Staff Scientist, Ludwig-Maximilians-Universität (LMU), Munich and the Laboratory for Functional Genome Analysis (LAFUGA)
Discover how experts are utilizing technologies such as qPCR, digital PCR and NGS to accelerate the development of liquid biopsies into a leading non-invasive diagnostic test for cancer. Talks focus on circulating biomarkers such as cell-free DNA, (cfDNA), circulating tumor cells (CTCs), and extracellular vesicles (EVs).
“I met leading companies/biomarker developers at one place sharing cutting edge technologies”.
“Interesting mixture of topics; possibility to discuss with the attendees”
“Learned a lot from some of the talks”
THURSDAY 10TH OCTOBER 2019 – LIQUID BIOPSIES; TECHNIQUES AND TECHNOLOGIES
Keynote Address: A universal biomarker for cancer: Are we there yet?
MATT TRAU, Professor of Chemistry, Deputy Director and Co-Founder of AIBN, University of Queensland, Australia
Epigenetic reprogramming in cancer genomes creates a distinct methylation landscape encompassing clustered methylation at regulatory regions separated by large intergenic tracks of hypomethylated regions. This methylation landscape that we referred to as Methylscape is displayed by most cancer types, thus may serve as a universal cancer biomarker. We examine the effect of levels and genomic distribution of methylcytosines on the physicochemical properties of DNA to detect the Methylscape biomarker. We find that DNA polymeric behaviour is strongly affected by differential patterning of methylcytosine, leading to fundamental differences in DNA solvation and DNA-gold affinity between cancerous and normal genomes. We exploit these Methylscape differences to develop simple, highly sensitive and selective electrochemical or colorimetric one-step assays for the detection of cancer. These assays are quick, i.e., analysis time≤10minutes, and require minimal sample preparation and small DNA input from either solid or liquid biopsies.
Multiplexed Digital PCR – a complete workflow for the detection of mutations, therapeutic monitoring and resistance appearance
Id-Solutions will present the outcomes of their scientific partnership with Stilla Technologies to provide a complete solution for liquid biopsies analysis. This talk will cover the entire workflow from cfDNA isolation to DNA quantification and mutations quantitative detection by multiplexed Digital PCR. A focus will be done on: (1) EGFR mutations for targeted therapies on non-small cell lung cancer and (2) KRAS, NRAS and BRAF mutations for colorectal cancer anomalies.
DAVID HENAFF, R&D Manager, ID-Solutions, Stilla Technologies
Morning Refreshments / Poster Presentations / One-to-One Meetings
Considerations when analyzing cell-free tumor DNA
ANDERS STAHLBERG, Associate Professor, University of Gothenburg, Sweden
• Analysis of circulating cell-free tumor DNA (ctDNA) in liquid biopsies offers new means for early cancer diagnostics, real-time monitoring of treatment efficiency and detection of relapse. Despite its potential use ctDNA remains challenging to detect and to quantify as it represents only a small fraction of all cell-free DNA.
• We have developed SiMSen-Seq, that allows allele frequencies < 0.1% to be detected. SiMSen-Seq is simple to perform, flexible in multiplexing and requires minimal DNA input.
• Here, we discuss important considerations for ctDNA analysis in plasma, including all experimental steps from sampling to data interpretation. Furthermore, the use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine.
Standardized analysis of extracellcular vesicles in liquid biopsies
AN HENDRIX, Assistant Professor, University of Ghent, Belgium
The identification of extracellular vesicle (EV)-associated biomarkers is challenging owing to the complexity of liquid biopsies. We 1) performed quality control studies to identify the impact of (pre-) analytical variables on biomarker identification, 2) developed reference materials to ensure standardized EV measurements, and 3) created EV-TRACK to stimulate researchers to put experimental guidelines into practice. This combined expertise boosted the identification of bacterial EV in the systemic circulation of patients with intestinal barrier dysfunction.
