Liquid Biopsies and Minimally-Invasive Diagnostics 2016 Agenda

Liquid Biopsy Tests Move Towards Routine Patient Care and Management
Walter Koch, Vice President, Roche Molecular Systems, United States of America

Over the past decade several tissue-based companion diagnostic assays have gained FDA approval in conjunction with targeted cancer therapies, resulting in improved treatment options for many cancer patients.  Despite various international guidelines, not all patients are tested, due in part to limited tissue availability, or health status limits on acquiring a re-biopsy sample.  This has spurred development of so-called liquid biopsy tests which use body fluids such as plasma to ascertain tumor mutational status in circulating cell free DNA originating from tumors, or circulating tumor cells.  On June 1, 2016 the FDA-approved the cobas® EGFR Mutation Test v2 real-time PCR test for the qualitative detection of defined mutations of the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) patients. For the first time the test can be used with both formalin-fixed paraffin-embedded tumor tissue (FFPET) as well as circulating-free tumor DNA (cfDNA) from plasma derived from EDTA anti-coagulated peripheral whole blood.  The test is indicated as a companion diagnostic to aid in selecting NSCLC patients for treatment with the targeted therapies including TARCEVA®. This milestone heralds the first of many to come that will provide additional opportunities for cancer patients to be evaluated with blood samples for treatment with targeted therapies.  Analogous to HIV viral load testing, further clinical development will likely extend the use of such blood tests to allow monitoring of initial drug response as well as disease progression and/or development of specific resistance mutations.  Collectively these advances promise to provide important clinical information not easily obtained by repeat tissue biopsy approaches.

Promise of Circulating Tumor Cell and Cell-Free DNA Assays in the Oncology Clinic
Minetta Liu, Associate Professor and Chair, Oncology Research, Mayo Clinic, United States of America

Cell Free DNA, RNA and Exosome Liquid Biopsy Cancer Diagnostics
Michael Heller, Professor, Dept Bioengineering, University of California-San Diego, United States of America

Circulating cell free (ccf) DNA, RNA and exosomes are now considered important biomarkers for liquid biopsy cancer diagnostics, and also hold great promise for early cancer detection. Nevertheless, the isolation of these biomarkers from patient samples requires relatively complex, time consuming and expensive procedures which greatly limits their practical use for most cancer diagnostic applications. New AC dielectrophoretic (DEP) microarray/chip devices now allow 15-20-minute isolation of cancer related ccf-DNA, RNA and exosome biomarkers from 20-50ul of blood, plasma or serum. After isolation of the biomarkers, specific fluorescent dyes can be used first to simultaneously detect the different biomarker levels directly on the chip (in-situ). In a subsequent step, immunofluorescent analysis can be carried out to identify specific protein biomarkers on the exosomes. Finally, the ccf-DNA and RNA (mRNAs and miRNAs release from the exosomes) can be eluted from the DEP chip, and PCR and sequencing analysis carried out to identify the cancer-related point mutations and other polymorphisms, as well as to further verify the tissue origin of the biomarkers. In the case of our Chronic Lymphocytic Leukemia clinical studies, final PCR and DNA sequencing results for the CLL related ccf-DNA isolated by DEP were found to be exactly comparable to two much more complex and time consuming “gold standard” procedures. In the case of glioblastoma exosomes isolated from plasma, exosome-specific surface and interior proteins CD63 and TSG101 could be detected by immunofluorescence, and mutated EGFRvlll mRNA was detected by RT-PCR. Finally, the exosomal related protein biomarker Glypican-1 could be isolated from pancreatic cancer patient plasma samples by DEP and detected on-chip by immunofluorescence. Thus, DEP represents a powerful new minimally invasive technology for cancer diagnostics that is particularly well suited for the rapid isolation of cell free nucleic acid and exosomes.

