Clinical Application of Cell-Free DNA

The quest for validation of circulating cell-free DNA for monitoring treatment response, managing drug resistance and enabling early diagnosis has led to the rapid rise of this industry and importance in the medical community. The use of circulating cell-free DNA to replace tissue biopsy in solid cancers is controversial, but it is clear that it will be an essential tool for monitoring disease in blood borne cancers. The field of maternal and fetal medicine has been greatly advanced by the use of circulating fetal DNA, and will provide lessons for the oncology field for clinical and analytical validation and facing regulatory hurdles.

Scientific Advisory Board
Luis A. Diaz, M.D., Associate Professor, Oncology, Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Conference Chairman
Maximilian Diehn, M.D., Ph.D., Assistant Professor, Radiation Oncology, Stanford Cancer Institute, Institute for Stem Cell Biology & Regenerative Medicine, Stanford University
David S. B. Hoon, MSc, Ph.D., Chief of Scientific Intelligence, Director, Molecular Oncology, Director, JWCI Sequencing Center, John Wayne Cancer Institute


SC3: Translating CTCs for Clinical Use

SC5: Establishing the Value of Diagnostic Tests

*Separate registration required


10:30 am Registration

11:15 PLENARY KEYNOTE SESSION: Click here for details

12:50 pm Luncheon Presentation: Sensitive Assays to Detect Mutant ESR1 in Plasma Circulating Tumor DNA

Gaorav Gupta, Ph.D., Assistant Professor, University of North Carolina at Chapel Hill

Mutant estrogen receptor (ESR1) has emerged as a clinically significant mechanism for acquired resistance to endocrine therapy in metastatic breast cancer (MBC). ESR1 mutations are rarely identified in untreated primary breast cancers, but are present in up to 30% of patients with estrogen receptor positive (ER+) MBC who have progressed on multiple prior lines of endocrine therapy. Several of the most commonly identified mutations cluster in the ligand-binding domain of ESR1 – including D538G and Y537S/N/C – and have been postulated to confer endocrine therapy resistance due to ligand-independent activation and/or reduced affinity for estrogen receptor antagonists. Reports have also described additional ESR1 mutations in other parts of the gene that are less prevalent but may also have a clinically significant role in mediating therapeutic resistance. Here, we describe the development and implementation of a multiplexed digital droplet PCR ESR1 assay to perform absolute quantification of variant alleles in ctDNA from a cohort of patient samples with MBC. We demonstrate the ability to detect changes in allelic burden with longitudinal sampling from the same sample. We also report the development and validation of a custom ThunderBolts Open Source assay on the RainDrop platform to screen for additional mutations in the entire ESR1 coding region. Highly sensitive, specific, and accurate assays to detect mutant ESR1 alleles in plasma ctDNA – such as the assays described here – will likely facilitate the optimal and personalized management of patients with ER+ MBC.

1:25 Ice Cream and Cookie Break in the Exhibit Hall with Poster Viewing


1:50 Chairperson’s Opening Remarks

Maximilian Diehn, M.D., Ph.D., Assistant Professor, Radiation Oncology, Stanford Cancer Institute, Institute for Stem Cell Biology & Regenerative Medicine, Stanford University

2:00 KEYNOTE PRESENTATION: A Regulatory Perspective on Tumor Gene Panels

Eunice_Lee Eunice Lee, Ph.D., Chief, Molecular Pathology and Cytology Branch, Division of Molecular Genetics and Pathology, Office of In Vitro Diagnostics and Radiological Health, Center for Devices and Radiological Health, US Food and Drug Administration

High throughput assays are rapidly being employed for oncology applications in the clinical setting. This has introduced new challenges to the regulatory paradigm for in vitro diagnostic devices (IVDs). An overview of the regulatory landscape for IVDs will be provided, including companion and complementary diagnostics. The complexities associated with tumor gene panels intended to evaluate liquid biopsies will also be discussed, highlighting considerations for validating the performance of the assays.

2:30 Multipanel Massive Parallel Sequencing cfDNA in Monitoring Cutaneous Melanoma Progression

Dave_HoonDavid S. B. Hoon, Ph.D., MSc, Chief of Scientific Intelligence; Director, Molecular Oncology; Director, JWCI Sequencing Center, John Wayne Cancer Institute

Analysis of cfDNA gene mutation panel in melanoma patients serial bleeds over several years of follow-up can be very informative on events ongoing during tumor progression. The highly sensitive and approach of multiple gene mutation panel assessment by MPS of cfDNA provides a very comprehensive analysis to allow correlation to real-time clinical events of patients. These retrospective studies demonstrate that cfDNA mutations can arise at different time points during tumor progression. This approach of precision medicine monitoring melanoma progression allows a more accurate real-time assessment of what is transpiring in the patient.

