Circulating Biomarkers: Cell-Free Nucleic Acids, Proteins and Rare Circulating Cells Agenda

Blood-based Testing for Somatic Alterations in Non-Small Cell Lung Cancer
Walter Koch, Vice President, Roche Molecular Systems, United States of America

For cancer patients with inaccessible or unavailable tumor tissue biopsies, an unmet Medical need exists for a surrogate for tissue testing.   New ultra-sensitive PCR and NGS technologies allow analysis of tumor derived nucleic acids and cells in blood with potential applications to therapy selection for targeted therapeutics, assessment of initial therapy response, monitoring for disease progression during treatment, and identification of drug resistance mechanisms that can suggest further treatment options.  This presentation will cover clinical development of a FDA approved assay detecting EGFR mutations in plasma from NSCLC patients to highlight what is possible, what has been achieved, and what challenges remain before Liquid Biopsy applications find routine clinical use.

Exosomes and Their Potential as Therapeutics
Jan Lötvall, Professor Krefting Research Centre, University of Gothenburg, Chief Scientist, Codiak BioSciences; Founding President of ISEV, United States of America

This presentation will discuss the mechanisms by which exosomes can exert their effects in recipient cells, discussing their RNA and protein cargo. Further, the talk will discuss the overall vesicular secretome, including subgroups of extracellular vesicles, such as microvesicles, exosomes, and subpopulations within each group. Lastly, the talk will discuss opportunities of high jacking the exosome cell-to-cell communication pathways to develop the next generation of biologicals with the aim to cure complicated diseases.

Diagnosis of Ischemic Heart Disease Using Plasma Extracellular Vesicles
Dominique PV de Kleijn, Professor Experimental Vascular Surgery, Professor Netherlands Heart Institute, University Medical Center Utrecht, The Netherlands, Netherlands

Cardiovascular Disease (CVD) is with the cardiovascular events of Ischemic Heart Disease and Stroke, the number 1 and 2 cause of death in the world and expect to increase especially in Asia. Ischemic heart disease (IHD) comprises 3 entities: stable coronary artery disease (SCAD), unstable angina (UA) and myocardial infarction (MI). Because IHD is associated with an increased risk of adverse clinical events such as heart failure and death, early recognition of IHD is of utmost importance. However, to diagnosis IHD is challenging, as many patients present with atypical symptoms. It is known that women have a different symptom sensation than men. Troponins are the main diagnostic tool for detection of MI. Blood biomarkers for SCAD (typically causing stable angina) and UA, however, are not available. These diagnoses frequently require hospital visits/admissions for time-consuming and costly (non)invasive tests. We show that the plasma extracellular vesicle content can be used as an accurate source for early diagnosis of SCAD and UA.

New Tools for Liquid Biopsies: Microfluidic Platforms for the Efficient Isolation and Molecular Profiling of Circulating Tumor Cells (CTCs), Cell Free DNA (cfDNA) and Nanovesicles (Exosomes)
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America

Liquid biopsies are generating great interest within the biomedical community due to the simplicity for securing important biomarkers to manage complex diseases, such as many of the cancer-related diseases. These circulating markers consist of CTCs, cfDNA and exosomes. We are developing a suite of microfluidic devices that are can process whole blood directly and engineered to efficiently search for a variety of disease-associated liquid biopsy markers from divergent subpopulations comprising the tumor microenvironment that can supply complementary clinical information. Each microfluidic device can isolate the target with recovery >90% and sufficient purity (>80%) to enable downstream molecular analysis of the particular biomarker. The microfluidic devices are made from thermoplastics via injection molding to allow for mass-production of devices with tight compliancy to accommodate clinical implementation. In this presentation, information will be shared on the operational parameters of these devices for the selection of liquid biopsy markers, and the downstream molecular information that can be garnered from the isolated markers in diseases such as colorectal, ovarian, breast, pancreatic and prostate cancers as well as some of the liquid-based cancers (acute myeloid leukemia).

