Circulating Cell-Free DNA

The development of noninvasive methods to detect and monitor tumors continues to be a bottleneck in oncology research. Circulating cell-free DNA shows great promise, but still requires improvements and additional research before it becomes standard practice. At Cambridge Healthtech Institute’s Fifth Annual Circulating Cell-Free DNA, leading researchers from academia and industry will come together to address advances as well as existing challenges in this rapidly growing field. This year’s event will place emphasis on early detection, next steps in monitoring treatment response, and making cfDNA standard clinical practice.

Thursday, February 15

7:00 am Registration Open and Morning Coffee

CHALLENGES AND SOLUTIONS FOR EARLY DETECTION

8:25 Chairperson’s Opening Remarks

Abhijit Patel, M.D., Ph.D., Associate Professor, Yale University School of Medicine

8:30 Eliminating Barriers in NGS Sample Preparation, Yet Catching the Needles-in-a-Haystack

Mike_MakG. Mike Makrigiorgos, Ph.D., Professor, Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School

With the continuous reduction in sequencing costs, sample preparation for targeted re-sequencing presents a bottle-neck to efficient sequencing. We provide novel mutation enrichment-based sample preparation methods that (a) provide higher sensitivity for detection of rare mutations, and (b) reduce the cost of both sample preparation and re-sequencing by orders of magnitude. Application in circulating DNA from clinical cancer samples will be presented.

9:00 Tracking Therapeutic Response Using an NGS-Based ctDNA Assay

Abhijit_PatelAbhijit Patel, M.D., Ph.D., Associate Professor, Yale University School of Medicine

Our group has developed a simple, inexpensive, and ultrasensitive NGS-based assay that enables highly multiplexed measurement of mutant ctDNA. Data will be presented from ongoing studies to establish the clinical utility of this technology, with a focus on monitoring of therapeutic response.

9:30 Multiplexed Enrichment of Rare Sequence Variants in Tumor and Cell-Free DNA

David_ZhangDavid Yu Zhang, Ph.D., Ted Law Jr., Assistant Professor, Bioengineering, Rice University

Rare DNA sequence variants hold important clinical and biological information, but are challenging for existing methods (e.g. PCR, NGS) to profile in an inexpensive, multiplexed, simple-to-implement, and sequence-general way. Here, we present Blocker Displacement Amplification (BDA), a temperature-robust PCR method that selectively amplifies all sequence variants within a roughly 20nt window by 1000-fold over wildtype sequences, allowing easy detection and quantitation of hundreds of potentials variants originally at <0.1% allele frequency.

10:00cfDNA Challenges and Solutions for Early Detection and Monitoring

Brian Haynes, Ph.D., Associate Director, Bioinformatics, Asuragen, Inc.
NGS analysis of ctDNA is highly centralized, owing to cumbersome workflows, complex assays, and a lack of high-performance kitted solutions. With the benefit of patient-like reference materials for assay familiarization and methods optimization, we developed an ultrasensitive targeted amplicon NGS workflow for profiling somatic mutations in cfDNA. We applied our NGS technology to a cohort of more than 50 cancers with matched plasma and tissue specimens and achieved accurate detection of variants down to 0.1% allele frequency. 

Stratec NEW 10:15 Automated Solution for the Isolation of cfDNA from 4ml of Blood and the Stop-Primer Methode for qPCR Analysis

Christoph Mauracher, Ph.D., Managing Director, STRATEC Consumables GmbH

Circulating cell-free DNA (cfDNA) is of interest in many applications (e.g. fetal DNA in maternal plasma, or liquid biopsies). We developed an automated cfDNA extraction method for the InviGenius Plus which can be combined with our priority Stop-Primer qPCR methode for the analysis of cancer genes.

10:30 Coffee Break in the Exhibit Hall with Poster Viewing

CHALLENGES AND SOLUTIONS FOR EARLY DETECTION

11:15 Real World Experience: Concordance of Genomic Alterations by Next-Generation Sequencing in Tumor Tissue versus Circulating Tumor DNA

Young_Kwang_ChaeYoung Kwang Chae, M.D., MPH, MBA., Assistant Professor of Medicine, Feinberg School of Medicine, Northwestern University

The concordance of genomic alterations between two commercially available ctDNA and tissue biopsies was compared in patients with cancer using paired next-generation sequencing tissue and ctDNA biopsies. Across all genes, concordance between the two platforms was approximately 90%. When only considering genomic alterations in either assay (e.g., excluding wild type/wild type genes), concordance was in the range of 10-20%. Concordant mutations were associated with significantly higher variant allele frequency.