Advantages of droplet digital PCR for clinical use and the road to diagnostics
Digital PCR has been rapidly adopted by the research community for the detection and quantification of specific genetic markers. This adoption has mostly been driven by the high sensitivity and accuracy of the method in applications where other quantification methods such as RT-qPCR or next generation sequencing fell short. However, some other characteristics inherent to digital PCR may also be of importance in the adoption of digital PCR for clinical use. Digital PCR provides an absolute quantification method, simplifying the comparison of quantitative measurements across samples, users and instruments. Furthermore, digital PCR is robust and highly reproducible. In this presentation I will discuss and illustrate some examples of the benefits of droplet digital PCR for use in a clinical diagnostic setting.
KOEN DE GELAS, Regional Sales Specialist ddPCR, Bio-Rad, Northern Europe
Innovation in Research: From Protein Array to Companion Diagnostics to Profiling of Adverse Events
DOLORES CAHILL, Professor of Translational Science, University College Dublin, Ireland
Overview of profiling the autoantibody repertoire on high content protein arrays and using this approach to identify biomarkers, and develop diagnostics and companion diagnostics.
Clinical utility of CTC and ctDNA in metastatic and neo/adjuvant setting of breast cancer
JEAN-YVES PIERGA, Institut Curie, Department of Medical Oncology and Circulating Tumor Biomarkers Laboratory, SiRIC, Université Paris Descartes, France
Circulating tumor cells (CTCs) have been identified as potential blood-based biomarker capable of providing prognostic and predictive information in breast cancer. Their applications include early diagnosis, prognostic assessment, detection of minimal residual disease, early detection of cancer relapse and management of metastatic disease. The clinical validity of the CTCbased liquid biopsy has been already assessed by numerous studies. Pooled analysis of large clinical data set in adjuvant, neoadjuvant and metastatic setting are now available. Recent results of clinical trial could support their clinical utility. The potential applications for circulating tumour DNA (ctDNA) in early and metastatic setting include prognosis assessment before treatment initiation, early assessment of treatment efficacy and help to guide personalized therapies.
Liquid Biopsy in Oncology Clinical Trials: Present and Future
IWANKA KOZAREWA, Associate Principal Scientist, Translational Medicine, AstraZeneca, UK
• Liquid biopsy applications in clinical trials
• Challenges and advantages of using liquid biopsy material
• Future of liquid biopsy sequencing in Oncology
Afternoon Refreshments / Poster Presentations / One-to-One Meetings
Translating liquid biopsies into routine care
PANEL DISCUSSION – Speakers include:
• KATARZYNA WITKOWSKA
Commercial Partnerships Manager, Genomics England, UK
• KAREN SPINK
Innovation Lead – Precision Medicine, Innovate UK
• ALEXANDRE HARLÉ
Assistant Professor, Molecular Biologist, Institut de Cancérologie de Lorraine, France
• IWANKA KOZAREWA
Senior Scientist, AstraZeneca, UK
• BERNHARD POLZER
Head Cellular and Molecular Diagnostics, Division Personalized Tumor Therapy, Fraunhofer-Institute for
Toxicology and Experimental Medicine ITEM-R, Germany
• SIMON LUCAS
Innovation Field Explorer, Merck KGaA, Germany
Networking Drinks Reception
FRIDAY 11TH OCTOBER 2019 – CLINICAL APPLICATIONS
Keynote Address: A Liquid Biopsy Assay to detect Cancer
DIANA ANDERSON, Established Chair, Biomedical Sciences, University of Bradford, UK
There appeared to be no single test to identify cancer in general, but we have developed such an assay. In this modified patented Comet assay, we investigated peripheral lymphocytes from blood of 208 individuals, known as the Lymphocyte Genome Sensitivity (LGS) test. Ninety four individuals were controls. All cancers tested exhibited comparable responses. Analyses of Receiver Operating Characteristic (ROC) curves, of mean log Olive tail moments for cancer alone versus controls alone, the area under the curve was 0.93. By varying the threshold for test positivity, its sensitivity or specificity can approach 100% whilst maintaining acceptable
complementary measures. Since lymphocytes in blood only are examined, and the test has been repeated with over 900 individuals with equally valid responses, this is a useful Liquid Biopsy assay.
Liquid biopsies on the road to clinical utility
RALPH GRAESER, Senior Translational Medicine Expert, Boehringer Ingelheim
• Use of liquid biopsies in the clinic: diagnostic, prognostic, predictive?