Diagnosis of Ischemic Heart Disease Using Plasma Extracellular Vesicle Sub-fractions
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 diagnose 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. Using 4 plasma fractions, we show that the plasma extracellular vesicle content in these 4 fractions can be used as an accurate source for early diagnosis of SCAD and UA.

Digital Quantification of Biomarkers in Liquid Biopsies Using the Integrated Comprehensive Droplet Digital Detection (IC 3D) System
Weian Zhao, Associate Professor, Department of Biomedical Engineering, University of California-Irvine, United States of America

We will present a new platform technology that could detect rare blood biomarkers including infections, CTCs, miRNA and exosomes with extremely high sensitivity, specificity, throughput and minimal sample processing.

Circulating Tumor DNA in Advanced-Stage Cancer Patients – Somatic Genomic Landscape and its Clinical Utility
AmirAli Talasaz, Co-Founder, President & COO, Guardant Health, United States of America

Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) enables non-invasive profiling of solid tumor cancers. Liquid biopsy studies to date have been limited to modest-size cohorts and case studies. Somatic genomic profiles of over 15,000 patients with advanced-stage clinical cancer were determined by a highly accurate, deep-coverage ctDNA NGS test targeting 70 genes (Guardant360). Frequencies of somatic ctDNA alterations per gene were compared to those previously described in tissue sequencing projects (e.g., TCGA). Accuracy of ctDNA sequencing (PPV) was assessed by comparing with matched tissue tests. Different classes of clinical outcome benefits have been observed by liquid biopsy by detecting actionable mutations in cases with tissue QNS or actionable mutations emerging at time of progression or under-genotyped tumors.

Integrated Microfluidic Systems for the Isolation of CTCs, cfDNA and Exosomes
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America

Could Precision Medicine be Tailor-Made for Metastatic Head and Neck Cancers
Chamindie Punyadeera, Associate Professor, Institute of Health and Biomedical Innovation, Queensland University of Technology, Australia

Profile CTC Stiffness by Mechanical Separation Chips to Better Predict Breast Cancer Metastasis
Lidong Qin, Professor and CPRIT Scholar, Houston Methodist Research Institute, United States of America

Detection of Circulating Tumor Cells (CTCs) has emerged as a promising minimally invasive prognostic tool to stage cancer metastasis; however, the identification and characterization of CTCs require extremely sensitive and specialized analytical methods, as CTCs are rare, heterogeneous, and associated with a complexity of blood sample matrix. To combat the current CTC detection limitations, we plan to incorporate the filtration method based on a size exclusion capability of microfluidics chips with cocktails of biomarker-specific antibodies to stain and image CTCs. By observing the cell distribution on the chip based on CTCs’ capability in crossing micro-barrier arrays, we can profile the captured CTCs’ stiffness and correlate it to the cancer metastasis potential. We expect to provide a better cancer prognosis tool than counting cells.

Clinical Applications of NanoVelcro Rare-Cell Assays for Detection and Characterization of Circulating Tumor Cells
Hsian-Rong Tseng, Professor, Crump Institute for Molecular Imaging, California NanoSystems Institute, University of California-Los Angeles, United States of America

Circulating tumor cell (CTC) is regarded as a liquid biopsy of tumor, allowing non-invasive, repetitive, and systemic sampling of disease. Although detecting and enumerating CTCs is of prognostic significance in metastatic cancer, it is conceivable that performing molecular and functional characterization on CTCs will reveal unprecedented insight into the pathogenic mechanisms driving lethal disease. Nanomaterial-embedded cancer diagnostic platforms, i.e., NanoVelcro CTC Assays represent a unique rare-cell sorting method that enables detection isolation, and characterization of CTCs in peripheral blood, providing an opportunity to noninvasively monitor disease progression in individual cancer patients. Over the past decade, a series of NanoVelcro CTC Assays has been demonstrated for exploring the full potential of CTCs as a clinical biomarker, including CTC enumeration, phenotyping, genotyping and expression profiling. In this presentation, Dr. Tseng will briefly introduce the development of three generations of NanoVelcro CTC Assays, and highlight the clinical applications of each generation for various types of solid cancers, including prostate cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, and melanoma.