Saliva Liquid Biopsy (sLB)
David Wong, Felix and Mildred Yip Endowed Chair in Dentistry; Director for UCLA Center for Oral/Head & Neck Oncology Research, University of California-Los Angeles, United States of America

Liquid biopsy is a rapidly emerging field to address this unmet clinical need as diagnostics based on cell-free circulating tumor DNA (ctDNA) can be a surrogate for the entire tumor genome. The use of ctDNA via liquid biopsy will facilitate analysis of tumor genomics that is urgently needed for molecular targeted therapy. Currently, most targeted approaches are based on PCR and/or next generation sequencing (NGS) for liquid biopsy applications with performance concordance in the 70-80% range with biopsy-based genotyping.  “Electric Field Induced Release and Measurement (EFIRM)” is an emerging liquid biopsy technology that provides the most accurate detection that can assist clinical treatment decisions for the most common subtype of lung cancer, non-small cell lung cancer (NSCLC), with tyrosine kinase inhibitors (TKI) that can extend the disease progress free survival period of these patients. EFIRM can detection ctDNA at single copy level whereas ddPCR detects ctDNA a minimum of 10 copy number. In addition EFIRM requires only 40 µl of sample volume, no sample processing, reaction time is 15min and can be performed at the point-of-care or high throughput reference lab using plasma or saliva. In two blinded independent clinical studies, EFIRM detects actionable EGFR mutations in NSCLC patients with >95% concordance with biopsy-based genotyping. EFIRM is minimally/ non-invasive detecting the most common EGFR gene mutations that are treatable with TKI such as Gefitinib or Erlotinib to effectively extend the progression free survival of lung cancer patients.

EVs Add New Dimensions to Cancer Clinic
Xandra Breakefield, Professor, Mass General Hospital (MGH)/Harvard Medical School, United States of America

Critical issues in cancer treatment are early detection, personalized data on gene drivers, and new therapies. In the case of both brain tumors, which are not easily accessible, and peripheral tumors, which may metastasize all over the body, we need to obtain clinically critical information from collective biofluids.  Extracellular vesicles (EVs) have become a clinically relevant resource of biomarker information by providing a means to sensitively detect mutant proteins, DNA and RNA in biofluids, including blood, cerebral spinal fluid (CSF) and urine samples.  Assays and devices to glean this information are emerging as products for clinical analyses. Waiting in the wings, and about to erupt, is the potential to use EVs as therapeutic delivery vehicles which can be derived from the patients’ own cells and target tumors throughout the body.

Plasma RNA Signatures of Cardiac Remodeling in Heart Failure Play a Role in Disease Pathogenesis
Saumya Das, Assistant Professor in Medicine, Beth Israel Deaconess Medical Center and Mass General Hospital, United States of America

In the modern era of revascularization, acute mortality from coronary artery disease has decreased. However, adverse cardiac remodeling associated with ischemic heart disease combined with the explosion of cardiometabolic diseases has led to an epidemic of heart failure. There is a large unmet need to discover novel prognostic biomarkers and functional mediators of cardiac remodeling that may be exploited for novel therapies. Plasma extracellular microRNAs have been characterized as diagnostic biomarkers in cardiovascular diseases. Our work has identified plasma microRNA signatures of cardiac remodeling in patients with chronic heart failure and in patients post myocardial infarction. Bioinformatics and network analyses combined with experimental work has demonstrated that the microRNA components of these signatures are highly interconnected and regulate pathways of inflammation, cell growth and fibrosis. Finally, the use of next generation sequencing techniques to expand the repertoire the plasma RNA biomarkers has identified non-miRNA small RNAs, including snoRNAs, Y-RNAs and tRNA fragments as possible novel biomarkers of cardiovascular diseases.

