Circulating cell-free DNA has huge potential to change the way we diagnose, treat, and monitor patients, but there is still much to do to improve assays for routine clinical use. Cambridge Healthtech Institute’s Third Annual Enabling Technologies for Cell-Free DNA will examine the current state of cfDNA technologies and the road toward bringing these technologies to the clinic. This year’s event will focus on the latest technologies being developed, methods for improved sensitivity, specificity, and characterization, and extracting and isolating cfDNA from multiple sample types. Assay and data standardization will also be addressed.

Final Agenda

WEDNESDAY, 12 APRIL

12:30 Registration

Advances in Technologies for cfDNA Mutation Testing and Analysis

13:30 Chairperson’s Opening Remarks

Christa Noehammer, Ph.D., Senior Scientist, Molecular Diagnostics, Austrian Institute of Technology GmbH, Austria

13:35 KEYNOTE PRESENTATION: Quantitative Nuclease-Assisted Minor-Allele Enrichment Simplifies Circulating DNA Analysis

G. Mike Makrigiorgos, Ph.D., Professor, Radiation Oncology, Dana Farber and Harvard Medical School, United States

Circulating DNA analysis presents both opportunities and challenges in harnessing the potential of circulating tumor biomarkers. We present a powerful new development qNaME-PrO-NGS that combines mutation enrichment as well as strict quantification of original mutation abundance by use of molecular barcoding technology. qNaME-PrO enables 100-fold cheaper sequencing on existing sequencing platforms. We present applications in tracing circulating tumor DNA following sequencing of tumor and cfDNA in clinical cancer samples.

14:05 Circulating DNA Analysis as the Next-Generation Diagnostic in Oncology

Alain R. Thierry, Ph.D., Senior Investigator, Research Institute in Oncology of Montpellier, INSERM, France

Based upon observations on circulating cell-free DNA (cfDNA) structural and origin features, we developed an Allele Specific-qPCR-based method with blocker (termed Intplex) which is the first multiplexed test for cfDNA. The Intplex test can be adapted to all mutations, genes or cancers and enables rapid, highly sensitive, cost effective and repetitive analysis. It offers the opportunity in detecting quantitatively and dynamically mutation. We showed the first clinical validation and utility of the analysis of cfDNA in oncology and demonstrated efficient tracking of emergence of resistance clone under cancer treatment. Studies of the evaluation of the cfDNA analysis for following up sage 2-3 cancer patients are ongoing expanding the scope of personalized cancer medicine.

14:35 Simple, Multiplexed, PCR-Based Barcoding of DNA for Ultra-Sensitive Mutation Detection by NGS: SiMSen-Seq

Anders Ståhlberg, Associate Professor, Sahlgrenska Cancer Center, University of Gothenburg, Sweden

Several barcoding strategies have been reported but all require long and complex library preparation protocols. SiMSen-Seq was developed to generate barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (~4 hour) library construction protocol. SiMSen-Seq allows detection of variant alleles at <0.1% frequency with easy customization of library content and a protocol that can be implemented in any molecular biology laboratory.

15:15 Refreshment Break in the Exhibit Hall with Poster Viewing

16:15 Screening for Circulating RAS/RAF Mutations by Multiplex Digital PCR

Rikke Fredslund Andersen, Ph.D., Molecular Biologist, Clinical Biochemistry, Vejle Hospital, Denmark

A multiplex digital PCR method of screening for 31 mutations in the KRAS, NRAS, BRAF, and PIK3CA genes in the plasma will be presented. The method combines a high sensitivity with the ability to analyze for several mutations at a time and could be a step towards routine clinical application of liquid biopsies.

16:45 Non-Invasive Prenatal Testing of Monogenic Disorders by Droplet Digital PCR Combined with Uniformly Most Powerful Likehood Ratio Test

Juliette Nectoux, Pharm.D., Ph.D., Molecular Geneticist, Molecular Genetics, Cochin Hospital, France

We propose a digital PCR approach based on the evaluation of the Uniformly Most Powerful Likehood Ratio Test (UMP LRT) that determines if the dosages of the mutant and wild-type alleles of a disease-causing gene are balanced or unbalanced in maternal plasma. When applied to the testing of women heterozygous for a disease causing mutation, digital UMP LRT allows the fetal genotype to be deduced. This proof-of-concept study of non-invasive droplet dPCR for the analysis of both paternally and maternally inherited fetal alleles demonstrates that NIPT for single-gene disorders is now becoming achievable.

17:15 Nucleic Acid Quantification Technologies and Software Tools

Stefan Rödiger, Ph.D., Group Leader, Institute of Biotechnology, Brandenburg University of Technology Cottbus, Germany

Nucleic acids such as cfDNA have huge potential for diagnose, treatment, and monitoring of patients. However, there is a need for improved assays for routine clinical use. The talk will examine the perspective of a researcher working towards clinical hard and software applications, address challenges we faced with the qPCR methodology and lessons we learned for novel technologies such as digital PCR. In particular, assays and data standardization are briefly addressed in the context of reproducible analysis.