SuperSelective Primers for Multiplex Real-time PCR Assays that assess the Abundance of Rare Mutations Associated with Cancer
Fred Kramer, Professor of Microbiology, Biochemistry & Molecular Genetics, New Jersey Medical School Rutgers University, United States of America

Real-time multiplex PCR assays are potentially the most rapid, most sensitive, and least expensive way to assess the abundance of mutant DNA fragments present in liquid biopsies; provided that a way is found to selectively amplify rare mutant fragments without amplifying abundant related wild-type fragments; and provided that the amplicons generated from different mutants that occur in the same or an adjacent codon are prevented from forming heteroduplexes that interfere with exponential amplification, obscuring the threshold values of the rarer mutants.  “SuperSelective” PCR primers, due to their unique design, are extraordinarily specific, able to selectively initiate the synthesis of amplicons on ten mutant DNA fragments in the presence of 1,000,000 wild-type DNA fragments, even though the only difference between the mutant and the wild-type is a single-nucleotide polymorphism.  Each SuperSelective primer that is specific for a particular mutation possesses a unique 5' tag sequence that is incorporated into the resulting amplicons and detected in real-time by differently colored molecular beacon probes.  Each SuperSelective primer specific for a particular mutation also possesses a unique “bridge” sequence that assures that each primer only copies its intended amplicon, and that creates a single-stranded bubble in heteroduplexes that enables the rarest amplicons to be independently exponentially amplified.  And finally, the inclusion of primers for a wild-type reference gene fragment, enables the abundance of each type of mutant DNA fragment to be assessed (without measuring the amount of DNA in the sample) by determining the difference between its threshold value and the threshold value of the reference gene.

Cell-Free DNA: Potential Pan-Organ Marker for Organ Transplantation
John Sninsky, Chief Scientific Officer, CareDx
Marica Grskovic, , CareDx, United States of America

Patients who have undergone organ transplants require frequent monitoring to evaluate the organ’s graft status and to modulate a complex regimen of immunosuppressive medications to address short and long term clinical needs. Sub-optimal dosing and the threat of allograft rejection must be balanced with excessive dosing and increased risks of infections and cancer. A significant unmet medical need exists for clinical diagnostic tools to permit surveillance management of transplant patients and improve the long-term outcomes of immunosuppressive therapy. Cell-free DNA (cfDNA) has been described as a biomarker for prenatal testing, cancer, and organ transplantation, each of which presents different clinical and technological challenges. cfDNA circulating in the plasma of transplant patients represents a mixture of residual nucleosomal-protected genomic regions released from dying cells of allograft (“transgenome”) and recipient cfDNA. The genomes of the organ donor and allograft recipient are distinguishable by next generation sequencing. Longitudinal samples from heart and kidney transplant patients showed significantly higher donor-derived cfDNA levels prior to biopsy-confirmed rejection which were reduced following adjustments to immunosuppressive therapy. The cfDNA compartment also harbors information about specific infectious diseases and somatic mutations associated with cancer development.  The role of Big Data in tracking organ transplantation outcomes will be discussed.

CSF microRNA Profile as a Liquid Biopsy Platform to Assess Glioblastoma Tumor Burden
Clark Chen, Co-Director, University of California-San Diego, United States of America

Glioblastoma is the most common form of primary brain neoplasm and remains one of the deadliest of human cancers.  Clinical care for glioblastoma patients is largely limited by the absence of biomarker for assessment of tumor burden or therapeutic response. The discovery that glioblastoma cells secrete extra-cellular vesicles (EVs) containing tumor specific genetic material has opened a new platform for biomarker development.  These EVs transgress anatomic compartments and can be detected in clinical bio-fluids. The EVs shelter the tumor-specific genetic material from the extracellular environment that is replete with RNAses and preserve the integrity of these materials. Importantly, the genetic materials within EVs appear highly enriched for RNAs in the size range of microRNAs (miRNAs). The talk will focus on using cerebrospinal fluid (CSF) derived exosomal miRNA signature as a liquid biopsy platform for assessing glioblastoma tumor burden. Issues pertaining to vesicle quantitation, normalization, optimal specimen storage condition, and influence of microenvironment will be discussed.