Microfluidic Enrichment for Single Cancer Cell Analysis
Chwee Teck Lim, NUS Society Chair Professor, Department of Biomedical Engineering, Institute for Health Innovation & Technology (iHealthtech), Mechanobiology Institute, National University of Singapore, Singapore

Tumor heterogeneity is currently a major hindrance to cancer diagnosis and treatment. Here, microfluidic technology is used to enrich and enable probing of the molecular heterogeneity of single circulating tumor cells so as to obtain patient derived information for the personalized treatment of cancer patients.

Tumor heterogeneity is a general trait of cancer which has proven to be a major hindrance in cancer classification, diagnosis and treatment. Cancer can be classified loosely into a few distinct sub-types, but recent technological advances have begun to reveal the true extent of its heterogeneity. Single cell analysis is emerging as an important approach to detect variations in morphology or genetic, proteomic and molecular expression.  Here, we will present several novel microfluidic technologies to probe the heterogeneity of cancer patient derived circulating tumor cells (CTCs).  These include detecting the proteolytic capability of each CTC to obtain hint of their invasiveness as well as identify unique actionable key driver mutations with aim of improving anticancer therapy.  It is hope that this single cell analysis approach will not only lead to more precised treatment of cancer patients from the individually derived information of these tumor cells, but will also aid in the development of better drugs to combat this disease.

Closed Microfluidic PCR-based Surface Plasmon Resonance Biosensor for Multiple Detection of Circulating Tumor DNAs
Sehyun Shin, Professor & Director, Nano-Biofluignostic Engineering Research Center, Korea University and Anam/Guro Hospital of Korea University, Korea South

Circulating tumor DNA (ctDNA) has been demonstrated as the most promising biomarker for non-invasive assessment of cancer as well as the most accurate predictor of cancer treatment responses. However, there are several hundreds of tumor DNAs even for one organ cancer (i.e., Lung cancer) and thus multiplexing is highly required for cancer detection from blood. The conventional techniques have been faced critical limits including multiplexing and cost and thus innovative technologies are highly required. Here, we present a highly precise and selective assay for detecting epidermal growth factor receptor (EGFR) mutations in plasma (or liquid biopsy) using DNA-DNA hybridization and Au nanoparticle probe with a lab-made surface plasmon resonance (SPR) sensometry. Targeted DNAs are amplified in a closed-loop microfluidic PCR module consisting of three different temperature regions. We prepared wild type EGFR, EGFR mutants including point mutation and deletion. Linker DNAs coated on a sensor surface of SPR captured different DNA types. Due to characteristics of SPRi, the whole assay process was monitored in real-time and completed within an hour. This study as a proof of concept can be further expanded into high degree of multiplexing detection of major and known ctDNAs, which could provide a solution for clinical unmet needs in cancer treatment and early detection.

Microfluidics for the Interrogation of Circulating Biomarkers in Glioblastoma Patients
Shannon Stott, Assistant Professor, Massachusetts General Hospital & Harvard Medical School, United States of America

Clinically, there is a dire need to diagnose and monitor brain tumor recurrence and to detect mutations in real time to guide patient treatment. A blood-based ‘liquid biopsy’ that captures and analyzes both circulating tumor cells (CTCs) and extracellular vesicles (EVs) would be an ideal approach to better predict tumor response in glioblastoma patients without the need for highly invasive brain surgery. Through these blood-on-a-chip assays, we aim to gain a better understanding of when these important tumor derived CTCs and extracellular vesicles are released and how we can exploit their molecular content to better guide patient treatment. Working in partnership with Dr. Brian Nahed at MGH, we have used our microfluidic technologies to isolate CTCs and EVs from the blood of patients with advanced glioblastoma multiforme. In this talk, data will be presented on our technological approach as well as our effort to interrogate their molecular content using next generation RNA sequencing. Through the microfluidic isolation of blood based biomarkers from patients, our goal is to obtain complementary data to the current standard of care to help better guide treatment.

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.

Will the Liquid Biopsy Ever Replace Solid Tumor Biopsies?
Phil Stephens, Chief Scientific Officer, Foundation Medicine, United States of America

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.

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.

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.