Liquid Biopsies 2019 Agenda

Mt-SEA Pipeline: High Throughput Flow Cytometric Analysis of Exosomes in Clinical Biofluids
Jennifer Jones, NIH Stadtman Investigator, Head of Transnational Nanobiology, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, United States of America

Because Extracellular Vesicles (EVs) carry surface receptors that are characteristic of their cells of origin, EVs have tremendous potential as non-invasive biomarkers for diagnosis, risk-stratification, treatment selection, and treatment monitoring. We developed a first-in-class pipeline to characterize EV heterogeneity and provide high-sensitivity quantification of informative EVs in biofluids before, during, and after treatment. By combining multiplex assays with high-resolution, single EV flow cytometric methods together into a Mutiplex-to-Single EV Analysis (Mt-SEA) pipeline, we are able to characterize a broad range of relevant EV subsets, while also accurately measuring the concentration of specific EV populations.  Detection of tumor-associated EVs and detection of EV repertoire changes during treatment paves the way to future evaluation of EVs as as biomarkers for use in personalized, adaptive therapies.

Disease-Specific Vascular Markers for Cancer, Atherosclerosis and Brain Diseases
Erkki Ruoslahti, Distinguished Professor and Former President, Sanford Burnham Prebys Medical Discovery Institute, United States of America

We study peptides that home to specific targets in the body. We discover the homing peptides by screening phage libraries for peptides that direct homing of phages to the target tissue in vivo. The homing peptides, which usually bind to receptors in the vessels of the target tissue, can be used to selectively deliver diagnostic probes and drugs to the target. The latest development is the discovery of tumor-penetrating peptides. These peptides activate an endocytic bulk transport pathway we have dubbed the CendR pathway, which can enhance the exit from blood and tissue penetration co-administered compounds even without chemical coupling to the peptide (Ruoslahti, Adv Mater. (2012) 24 3747). We have also identified peptides specific for atherosclerotic plaques, accumulate at the site of a brain injury or recognize the blood vessels in Alzheimer’s disease brain. Our homing frequency have an intrinsic biological active I the disease they target. Examples of the peptide discovery process from initial screening to clinical trials will be given.

Precision Medicine Using Liquid Biopsies: A New Paradigm for Managing Cancer Diseases
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America

Precision medicine seeks to match patients to appropriate therapies that optimize clinical outcome from molecular signatures of their disease. These molecular signatures can be secured from circulating markers found in blood (i.e., liquid biopsies). The marker types include circulating tumor cells (CTCs), cell-free DNA (cfDNA), and nanoscale vesicles (exosomes). Unfortunately, many disease-associated blood markers are a vast minority in a mixed population making them difficult to analyze due to deficiencies in current technologies used for their isolation. To address this deficiency, we are generating innovative microfluidic tools for selecting circulating markers from whole blood and determining the presence/absence of disease-specific molecular signatures secured from the liquid biopsy markers to guide therapy for a patient. The microfluidics can process whole blood (=1 mL) and search for CTCs, cfDNA, or exosomes and make them available for downstream molecular processing. The microfluidics are made in plastics via injection molding, making them particularly attractive for clinical implementation, which demands low-cost disposables. In this keynote address, I will discuss our microfluidic platforms for CTC, cfDNA, and exosome isolation. The exosome chip consists of 1.4 million pillars that contain surface-immobilized antibodies directed against antigens from cancer cells that are epithelial based (EpCAM) and those with a mesenchymal phenotype (fibrobast activation protein alpha, FAPa). I will then talk extensively about our exosome isolation chip, and its use in several clinical examples and securing molecular information from the affinity-selected exosomes.

Exosomes: Novel Opportunities for the Diagnosis and Therapy of Cancer
Lucia Languino, Professor of Cancer Biology, Thomas Jefferson University, United States of America

Our studies have revealed a role for Integrins in mediating extracellular vesicle (EV) functions.  We have demonstrated a cross-talk between prostate cancer cells and monocytes that promotes pro-tumorigenic M2 polarization.  We have shown that this cross-talk is mediated by the alphavß6 integrin in EVs and have suggested that inhibition of this integrin and its downstream effectors might offer a novel immune – based therapeutic strategy in prostate cancer.  We also demonstrate that the EVs circulating in plasma from prostate cancer patients contain higher levels of a different integrin, alphavß3, and CD9 as compared to plasma EVs from age-matched subjects who are not affected by cancer.  We show that the EVs present in plasma from prostate cancer patients contain higher levels of alphavß3 and CD9 as compared to plasma EVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the alphavß3 integrin is expressed in a subset of EVs characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2.  Our results suggest that detecting alphavß3 integrin in prostate cancer patient EVs could be a clinically useful and non-invasive biomarker to follow prostate cancer progression.