11:45 Biomarkers and Technologies for Early Detection

Michael J. Heller, Ph.D., Distinguished Scientist, OHSU Knight Cancer Early Detection Advanced Research Center (CEDAR); Professor Emeritus, Departments of Nanoengineering and Bioengineering, University of California, San Diego

Point of care (POC) devices for cancer and other molecular diagnostics still present considerable challenges. Cell-free (cf) DNA and exosomal RNA and proteins are now regarded as important biomarkers for liquid biopsy cancer diagnostics, therapy monitoring and hold promise for early cancer detection. New electrokinetic technologies will provide powerful seamless sample to answer devices that will hopefully meet the challenges for liquid biopsy diagnostics and early detection.

Stilla12:15 pm Single Cell Analysis with the Naica System

Laura Cavé, Applications Specialist, Applications & Sales, Stilla Technologies

The Naica System uniquely allows users to image droplets and their contents, both pre- and post-amplification, as well as recover droplets for downstream analysis. We have taken advantage of these features to develop exciting new applications for single cell analysis in mammalian cells and bacteria.

12:30 Session Break

Bio-Rad(1) 12:40 Luncheon Presentation: Inroads into Cancer Patient Care with Droplet Digital PCR

George Karlin-Neumann, Ph.D., Director, Scientific Affairs, Digital Biology Center, Bio-Rad Laboratories

Over the past 5 years, Droplet DigitalTM PCR technology has proven to be a highly sensitive and accurate means for the detection and quantification of nucleic acid markers in 1000’s of labs worldwide. Its widespread adoption in liquid biopsy studies and its demonstrated interlab reproducibility have propelled it into clinical use for cancer patients in cutting-edge diagnostic labs. Where biomarkers of interest are known, ddPCR provides affordable and rapid answers to aid in clinical decision-making.

1:15 Session Break

MONITORING RESIDUAL DISEASE AND THERAPEUTIC RESPONSE

1:55 Chairperson’s 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 The Role of ctDNA in Immunotherapy

Razelle_KurzrockRazelle Kurzrock, Ph.D., M.D., Director, Center for Personalized Cancer Therapy, University of California

Blood-derived circulating tumor DNA (ctDNA) permits genomic sequencing of DNA shed from multiple sites. High ctDNA alteration numbers correlated with improved outcomes post-checkpoint inhibitors (variants of unknown significance ((VUS) >3 versus ≤3), stable disease ≥6 months/partial/complete responses (PR/CR), 45% versus 15%; p = 0.014); median progression-free survival, 23 versus 2.3 months (p = 0.0004) (2-month landmark analysis, responders versus non-responders, VUS>3) Therefore, hyper-mutated ctDNA predicts better outcome after immunotherapy.

2:30 Early Detection of Molecular Residual Disease in Localized Lung Cancer via Circulating Tumor DNA Profiling

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

Circulating tumor DNA (ctDNA) represents a promising biomarker for sensitive, specific, and dynamic detection of disease burden in cancer patients. In this presentation I will describe application of the high throughput sequencing-based Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) method to detection of ctDNA molecular residual disease following curative-intent therapy in early stage lung cancer.

3:00 Cell Free Tumor Derived DNA in the Management of Human Tumors

Chetan_BettegowdaChetan Bettegowda, M.D., Ph.D., Assistant Professor, Neurosurgery and Oncology, Johns Hopkins University School of Medicine

There are no clinically utilized biomarkers for the majority of central nervous system malignancies. Brain tumors infrequently shed tumor-derived DNA into the circulation. This talk will describe ongoing efforts to utilize cerebrospinal fluid as a reservoir for tumor derived DNA and potential applications of such an approach.

3:30 Refreshment Break and Poster Competition Winner Announced in the Exhibit Hall

4:15 Dynamic Monitoring of Circulating Tumor DNA in Non-Hodgkin Lymphoma

Christopher Melani, M.D., Staff Clinician, Center for Cancer Research, National Cancer Institute

Next-generation sequencing-based assays detect circulating tumor DNA allowing assessment of tumor dynamics in blood. Monitoring ctDNA encoding immunoglobulin receptors detects recurrent disease prior to scans in diffuse large B-cell lymphoma. “Liquid biopsies” of ctDNA for somatic mutations address tumor heterogeneity, clonal evolution, and mechanisms of resistance to guide precision treatment. I will describe applications of ctDNA in lymphoma including real-time analysis of tumor dynamics, early disease detection, and precision-directed treatment.

4:45 Early Response Monitoring with Circulating Tumor DNA in Lung Cancer

Hatim_HusainHatim Husain, M.D., Assistant Professor of Medicine, Division of Medical Oncology, Department of Medicine, University of California, San Diego

Non-invasive drug response biomarkers for early assessment of tumor response can enable adaptive therapeutic decision-making for individualized patient treatment and proof-of-concept for target inhibition of tumor cells by investigational drugs. Findings suggest that tyrosine kinase inhibitors induced tumor apoptosis within days of initial patient dosing. We demonstrate that early monitoring of ctDNA by non-invasive sampling provides temporal and quantitative dissection of early early tumor response.