3:00 Monitoring of Circulating Tumor DNA in Diffuse Large B-Cell Lymphoma

Mark_RoschewskiMark Roschewski, M.D., Staff Clinician, Commissioned Corps, United States Public Health Service, Center for Cancer Research, Lymphoid Malignancies Branch, National Cancer Institute, National Institutes of Health

Monitoring treatment response in diffuse large B-cell lymphoma (DLBCL) relies on imaging scans that do not improve survival. Cell-free circulating tumor DNA (ctDNA) can be detected in patients with DLBCL. Next-generation sequencing (NGS)-based assays of ctDNA provide biologic information previously inaccessible and predict early treatment failure and disease relapse prior to CT scans in DLBCL. Will discuss clinical and research applications of ctDNA monitoring in DLBCL.

 3:30 Optimal Optical – Single-Molecule Fluorescence of Cell-Free DNA
Samuel Stavis, Ph.D., Project Leader, CNST, NIST
We optimize a method of using single-molecule fluorescence to measure the size distribution of cell-free DNA. We test the limits of labeling short fragments of DNA with high-contrast fluorescent dyes, develop high-efficiency protocols for sample preparation, and perform high-throughput measurements with a modern fluorescence microscope. With further development, we believe that our approach can maximize the output of useful clinical information from an input of finite measurement resources.

4:00 Refreshment Break in the Exhibit Hall with Poster Viewing


4:40 Chairperson’s Opening Remarks

Luis A. Diaz, M.D., Associate Professor, Oncology, Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Conference Chairman

4:45 Analysis of cfDNA to Monitor Metastatic CRC Patients and Manage Drug Resistance

Scott_KopetzScott Kopetz, M.D., Ph.D., FACP, Associate Professor, Gastrointestinal Medical Oncology, University of Texas MD Anderson Cancer Center

Circulating cell-free DNA provides an opportunity for rapid interrogation of patient responses for metastatic colorectal cancer patients. Mechanisms of resistance and heterogeneity can also be interrogated with this methodology. This lecture will provide illustrative cases and discussion of the opportunities and hurdles for implementation.

5:15 Liquid Biopsy Approaches for Characterizing Cancer Genomes

Victor_VelculescuVictor Velculescu, M.D., Ph.D., Professor, Oncology; Co-Director, Cancer Biology, Johns Hopkins Sidney Kimmel Cancer Center; Co-Founder, Personal Genome Diagnostics

Analyses of cancer genomes have revealed mechanisms underlying tumorigenesis and new avenues for therapeutic intervention. In this presentation, I will discuss lessons learned through the characterization of cancer genome landscapes, challenges in translating these analyses to the clinic, and new technologies that have emerged to analyze molecular alterations in the circulation of cancer patients as cell-free tumor DNA. These approaches have important implications for non-invasive detection and monitoring of human cancer, therapeutic stratification, and identification of mechanisms of resistance to targeted therapies.

5:45 Evaluation of Circulating DNA Analysis in Oncology: Multi-Center Clinical Trials in Europe and Efforts to Standardize Collection of Material and Interpretation of Results

Alain_ThierryAlain R. Thierry, M.D., Director, Research, Research Institute in Oncology of Montpellier, INSERM

Following critical observations in the past 10 years, numerous studies of clinical validation and clinical utility of circulating DNA analysis, especially in oncology, are ongoing. However, no consensus is made in the field toward setting guidelines and better Standard Operating Procedures (SOP). A comprehensive set of rigid criteria outlining the blood collection, handling, plasma preparation, cirDNA extraction and storage conditions are needed. We will discuss our experience in leading various multicenter, prospective, blinded assays.

6:15 Close of Day

6:00 Dinner Short Course Registration


SC14: Liquid Biopsy: Clinical Applications and Business Considerations

*Separate registration required


7:30 am Problem-Solving Breakout Discussions with Continental Breakfast

Cell-Free DNA in Liquid Biopsies

Daniel Wetterskog, Ph.D., Senior Scientific Officer, Centre for Evolution and Cancer, Translational Biology of Urological Cancers Team, Institute of Cancer Research


  • What information about a disease state can be gathered by studying cell-free DNA in liquid biopsies?
  • How can we determine from which tissues of origin/tumour sites cell-free DNA is represented in liquid biopsies?
  • Is there a need for improvements of existing techniques or novel techniques in studying cell-free DNA in liquid biopsies?
  • How can we minimize false negative and positive results?


