Biofluid Biopsies Agenda

Conference Registration and Breakfast

Coffee Break, Networking, Exhibit and Poster Viewing

Detection of Circulating Tumor DNA by Deep Sequencing
Maximilian Diehn, Assistant Professor, Stanford University School of Medicine, United States of America

I will describe the development and application of CAPP-Seq, a deep sequencing-based method for ultra-sensitive and specific detection of circulating tumor DNA that is broadly applicable.

Networking Lunch in the Exhibit Hall and Poster Viewing

Technology Spotlight:
Droplet Digital PCR and the Liquid Biopsy
Dawne Shelton, Senior Scientist, Bio-Rad Laboratories Digital Biology Center

Targeted therapies in many forms of cancer today have allowed unprecedented progress in the treatment of disease. Despite these advances, we currently lack simple, highly sensitive, yet inexpensive methods to follow the progression of disease with associated genomic markers. Some of the current major limitations are: 1) methods to detect, with confidence, mutational loads below 10%, 2) limited amounts of sample (pg-ng range) per biological specimen, 3) diagnostic turnaround time, and 4) cost. Droplet digital PCR is an inexpensive, rapid, and sensitive method for detecting a wide range of mutational loads in FFPE, cell free DNA and tissue. Absolute counting of target molecules enables precise measurement of input and accurate mutational loads without bias. We’ll summarize work covering the tracking of cell-free tumor DNA during  patient progression of disease and treatment monitoring, early detection in blood,  detection of copy number amplifications in blood, as well as the detection of KRAS mutations using a multiplex screening strategy.

Panel Discussion
Klaus Pantel, Chairman, Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Germany

Panelists:

Mike Heller, PhD -- University of California, San Diego
Thomas Krahn, PhD -- Bayer Pharma AG
Klaus Lücke, PhD -- Gilupi GmbH
Dave Hoon, PhD -- John Wayne Cancer Institute
Dolores DiVizio, MD PhD -- Cedars-Sinai Los Angeles
Max Diehn, MD PhD -- Stanford University

Capturing and Molecular Analysis of CTCs: Potential as Predictive Biomarkers in Clinical Studies
Thomas Krahn, Head of Global Biomarker Research Department, Bayer Pharma Ag, Germany

Obtaining tumor tissue from cancer patients for diagnosis and biomarker assessment for patient selection is challenging. Circulating tumor cells (CTCs) may represent an attractive alternative as a “liquid biopsy” providing real-time information about the patient’s current disease state as well as response to treatment. However, capturing these rare cells from body fluids is a major challenge that still needs improvement. We are exploring technologies to capture larger amounts of tumor cells and to use their molecular profile to predict response to treatment in early clinical trials.  We will present data of a study comparing different CTC capturing technologies. For optimal comparison, CTC preparations from the same patient were used. The required high amounts of sample aliquots and CTCs were obtained e.g. by apheresis.  Today’s standard, the CellSearch® system by Veridex was performed and compared with other antigen-based methods and antigen independent methods. Downstream analysis of the isolated CTCs is performed by highly sensitive protein analysis technologies. Limited capture efficiency is tried to be tackled by cultivation of CTCs. Examples of predictive biomarkers based on CTCs capturing and subsequent molecular analysis will be shared and discussed.

Recovery of Pure Tumor Cell Populations using the DEPArray System: Enabling Molecular Profiling and NGS applications to CTCs and FFPE
Farideh Bischoff, Executive Director, Silicon Biosystems Inc, United States of America

Silicon Biosystems has combined the ability to manipulate individual cells using DEP (dielectrophoresis) technology with high quality image-based cell selection to create the DEPArray™ System. This automated platform enables identification and recovery of specific individual cells of interest from complex, heterogeneous samples; enabling analysis of pure cell populations.  Given that molecular testing of CTCs and FFPE tissue can often be compromised due to specimen size, heterogeneity, and/or the percentage of normal cells present, we have developed methods that enable reliable recovery of pure tumor cell populations that are amenable to downstream molecular profiling and NGS.  We will present results from case studies to support these claims.