Tumor-derived Exosomes Contribute to a Pro-inflammatory Tumor Microenvironment through the Stimulation of Chemokines and Cytokines in Mesenchymal Stromal Cells and Macrophages
Yves A. DeClerck, Professor of Pediatrics and Biochemistry & Molecular Medicine, Children’s Hospital Los Angeles, University of Southern California, United States of America

Exosomes are members of a large family of extracellular vesicles involved in cell-cell communication that play important regulatory functions in physiological and pathological conditions. Using neuroblastoma (NB), the second most common solid tumor in children and a cancer that is highly metastatic to the bone marrow and the liver as a model, we show that NB-derived exosomes contribute to the stimulation of several protumorigenic cytokines and chemokines (IL-6, IL-8, MCP-1, VEGF) by mesenchymal stromal cells (MSC) and macrophages via ERK1/2 activation.  In vivo NB-derived exosomes are also preferentially captured by macrophages at the site of metastasis (bone marrow and liver).  Altogether these cytokines promotes the proliferation of tumor cells, their resistance to chemotherapy and immune escape via STAT3 and ERK1/2 activation.

Nanodiagnostic Tool for Comprehensive Assessment of Cancer in Blood
Shan Wang, Leland T. Edwards Professor in the School of Engineering, Stanford University, United States of America

Blood borne biomarkers, such as secreted proteins, circulating tumor DNA (ctDNA) and circulating tumor cell (CTC), have garnered much attention as minimally invasive cancer biomarkers for the “liquid biopsy” of tumors over the past decade. In this talk, we will describe three magnetic-nanoparticle-based diagnostic tools that have been used to assess cancer in peripheral blood successfully: 1) Stanford spin-off MagArray Inc. has launched REVEAL, a cancer-specific blood test that may aid clinicians in characterizing indeterminate pulmonary nodules (4-30mm) in current smokers aged 25-85. The REVEAL Score is calculated using an algorithm based on the measurement of 3 clinical factors and 3 blood proteins associated with the presence of lung cancer. The assay is optimized to rule out lung cancer with a negative predictive value (NPV) of = 95%.  2) We have developed sequence-specific capture of hypermethylated or mutated genes of interest from cfDNA for early detection of NSCLC and colorectal cancer (CRC). With downstream analysis of PCR products on giant magnetoresistive (GMR) biosensors and high-resolution melt analysis for a highly multiplexable assay, we have achieved a sensitivity of 0.01% mutant/methylated allele fractions. Our optimized assay has the ability to detect down to 16 pg (~4 genome equivalents) of hypermethylated DNA spiked-in to 3.5 mL of plasma. 3) We have utilized the MagSifter and immunocytochemistry combined with automated image analysis and machine learning techniques (random forest, transfer learning) for the enumeration and diagnosis/prognosis of NSCLC derived CTCs in patient samples, which are well suited for therapy selection and monitoring.

Biological Functions and Methods of Detection of Large Oncosomes in Cancer
Dolores Di Vizio, Professor, Cedars Sinai Medical Center, United States of America

Extracellular Vesicles (EVs) are important mediators of intercellular communication pathways that lead to tumor progression, and potential sources for discovery of novel cancer biomarkers. Recent studies have shed light on the existence of different populations of cancer-derived EVs. These heterogeneous EV populations exhibit distinct molecular cargo, thus pointing to the possibility that the various EV populations might play diverse roles in cancer and that this does not happen randomly. However, data attributing cancer specific intercellular functions to given populations of EVs are still limited. A deeper functional, biochemical and molecular characterization of the various EV classes might identify more selective clinical markers, and significantly advance our knowledge of the pathogenesis and disease progression of many cancer types. We demonstrated that atypically large EVs can be shed from highly migratory and metastatic cancer cells. These EVs, named large oncosomes, play distinct functions and contain a specific repertoire of molecules that can be used for detection of tumor-derived cargo in plasma.