Integration of cfDNA into Clinical Trial Designs in Adjuvant Therapy

Scott Kopetz, M.D., Ph.D., FACP, Associate Professor, Gastrointestinal Medical Oncology, University of Texas MD Anderson Cancer Center


  • What study designs are possible with the use of cfDNA in the adjuvant setting?
  • What is required for establishing surrogacy for a DFS or OS endpoint?
  • What are the technical considerations for wide scale deployment of a test for minimal residual disease?



8:10 Chairperson’s Opening Remarks

David S. B. Hoon, Ph.D., MSc, Chief of Scientific Intelligence, Director, Molecular Oncology, Director, JWCI Sequencing Center, John Wayne Cancer Institute

Natera8:15 Novel Methods for Detecting CNV at Tumor Fractions < 1%

Solomon Moshkevich, Vice President, Product & Strategy, Natera

Most methods available today for measuring Copy Number Variations (CNV) are limited below tumor VAF of 20%, for single copy gains or losses. Some laboratories make claims about Limits of Detection (LOD) in the context of significant amplification levels (8-50x), but these claims can be misleading if one wants to detect a single copy deletion. Natera has developed a novel method for detection of CNV that demonstrates LOD for single copy gains/losses down to <1%.

8:30 Clinical Applications of Tracking Plasma AR Aberrations in Castration-Resistant Prostate Cancer

Daniel_WetterskogDaniel Wetterskog, Ph.D., Senior Scientific Officer, Centre for Evolution and Cancer, Translational Biology of Urological Cancers Team, Institute of Cancer Research

Our group has been using plasma to interrogate resistance in castration-resistant prostate cancer (CRPC) and develop biomarkers for selecting treatment. Using targeted next-generation sequencing of cfDNA from sequential plasma samples we identified AR mutations emerging with resistance to abiraterone. We also identified a strong association between plasma AR aberrations and resistance to abiraterone in heavily-pretreated CRPC patients. Our data support the clinical utility of cfDNA studies in metastatic prostate cancer.

9:00 Genomic Alterations in Cell-Free DNA and Enzalutamide Resistance in Castration-Resistant Prostate Cancer

Alexander W. Wyatt, Ph.D., Senior Research Scientist, Vancouver Prostate Centre, Assistant Professor, University of British Columbia

The molecular landscape underpinning response to the androgen receptor (AR) antagonist enzalutamide in metastatic castration-resistant prostate cancer (mCRPC) patients is undefined. We collected temporal cfDNA samples from 65 mCRPC patients receiving enzalutamide and performed deep sequencing. AR amplification was associated with primary resistance, as was heavily mutated AR. AR mutations exhibited clonal selection during treatment. At progression cfDNA sequencing revealed mutations or copy number changes in all patients tested, including clinically-actionable alterations in DNA damage repair genes and PI3K pathway genes.

9:30 Transitioning from Research-to Clinical-Grade Sequencing: Organ Transplant Use Case

John Sninsky, Ph.D., CSO, Research & Development, CareDx, Inc.

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. A clinical-grade cfDNA NGS assay was developed to accurately and reproducibly monitor the levels of the “transgenome”. Among key elements of the clinical-grade assay that will be discussed are the use of method proficiency testing, independently sourced analytical reference material and best-in-class bioinformatics and laboratory information management.

Celsee Diagnostics
10:00 Coffee Break in the Exhibit Hallwith Poster Viewing

10:50 Characterizing Clonal Evolution and Treatment Resistance Using Circulating Tumor DNA Analysis

Muhammed_MurtazaMuhammed Murtaza, Ph.D., MBBS., Assistant Professor and Co-Director, Center for Noninvasive Diagnostics, Translational Genomics Research Institute

Analysis of circulating tumor DNA is moving rapidly towards clinical applications such as noninvasive tumor genotyping, monitoring of tumor burden and tracking clonal evolution. However, there is little evidence evaluating the extent of tumor heterogeneity that can be captured in plasma DNA. In this presentation, I will share insights gained from extensive comparison between multi-regional tumor biopsies and serial plasma samples in a patient with breast cancer.

11:20 Circulating Cell-Free DNA: A Novel Gateway to the Genome of Hodgkin/Reed-Sternberg Cells in Hodgkin’s Lymphoma?