Coffee Break, Networking, Exhibit and Poster Viewing

MicroRNAs as Non-Invasive Biomarkers
Martin Beaulieu, Director, Regulus Therapeutics Inc, United States of America

Recent studies show that miRNAs are released from tissues into the circulation and the levels of specific miRNAs can be used as surrogates for the disease tissues. miRNAs are highly stable in the circulation due to the fact that they are protein bound and/or encapsulated in vesicles. We have developed and optimized a high-throughput platform that consistently measures over a hundred miRNAs from less than 200 microliters of serum, plasma and whole blood.  We also developed robust methods enabling miRNA profiling from other biofluid types such cerebrospinal fluid and urine.  Using this optimized platform, we conducted large-scale profiling studies to identify miRNA biomarkers in a wide variety of disease conditions.  We found distinct microRNA signatures distinguishing patients with multiple sclerosis from normal volunteers, patients with high risk of metastasis of colorectal cancer and other malignant diseases. We are also developing a microRNA signature that may be both prognostic and predictive for patients with rheumatoid arthritis.

Networking Reception: Premium Beers, California Wines, and Dinner for All Speakers, Delegates, and Exhibitors. Enjoy an Awesome Evening and Network with your Fellow Delegates.

Close of Day 1 of the Conference

Breakfast and Networking

CTC Applications in Clinical Development: CTC Enumeration, Biomarker Status and Single Cell Analysis
Edith Szafer-Glusman, Senior Research Associate, Genentech Inc, United States of America

Technology Spotlight:
Detection and Quantitative Monitoring of Circulating Tumor DNA in Cancer Patients
Jason Poole, Director, Trovagene Inc

An optimized isolation technique for circulating tumor DNA (ctDNA) makes it possible to detect systemically derived ctDNA in plasma and urine. Using a small footprint capture and enrichment technique, we demonstrate the analytical detection and quantification of these tumor fragments down to a sensitivity of less than 0.01%, creating a non-invasive cancer mutation detection platform using massively parallel sequencing techniques. We are using this proprietary technology platform to establish a quantitative correlation between ctDNA and early detection of therapeutic response, resistance and disease recurrence.

Coffee Break, Networking, Exhibit and Poster Viewing

CTCs and ctDNA as Sample Types: Identification, Enumeration, and Biomarker Analysis, Including High Sensitivity Mutation Analysis Using the CEE-Selector Assay
Lyle Arnold, Chief Scientific Officer, Biocept, United States of America

CTCs and ctDNA promise to be a compelling alternative as well as complementary with solid tumor tissue as a sample type. At Biocept, we are using a patented platform called Cell Enrichment and Extraction (CEETM) for the testing of CTCs. This platform is based on a micro-fluidic capture channel, and associated instrumentation that is manufactured by the Company. We can readily capture, enumeration, and interrogate CTCs for surface proteins using immunocytochemistry (ICC), conduct fluorescent in-situ hybridization (FISH) analysis, and at the same time, interrogate samples for mutations using a unique PCR method called CEE-SelectorTM.  The CEE-SelectorTM, which has sensitivity of better than 1:10,000 (mutant:wild-type), is very effective for the analysis of genetic mutations associated with CTCs and ctDNA.  Using these methodologies, a CTC assay has been commercially launched for breast cancer, OncoCEE-BR, and a series of other assays for lung, gastric, colorectal, melanoma, and prostate cancer will be introduced in the next two years using a combination CTCs and ctDNA as the sample types.

In vivo Isolation of Circulating Tumor Cells as Liquid Biopsy – Overview of Potential Applications as Companion Diagnostic for Target Therapies
Klaus Lücke, CEO & Founder, GILUPI GmbH, Germany

Circulating tumor cells (CTCs) are a promising surrogate marker for disease progression and response to therapy as they can be used as liquid biopsy. CTCs indicate tumor cell extravasation. They are important prerequisites for formation of metastasis. Early disease diagnosis as well as disease monitoring can be provided by the detection and characterization of these rare cells. The GILUPI CellCollector™ offers medical personnel at any point-of-care with the unique opportunity to enrich CTCs in vivo.

Lunch, Networking in the Exhibit Hall, and Poster Viewing

Technology Spotlight:
Detecting Circulating Nucleic Acids by Fluid Biopsy™ and Profiling Somatic Tumors with Next-Gen DNA Sequencing
Jay Therrien, Vice President, Rain Dance Technologies

RainDance Technologies is enabling ground-breaking research with a portfolio of picodroplet-based products for elucidating genetic targets for disease risk predisposition, initial detection, pathology and residual disease research. Hear about how the new ThunderBolts™ Cancer Panel is being deployed to rapidly detect and cost-effectively profile tumor samples and the ultra-sensitive RainDrop™ Digital PCR System is being used for follow-up mutation validation. This combination is compatible with all types of DNA, including FFPE, tissue and plasma and features a streamlined workflow and low overall cost per sample.