5:15 Panel Discussion: Monitoring Residual Disease and Therapeutic Response

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

5:45 Reception in the Exhibit Hall with Poster Viewing

6:45 Close of Day

Friday, February 16

8:00 am Registration Open and Morning Coffee

LIQUID BIOPSIES BEYOND CTCs AND CTDNA

8:25 Chairperson’s Remarks

Filip Janku, MD, Ph.D., Associate Professor, Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center

8:30 Liquid Biopsies in Cancer: Cell-Free DNA and Beyond

Filip_JankuFilip Janku, M.D., Ph.D., Associate Professor, Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center

Molecular testing of liquid biopsies provide minimally invasive approaches for molecular diagnostics in cancer. Testing of plasma-derived cell-free DNA has been approved by regulatory agencies in selected indications and is widely accepted in clinical and translational research. Novel circulating sources of cancer DNA such as exosomal nucleic acids, nucleic acids from tumor educated platelets are being investigated.

9:00 pm Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA

Christina_Wood-BouwensChristina Wood-Bouwens, Cellular and Molecular Biology, Life Science Research Professional, Stanford University

Single-color digital droplet PCR enables absolute quantification of mutation-bearing cell-free molecules for a fraction of the cost of next generation sequencing; Our novel assay has a molecular limit of detection down to 3 mutation ctDNA molecules, and can used with non-amplified template. I will describe how this technology will be used to create customized mutation assays for individual cancer patients on a high-throughput scale, enabling rapid longitudinal monitoring of ctDNA.

9:30 Novel Nanosensing Technologies for Exosome Detection and Profiling

Cesar_CastroCesar M. Castro, M.D., Director, Cancer Program, MGH Center for Systems Biology, Hematology - Oncology, Massachusetts General Hospital/Harvard Medical School

Exosomes have emerged as highly promising cancer biomarkers because they’re abundant in biofluids, carry proteins and RNA reflecting parental cells, and are stable in circulation. Yet, reliable and sensitive approaches for analyzing exosomes and their molecular information are lacking. This talk will discuss novel nanotechnology sensing platforms developed to analyze exosomal proteins and RNAs directly from clinical specimens and discuss future developments to facilitate their translation into routine clinical use.

10:00 Poster Presentation: A Comprehensive Approach to Sequence-Oriented IsomiR Annotation (CASMIR): Demonstration with IsomiR Profiling in Colorectal Neoplasia

Wilson Wu, PhD., Principal Research Technologist, Gastroenterology & Hepatology, Mayo Clinic

10:30 Coffee Break in the Exhibit Hall with Poster Viewing

LIQUID BIOPSIES BEYOND CTCs AND CTDNA

11:15 Diversity of the Vesicular Secretome

Jan_LötvallJan Lötvall, Professor, Department of Internal Medicine, Göteborg University, Chief Scientist, Codiak BioSciences

All cells release multiple types of extracellular vesicles, that can shuttle proteins, RNA and DNA between cells. The released vesicles are traditionally divided into “exosomes” and “microvesicles”, but current data shows that cells release multiple types vesicles with different morphology and cargo, far beyond the traditional “exosomes” and “microvesicles”. This presentation will discuss the diversity of the “vesicular secretome”, and will suggest subgroupings far beyond the traditional nomenclature.

11:45 Highly Sensitive Tumor Detection and Classification Using Methylome Analysis of Plasma DNA

Daniel_De_CarvalhoDaniel De Carvalho, Ph.D., Associate Professor, Princess Margaret Cancer Centre, University of Toronto

Methylome analysis of cfDNA is highly sensitive and suitable for detecting circulating tumor DNA (ctDNA) in early stage patients. A machine-learning derived classifier using cfDNA methylomes was able to correctly classify plasma samples from patients with seven cancer types and healthy donors. Therefore, methylome analysis of cfDNA can be used for non-invasive early stage detection of ctDNA and robustly classify cancer types.

12:15 Exploiting the Biology of Exosomes for Diagnosis and Treatment of Cancer

Raghu_KalluriRaghu Kalluri, Ph.D., Professor, Chair, The University of Texas MD Anderson Cancer Center, Department of Cancer Biology

Exosomes are detected in the tumor microenvironment and emerging evidence suggests that they play a role in facilitating tumorigenesis by regulating angiogenesis, immunity and metastasis. Circulating exosomes could be used as liquid biopsies and non-invasive biomarkers to potentially inform on early detection and diagnosis of cancer patients. Exosomes can be used for the treatment of cancer. This lecture will highlight some of the recent advances in the area of exosomes biology and their utility in the diagnosis and treatment of cancer.

12:45 Close of Symposium

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