Carboxypeptidase E: A Biomarker for Detection of Cancer in Circulating Exosomes
Y. Peng Loh, Chief and Senior Investigator, Section on Cellular Neurobiology, Eunice Kennedy Shriver National Institute of Child health and Human Development, National Institutes of Health (NIH), United States of America

Tumor recurrence and metastasis are the major causes of death in cancer patients. Biomarkers that can predict tumor recurrence in cancer patients who are in the early pathology stage and able to receive curative resection will greatly improve survival. To facilitate early cancer diagnosis and as a companion diagnostic to follow the efficacy of the therapy, such biomarkers should be detected not only in resected tumors, but also in serum to provide a non-invasive assay. Accumulating evidence suggest that carboxypeptidase E (CPE), along with its N-terminal truncated derivative, could serve as a powerful prognostic biomarker for predicting recurrence and survival. Over-expression of carboxypeptidase E (CPE) mRNA is common in many different human cancer types including metastatic cervical cancer, renal (clear cell) carcinoma, Ewing sarcoma, glioblastoma and various types of astrocytomas and oligodendrogliomas.

Exploiting the Biology of Exosomes for the Diagnosis and Treatment of Pancreatic Cancer
Raghu Kalluri, Professor and Chairman, Department of Cancer Biology, University of Texas MD Anderson Cancer Center, United States of America

Novel Multiplexing and Precision Sensing Approaches for Liquid Biopsy
Sehyun Shin, Professor & Director, Nano-Biofluignostic Engineering Research Center, Korea University and Anam/Guro Hospital of Korea University, Korea South

Every year, there are about 899,000 new cases and 260,000 mortalities, comprising 6 percent of all cancer deaths globally. Cancer detection is a critical approach for preventing cancer deaths because cases caught early are often more treatable. The development of non-invasive methods to detect and monitor tumors remains a major challenge in oncology. Liquid biopsy such as circulating tumor cells (CTCs), exosomes and circulating tumor DNA (ctDNA) has the potential to provide information about cancers without invasive biopsy. It has been rapidly developing since it yields minimum invasiveness, easy repetitive sampling, and whole mutation representation. These circulating biomarkers can be easily obtained from biofluids such as saliva and blood. Although PCR and NGS (Next Generation Sequencing) based ctDNA detection methods have been introduced, there are still unmet needs to detect ctDNA for clinical applications. We presented a clampSPR (closed-loop amplification PCR-based Surface Plasmon Resonance) sensometry for detecting ctDNAs with multiplexing assay; detect biomolecules with label-free analysis via changes in the refractive index on the sensing film of the SPR sensor in real time. The proposed clampSPR system is similar to real time PCR. After a sample is loaded to the system, it is circulated through a closed-loop channel with passing three different temperature zones by an innovative microfluidic pumping system. Target genes in a sample are amplified as two folds per circulation. Furthermore, encoded hydrogel microparticles were added for multiplexing. This system is fast as 30 minutes for 40 cycles. In addition, it is portable, minimize the sample volume with low cost. Conclusively, our novel approaches can apply to clinical diagnosis of cancer with several advantages such as high sensitivity and multiplexing.

Large Oncosomes: New Frontiers for Cell-to-Cell Communication in 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 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. We show that DNA and RNA-Sequencing can identify tumor specific alterations in large oncosomes circulating in mice and patient plasma. Large-scale profile analyses demonstrate that large oncosomes represent a novel population of EVs enriched in tumor-derived molecules. Large oncosomes are thus valuable candidates for new biomarker profiles to be developed using tissue- and blood-based assays in combination.