Technology Spotlight:
Stabilization of Circulating Tumor Cells (CTC)
Pankaj Singhal, Senior Vice President, Corporate Development & Operations, Biomatrica

Detection of circulating tumor cells in blood has become an invaluable tool in the clinical diagnosis and monitoring of various cancers.  CTCs are rare, fragile and susceptible to degradation, which may immediately occur from the time of sample collection and prior to its arrival in clinical diagnostic laboratories.  Thus, a persistent critical need exists in the research, diagnostics and therapeutics fields for methods that improve cell integrity and longevity of CTCs.

Exosomes and Other Extracellular Vesicles as Future Extracellular RNA Biomarkers
Jan Lötvall, Professor, University of Gothenburg; Founding President of ISEV; Editor-in-Chief, Journal of Extracellular Vesicles, Sweden

Most cells have the capacity to release different types of extracellular vesicles, that typically have an intact cell membrane which protects the cytoplasmic vesicular cargo. These extracellular vesicles include exosomes, microvesicles as well as apoptotic bodies, where the exosomes are the true nano-sized vesicles, with a diameter of 40-100 nm. In 2007, we discovered that exosomes released from mast cells contain both microRNA and mRNA, which subsequently can be transferred from one cell to another to mediate biological effect. Subsequently, it has been shown that exosomes are present in all human body fluids investigated, including serum, plasma, saliva, semen and breast milk. Importantly, the RNA content in exosomes changes when the cells undergo for example oxidative stress, or are transformed to malignant cells.  Importantly, different extracellular vesicles contain different RNA species, as well documented in different studies using different models. Therefore, cell may mediate multiple RNA-mediated signals by shuttling also other extracellular vesicles than exosomes between cells. Also this aspect will be discussed during the presentation.  The exosomal RNA content is also being investigated as biomarkers for different diseases, including malignancies. Several companies are currently investigating the RNA content of exosomes and other extracellular vesicles, to diagnose diseases and to monitor the effects of therapy.  Lastly, exosomes or similar vesicles could be utilized to deliver therapeutic RNAi molecules to diseased cells / tissues.  Thus, siRNA or other RNAi molecules could at least in theory be loaded into exosomes and be delivered either directly into a tumor area to treat that tumor, or systemically if harboring targeting molecules.  Exosomal shuttle RNA promises to be one of the most exciting research field in biology and medicine over the decades to come.

Large Oncosomes and Other Extracellular Vesicles: A Source of Cancer-derived Circulating Markers
Dolores Di Vizio, Professor, Cedars Sinai Medical Center, United States of America

We recently discovered that rapidly migratory, “amoeboid” prostate cancer cells shed large (1-10µm diameter), bioactive extracellular vesicles (EVs), termed large oncosomes. Large oncosomes can be selectively isolated by differential centrifugation and/or by filtration, and analyzed by immuno-flow cytometry with specific size beads in vitro and in vivo. Their quantitation in the circulation reports metastatic disease in patients and animal models (Cancer Res. 2009; Am J Pathol. 2012). In order to profile large oncosomes at a molecular level, we performed next generation sequencing (whole genome and RNA-seq) and quantitative proteomics studies (LC-MS/MS SILAC). We demonstrated the feasibility of RNA-seq analysis of EVs purified from human platelet-poor plasma and have obtained a minimum of 50 million paired end reads per sample. After mapping to RefSeq-annotated human gene loci and quantification as FPKM (fragments per kilobase of exon per million fragments mapped), a number of transcripts were identified as differentially expressed between patients with breast cancer and normal subjects (FDR <0.05). We also performed whole genome paired-end sequencing (Illumina) of large oncosome-derived DNA and demonstrated that single nucleotide mutations, insertions/deletions, and translocations present in donor cells can be identified in large oncosomes. Finally, SILAC profiling of tumor cell-derived large oncosomes in comparison with smaller EVs including exosomes demonstrated enrichment, in large oncosomes, of proteins functionally involved in cell cycle regulation, anti-apoptosis, and cell motility. Among the classes of proteins often identified in EVs, a cohort of these “exosome” biomarkers were significantly enriched in large oncosomes. These findings identify large oncosomes as a novel class of EV that contains clinically relevant biomarkers.