17:45 Close of Day

THURSDAY, 13 April

Detection, Extraction, and Isolation of cfDNA

08:30 Registration and Morning Coffee

09:00 Chairperson’s Remarks

Alain R. Thierry, Ph.D., Senior Investigator, Research Institute in Oncology of Montpellier, INSERM, France

09:05 Impact of Extraction on cfDNA Analysis: Considerations for Sample and Process QC

Alison Devonshire, Ph.D., Science Leader, Nucleic Acid Metrology, LGC, UK

Isolation of cfDNA from biological matrices can have a dramatic impact on the limit of detection and precision of analytical techniques to measure low concentration minority genetic targets such as somatic mutations. This talk will present how control materials have been developed and can be applied to monitoring the efficiency of cfDNA recovery in patient samples and for quality control of different workflows using alternative extraction methods and analytical techniques.

09:35 The Importance of Determining the Limit of Detection of Non-Invasive Prenatal Testing Methods

Francesco Fiorentino, Ph.D., CEO, GENOMA Group, Italy

Several non-invasive prenatal testing (NIPT) methods that rely on quantification of fetal cell-free DNA (cfDNA) suggest a fetal fraction (FF) of 4% or greater for a reportable result. In this study we demonstrated that the minimum FF level necessary for accurate aneuploidy assessment should be related to the actual limit of detection (LOD) of each specific NIPT approach used, and not necessarily fixed at 4% for all cfDNA testing methodologies. This study also underscores the importance of testing samples with low FF.

10:05 Active Extraction of NGS Grade Nucleic Acids from Challenging Clinical Sample Types

Berwyn Clarke, Ph.D., Clinical Diagnostics and Pharmaceutical Industry Manager, Covaris, United Kingdom

Whilst there is great emphasis on NGS as tool for precision medicine and bioinformatic interpretation of NGS data to make it actionable, by comparison much less consideration is given to original sample processing to ensure results reflect the biological reality. Here we discuss a variety of methods to address this.

 10:20 Circulating Cell-Free DNA Pre-Analytics: Importance of Standardized Workflow for Liquid Biopsy Applications

Andrea Ullius, Ph.D., Scientist, PAXgene Product Development, QIAGEN GmbH

Proper, standardized preanalytical method is key to maximizing the accuracy of circulating cell-free DNA (ccfDNA) analysis by minimizing any negative effects on assays caused by the release of genomic DNA from blood cells, and providing reliable and reproducible extraction of the low abundance and short ccfDNA fragments.

 10:35 Coffee Break in the Exhibit Hall with Poster Viewing

11:20 Selected Poster Presentation: A Simple and Robust Real-Time qPCR Method for the Detection of PIK3CA H1047R Mutations

Nicholas R. Leslie, PhD, FRSB, Reader and Associate Professor, Institute of Biological Chemistry, Biophysics and Bioengineering,, Nasmyth Building, Heriot Watt University

Somatic activating mutations in the phosphoinositide 3 kinase (PI3K) catalytic subunit alpha (PIK3CA) have been reported in many human cancers and several PI3K inhibitors are in late stage clinical oncology trials. In most tumour types PIK3CA mutations predominantly cluster within two major hotspot genomic regions: exon 9 (E542K, E545K) and exon 20 (H1047R). Assessing the PIK3CA H1047R status is of particular relevance in breast cancer patients where it represents the most frequently occurring single base mutation in this cancer. Here we describe a highly effective method for PIK3CA H1047R point mutation detection based on a simple SYBR green real-time PCR. This method can effectively detect PIK3CA H1047R mutation by using a set of allele-specific primers that target the mutant variant in combination with a non-productive primer blocking wild-type amplification and an internal control reaction running in parallel. It has been successfully validated using genomic DNA extracted from cancer cell lines and breast cancer tissue biopsies. Overall this approach could become a promising tool for the analysis of PIK3CA H1047R status in cancer patients and given the simplicity of the technique it could potentially be performed in any laboratory with real-time PCR capability.

11:50 Development and Optimization of a Microfluidic System for DNA Extraction and Purification

Joana Carvalho, Ph.D. Student, Environment Monitoring, Security and Food Quality Control, International Iberian Nanotechnology Laboratory, Portugal

The main goal of this work is the development of a portable and integrated DNA analysis device for in situ applications, where is intended to include all the steps of DNA analysis in a single device as a micro total analysis system (µTAS). With this in mind, a microfluidic device has been developed for DNA extraction and purification, which is a critical step for the success of the following DNA analysis stages. The miniaturization of this method brings several advantages compared with the traditional ones, such as portability, and it is possible to adapt this technology for different purposes.