Peter_VandenberghePeter Vandenberghe, M.D., Ph.D., Head, Laboratory for the Cytogenetic and Molecular Diagnosis of Malignant Diseases, Center for Human Genetics, University Hospitals Leuven; Professor, Medicine, Human Genetics, KU Leuven

Non-invasive massive parallel sequencing of ccfDNA allows the identification of genomic imbalances in Hodgkin/Reed-Sternberg (HRS) cells in early and advanced stage classical Hodgkin’s lymphoma (cHL). Given the scarcity of HRS cells in cHL biopsies, this observation opens important new perspectives for genetic investigation of cHL, and may facilitate the development of targeted and precision therapy in cHL.

11:50 Dynamic Changes in Circulating Tumor DNA in Urine after Drug Administration

Hatim_HusainHatim Husain, M.D., Physician, Medical Oncology, University of California, San Diego

We have been able to monitor early drug response to anti-EGFR therapy to model tumor lysis. We will be presenting data on pretreatment kinetics and dynamic changes in ctDNA for patients across anti-EGFR therapies and other genotypes in lung cancer. The data presented will be evaluating urine as a modality for monitoring ctDNA.

12:20 pm Liquid Biopsy: Providing Answers that Matter

Veena_SinghVeena Singh, Senior Vice President, Senior Medical Director, Biocept

Liquid biopsies offer the opportunity to interrogate a number of different target sample types, including ctDNA and CTCs. At Biocept both ctDNA and CTCs are used for identifying medically actionable biomarkers to assist in the optimal treatment of patients. Learn more about biomarkers on blood and providing answers that matter.

12:50 Luncheon Presentation: Automation Solutions for Cell Free DNA

Jon Smith, Manager, Application Sciences, Tecan

Ensuring clean, uncontaminated collection of plasma prior to purification of cfDNA is critical to the success of downstream molecular diagnostic assays. Automation of plasma isolation and collection prior to subsequent downstream processing is key for increasing efficiency and reducing costs due to retesting. Here we demonstrate a suite of automation tools for optimizing plasma isolation and streamlining subsequent downstream sample processing.

Biological Dynamics1:20 Innovative Approach to Treatment Response Monitoring in Oncology Using AC Electrokinetics (ACE) Lab-on-Chip Platform

Raj Krishnan, Ph.D., CEO, Biological Dynamics, Inc.

A large number of blood-based nanodiagnostic technologies, such as cfDNA, ctDNA, and exosomes-based assays, are disrupting the healthcare space and driving the next generation of innovation. We have developed a proprietary platform that offers a novel, rapid, and affordable way to isolate and quantify nanoparticles directly from physiological solutions, such as whole blood, plasma or serum using AC Electrokinetics (ACE). These nanoparticles have been shown to be clinically relevant in a number of therapeutic areas, including monitoring treatment response in oncology, transplant health, and reproductive medicine.

1:50 Session Break


2:00 Moderator’s Remarks

John Beeler, Ph.D., Vice President, Business Development, Inivata


  • How much content is valuable? How many genes are necessary or ideal in a gene panel?
  • What are oncologists and patients looking for?
  • How are Doctors using data to make decision?
  • What is the ideal technology to use? Do you need a comprehensive panel upfront? Discussion of technology and utilization
  • How does cost factor in?
  • How does regulatory approval factor in?

Regulation: Abraham Tzou, M.D., Medical Officer, FDA

CLIA New York State: Stephanie Shulman, M.P.H., M.S., M.T. (ASCP), Director, Clinical Laboratory Evaluation Program, Wadsworth Center, New York State Department of Health 

Policy: Donna A. Messner, Ph.D., Senior Vice President, Center for Medical Technology Policy

Oncologist: Liquid Biopsy: Use in Clinical Trials

Barbara_ConleyBarbara A. Conley, M.D., Associate Director, Cancer Diagnosis Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (tentative)

Liquid biopsies offer the promise of interrogating a patient’s cancer without a biopsy, as well as being able to longitudinally sample the molecular profile. Potential uses for “liquid biopsy” in clinical trials include initial diagnosis, target-finding (predictive marker), pharmacodynamics assays and ascertaining development of resistance. Qualities of a fit for purpose liquid biopsy for use in clinical trials for cancer drug development will be presented.

Payer: Girish Putcha, M.D., Ph.D., Director, Laboratory Science, Palmetto GBA (MolDX)

Investor: Eli Casdin, Managing Member, Casdin Capital 

4:05 Close of Conference