Coffee Break, Networking, Exhibit and Poster Viewing

Extracellular Vesicles and RNA of the Cervico-vaginal Compartment in Health and Retroviral Infection
Kenneth Witwer, Associate Professor, Johns Hopkins University School of Medicine, United States of America

Molecular components of cervico-vaginal secretions may predispose to or protect against infection by HIV and other pathogens. Investigating cervico-vaginal lavage (CVL) samples from a sex trade worker cohort, nanoparticle tracking analysis indicated significant differences in extracellular vesicle abundance in samples from HIV-infected women compared with samples from uninfected individuals. Additionally, profiling of extracellular RNA in CVL and the extracellular vesicle fraction by a medium-throughput stem-loop/hydrolysis probe qPCR platform revealed that several small RNAs, including miR-223, were differentially expressed in health and HIV infection. Our work and a previous report showed that this miRNA hinders HIV replication in CD4+ T-cells and monocyte-lineage cells. To trace the path of specific RNAs from cellular origin to availability at the environmental interface, non-human primate RNA from tissue and vaginal swab samples were compared with lavage RNA. This work demonstrates that extracellular vesicles can be recovered from the vaginal compartment, carry cargo important in innate defenses against infection, and may provide markers of mucosal health.

Isolation, Visualization and Characterization of Exosomes Found in Human Follicular Fluid: A Potentially Important Mechanism for Intra-Follicular Cell-Cell Communication
Shlomit Kenigsberg, Senior Researcher, Create Fertility Centre, Canada

Follicular fluid (FF) provides part of the microenvironment that regulates oocyte development and thus, may play a critical role in oocyte fertilization and embryo development. Intercellular signaling between granulosa cells (GLC), cumulus cells (CM), and the oocyte is required for proper folliculogenesis, ovulation, and hormonal secretion. These signals can be mediated by small membranous vesicles called exosomes that are  50-150nm in size and contain a unique repertoire of proteins and microRNAs (miRNAs). The recently discovered tissue and fluid distributions of exosomes elude to their potential biological relevance. Evidence suggests their involvement in cell-cell signaling. Therefore, we hypothesize that exosomes mediate intercellular signaling within ovarian follicles. Our aim was to isolate and characterize both human FF containing exosomes and-exosomes secreted by granulosa cells in vitro, using a variety of biochemical and cell biology techniques. Body fluid extracellular particle isolation techniques, characterization and visualization, as well as their potential as biomarkers for oocyte quality, will be discussed.

Evaluation of EGFR Mutations in Plasma from NSCLC Patients: Utility in Managing Patients on TKI Therapy
Chris Karlovich, Associate Director, Molecular Diagnostics, Clovis Oncology, Inc., United States of America

We are utilizing blood-based molecular testing in patients who have become resistant to first generation tyrosine kinase inhibitors with the goal of enabling subsequent therapy without need for repeat lung biopsy. The utility of plasma-based EGFR mutational analysis will be described in the context of CO-1686, a novel third-generation TKI that selectively inhibits the EGFR activating and T790M resistance mutations in NSCLC patients.

Current Applications of Circulating Tumor DNA Analysis
Theresa Zhang, Vice President, Research Services, Personal Genome Diagnostics, United States of America

Recent technology advances have expanded our abilities to detect circulating tumor DNA from plasma of cancer patients, enabling a wide range of applications including identification of genetic determinants for targeted therapy, evaluation of early treatment response, monitoring of minimal residual disease and assessment of evolution of resistance in real time.  This talk will discuss novel applications of ctDNA analysis in clinical and pharmaceutical research settings and highlight the major challenges encountered.

Prospects for Noninvasive Liquid Biopsies in Cancer and Other Diseases
Charles Cantor, Chief Scientific Officer, Sequenom Inc, United States of America

It is now clear that body fluids like blood, urine and saliva contain diagnostically useful nucleic acid molecular markers from remote sites in the body. Noninvasive prenatal diagnostics detecting fetal DNA in maternal plasma has become the standard of care. In this lecture, I will show that similar diagnostics seem possible in cancer and eventually should become possible in many other diseases. Because the markers are present at very low concentrations all markers of interest must be analyzed simultaneously from one single sample. Next generation DNA sequencing works well for this application but is still too costly to make many repeat measurements.  Nucleic acid mass spectrometry is a great companion to NGS because these measurements are easy to implement and inexpensive. It should be possible to monitor patients frequently enough by liquid biopsies to create and test dynamical models how how therapy is affecting the disease process.

Close of the Conference.