endothelial cells in the prostate tumor microenvironment and thus angiogenesis.  Here in, we demonstrate that EV-alphavß6 is efficiently transferred to alphavß6-negative recipient human microvascular endothelial cells (HMEC1).  We also demonstrate the intracellular localization of ß6-GFP transferred via PrCa cell-derived EVs.  De novo alphavß6 expression in HMEC1 is not a result of a change in mRNA levels but is a consequence of EV-mediated transfer of alphavß6 from PrCa cells to HMEC1.  Incubation of HMEC1 with purified EV from PC3 cells, transfected with shRNA to ß6, results in a significant reduction in viability and motility of HMEC1.  Our results also demonstrate that EV-alphavß6 significantly augments tube formation of HMEC1.  At the molecular level, our results demonstrate that transfer of PrCa cell-derived EV-alphavß6 to HMEC1 causes down-regulation of STAT1, a molecule known to negatively regulate angiogenesis.  Overall, our study suggests that EV-mediated transfer of alphavß6 from PrCa cells to endothelial cells may regulate angiogenesis during PrCa progression.

Close of Conference