12:20 Enjoy Lunch on Your Own

CHARACTERIZATION OF cfDNA: ADVANCES IN PROFILING AND SAMPLE TYPES

14:20 Chairperson’s Remarks

Stefan Rödiger, Ph.D., Group Leader, Institute of Biotechnology, Brandenburg University of Technology Cottbus, Germany

14:25 Identification of Biologics Treatment-Specific Changes in miRNA Expression in Exosomes of Patients with Chronic Inflammatory Diseases

Jorg Tost, Ph.D., Director, Laboratory for Epigenetics & Environment, Centre National de Genotypage, CEA – Institut de Génomique, France

Exosomes have raised recently considerable interest, but many researchers hesitate to move into the field due to insufficient information about the experimental procedures. We present a detailed experimental, bioinformatic, and statistical procedure to identify a treatment-specific small RNA-signature. While applied to a chronic inflammatory disease, the procedure could also be applied to samples taken non-invasively from cancer patients under a genotype-driven treatment.

14:55 Study of Spatial and Temporal Tumor Heterogeneity Using Copy Number Profiling and Whole Exome Sequencing on cfDNA in Neuroblastoma

Mathieu Chicard, Molecular Biology Engineer, Translational Research in Pediatric Oncology, Institut Curie, France

Neuroblastoma is a paediatric tumor with a very wide heterogeneity. Genetically, copy number alterations and some mutations are observed but no recurrent biomarker is present. To study this heterogeneity, we compared the genomic copy number of tumor and cfDNA using microarray for 70 patients and we also performed WES on cfDNA for 19 patients. For each technique, we observed more alterations in cfDNA than in tumor.

15:25 Next-Generation Sequencing to Detect Tumor Mutations in Circulating DNA: Base-Position Error Rate (BPER) Methodology and Clinical Application in Lung Cancer Patients

Nicolas Pecuchet, M.D., Engineer, R&D Biosphere Technology, Dassault Systemes, France

I will present our latest technical development for mutation detection in circulating DNA using targeted next-generation sequencing: the Base-Position Error Rate method. I will emphasize on the clinical perspectives of the BPER method by presenting the results of a prospective study of ctDNA in advanced Non-Small Cell Lung Cancer patients.

15:55 Detection of the Paternally Inherited Fetal Alleles in the Maternal Plasma using Fast Temperature-Tolerant COLD PCR for the Prenatal Diagnosis of β-Thalassemia

Stefania Byrou, Ph.D. Student, Molecular Genetics Thalassaemia, Cyprus Institute of Neurology and Genetics, Cyprus

Fast Temperature-Tolerant COLD PCR is a rapid and inexpensive technique which allows the enrichment of a minor allele over the overwhelming background of a major allele. A variation of this technique was attempted from our group aiming the detection of paternally inherited fetal alleles in the maternal plasma samples of pregnancies at risk for β-thalassemia. Seventeen maternal plasma samples were analyzed in at least triplicate reactions for two single nucleotide polymorphisms (SNPs) and the fetal paternally inherited allele was correctly detected in 92.96% of reactions tested (66/71) for SNP1 and in 97.34% (73/75) for SNP2. Our results demonstrate the efficiency and sensitivity of Fast Temperature-Tolerant-COLD-PCR in detecting the minor paternally inherited alleles in the maternal plasma.

16:25 Refreshment Break

16:45 Accuracy and Clinical Value of Maternal Incidental Findings During Noninvasive Prenatal Testing for Fetal Aneuploidies

Joris Vermeesch, Ph.D.Ir, Professor, Human Genetics, Laboratory of Cytogenetics and Genome Research, KU Leuven, Belgium

Genome-wide sequencing of cell-free (cf)DNA of pregnant women aims to detect fetal chromosomal imbalances. Because the largest fraction of cfDNA consists of maternal rather than fetal DNA fragments, maternally derived copy-number variants (CNVs) are also measured. Despite their potential clinical relevance, current analyses do not interpret maternal CNVs. We explored the accuracy and clinical value of maternal CNV analysis. We demonstrate that interrogating the maternal CNV landscape can improve overall pregnancy management.

17:15 Circulating Biomarkers and Exosomes in Salivary Diagnostics

Christa Noehammer, Ph.D., Senior Scientist, Molecular Diagnostics, Austrian Institute of Technology GmbH, Austria

Our current focus is to investigate saliva for its suitability for any type of circulating biomarker diagnostics. Along these lines we will present proof of concept data for salivary DNA-methylation - and autoantibody-based biomarkers in a breast cancer patient cohort. Besides reporting on the evaluation of different exosome isolation approaches we will share results from genome-wide DNA-methylation analysis in serum - and saliva-derived exosomes from healthy individuals and present data obtained along genome-wide microRNA analysis from the same sample sources using both microarrays and microRNA sequencing.

17:45 Close of Conference