Liquid Biopsy in MBC: Real-time Evaluation of Tumor Heterogeneity and Molecular Evolution in Clinical Management
Massimo Cristofanilli, Professor of Medicine, Associate Director of Translational Research and Precision Medicine, Robert H Lurie Comprehensive Cancer Center, Northwestern University, United States of America

Liquid biopsy of solid tumors includes evaluation of both, circulating tumor cells (CTCs) and tumor-derived nucleic acid. Several methodologies demonstrated that both, CTCs enumeration and molecular analysis and, evaluation of circulating tumor DNA (ctDNA) provide a real-time assessment of advanced disease allowing for monitoring of disease heterogeneity. The clinical implementation of such technologies in solid tumors, particularly in breast cancer, demonstrated the ability to elucidate mechanisms of resistance to specific therapies and implement a more personalized, targeted approach to treatment. As such, is necessary to review the current status of liquid biopsy in clinical management of solid malignancies.

Circulating Biomarkers: The New Nanotube-CTC-Microarray Chip for Clinical Impact
Balaji Panchapakesan, Professor, Department of Mechanical Engineering, Worcester Polytechnic Institute (WPI), United States of America

The ability to detect cancer and its heterogeneity non-invasively in the earliest of stages can make a significant clinical impact. Therefore, biopsy of blood and other bodily fluids also called “liquid biopsy” is an emerging concept for non-invasive detection as opposed to surgical methods. Circulating biomarkers is a rapidly growing field of research. Circulating biomarkers include, circulating tumor cells (CTCs), exosomes, circulating tumor DNA (ctDNA), cell-free DNA and RNA (mRNA, miRNA, non-coding RNA, and other RNAs), circulating proteins and metabolites. There are over 400 different methods to capture and analyze circulating biomarkers. However, the clinical impacts of liquid biopsy are yet to be realized. We describe the nanotube-CTC-chip for capture and analysis of circulating tumor biomarkers. Using a micro-array technology, well established in the clinical domain, and utilizing nanosurfaces, we demonstrate the capture and isolation of invasive CTCs, CTC clusters and metastasis initiating cells (MICs). Further, these micro-arrays could be used with little modification to capture exosomes, ctDNA, cfDNA and other protein biomarkers in a single chip. One can also develop nanosequencing techniques using these micro-arrays suggesting a standardized platform for liquid biopsies.

Customized Circulating Tumor DNA Platform for Cancer Research
Cheng-Ho Jimmy Lin, Chief Scientific Officer, Oncology, Natera, United States of America

We have created a ctDNA assay custom-built for treatment monitoring and minimal residual disease (MRD) assessment. Our methodology identifies 16 unique, clonal, somatic variants individualized to each patient’s tumor, followed by multiplex PCR and ultra-deep sequencing for longitudinal ctDNA analysis of whole blood samples. In this talk, I will be presenting data across four cancer types -  including lung, colorectal, bladder, and breast, and show how determining ctDNA status longitudinally can be important for patient management and decision-making.

Multi-Omic Liquid Biopsy Platform
Sam Hanash, Director, McCombs Institute for Cancer Detection and Treatment, University of Texas MD Anderson Cancer Center, United States of America

Multiple technologies are currently being explored for liquid biopsy applications beyond genomics, including proteomics, metabolomics, immunomics and extra-cellular vesicles. The individual and combined contributions of these approaches will be presented.

New Molecular Approaches of Blood Biopsy To Monitor Melanoma Patients Progression During Treatment
David Hoon, Professor, Director Department of Translational Molecular Medicine, John Wayne Cancer Institute, United States of America

Will present new platforms to assess blood biopsy in melanoma patients receiving therapy. New approaches in assessing the utility of CTC and cfNA detection.

Extracellular Vesicles (EVs) For Liquid Biopsy
John Nolan, CEO, Cellarcus Biosciences, Inc., United States of America

Extracellular vesicles (EVs) are released by all cells, and carry molecular from the cell of origin, making them attractive targets for biomarker and liquid biopsy development. In particular, the possibility to interrogate EVs in biofluid to identify tumor-, stroma-, and immune-derived EVs could lead to new biomarkers for disease diagnosis and treatment. However, EVs are heterogeneous, small, and difficult to measure, and commonly used EV analysis techniques lack the specificity and sensitivity required for robust biomarkers. Flow cytometry (FC) is a powerful tool for analyzing single cells and there is interest to use FC to measure individual EVs, but the small size and dim optical signals produced by EVs challenge the sensitivity and resolution of conventional FC-based approaches. We have developed FC-based approaches to detect and measure EVs as small as ~75 nm and cargo to 30 molecules per vesicle. We are using these tools to understand the release of EVs from tumor cells and to detect them in biofluids for biomarker development.