• CTCs vs ctDNA: one or the other – or both?
• CANCER-id – a public-private partnership with the goal to standardize liquid biopsy protocols for clinical use
Exploiting RNA in liquid biopsies for precision medicine purposes
In contrast to general belief, a substantial part of the human transcriptome is abundantly present in the blood and other biofluids as extracellular messenger RNA, long non-coding RNAs, and various small RNAs, ready to be exploited. I will discuss various workflows for RNA sequencing of biofluid derived RNA, including probe-based target capture and unbiased total RNA library prep as sensitive RNA sequencing workflow to study thousands of mRNA and lncRNA genes in cell-free RNA from patients’ plasma and other biofluids. The resulting RNA profiles can also be deconvoluted to estimate the fraction of the cells and tissues that contribute to the extracellular RNA. Human biofluid RNA sequencing enables liquid biopsy guided precision oncology, such as therapy stratification, treatment response monitoring and early detection of relapse. I will also discuss the pre-analytical jungle of RNA targeted liquid biopsies and need for standardization, as part of the ongoing extracellular RNA quality control study. I will end with showcasing the Human Biofluid RNA Atlas, in which the extracellular transcriptome of 20 human biofluids was established, providing a solid foundation for exploiting biofluids for diagnostic purposes.
JO VANDESOMPELE, CSO, Biogazelle
Morning Refreshments / Poster Presentations / One-to-One Meetings
Novel digital PCR and mutation enrichment technologies for the analysis of clinically relevant DNA alterations in liquid biopsies
MIKE MAKRIGIORGOS, Professor of Radiation Oncology, Dana Farber Cancer Institute and Harvard Medical School, USA
With the increasing interest in treatment assessment using liquid biopsy and circulating DNA, sensitive and multiplexed detection of tumor-derived alterations in blood are desirable. We provide novel forms of digital PCR, as well as mutation enrichment-based real time PCR methods that (a) enable several orders of magnitude improvement of detecting mutations or microsatellite instability than currently possible; (b) are highly multiplex-able; (c) reduce cost of analysis. Application in circulating DNA from clinical cancer samples will be presented.
A targeted multi-analyte liquid biopsy to identify early stage gynecologic cancers
JOHN MARTIGNETTI, Professor, Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, and Director, Laboratory of Translational Research, Western Connecticut Health Network, USA
Overwhelmingly the power and success of “liquid biopsy” has been focused on issues related to advanced disease in already diagnosed patients under active treatment. We demonstrate an approach and the feasibility of using a targeted liquid biopsy approach for detecting early stage gynecologic cancers by combining analysis of both DNA and proteins.
CTC analysis: Latest advancements and clinical applications
EVI LIANIDOU, Professor of Analytical Chemistry – Clinical Chemistry, University of Athens, Greece
• Liquid biopsy provides a valuable source of biomarkers through simple and minimally invasive serial blood draws and represents a highly dynamic diagnostic, prognostic and theranostic tool for the management of cancer patients.
• Circulating tumor cells (CTCs) are major players in liquid biopsy and their molecular characterization offers
an exciting approach to monitor the efficacy of systemic therapies in real-time, unravel the biology of cancer cell dissemination, understand resistance to established therapies and identify gene targets and signalling pathways relevant to therapeutic interventions.
• Single-cell CTC analysis is a powerful tool to understand tumor heterogeneity and the mechanisms involved in cancer progression with potential implications for improving treatment strategies.
• This lecture will be focused on the latest developments in the detection and molecular characterization of CTCs, and their clinical applications in many types of cancer.
Prognostic and predictive value of circulating methylated DNA in metastatic colorectal cancer patients treated with regorafenib
LUDOVIC BARAULT, Senior Research Associate, Candiolo Cancer Institute, FPO-IRCCS, University of Turin, Candiolo (TO), Italy
• Regorafenib is associated with improved progression free survival (PFS) in a subset of metastatic colorectal cancer
(mCRC) patients and no biomarkers of efficacy are available for this drug which is often associated with many toxicities.
• We previously found an association between circulating methylated DNA and outcome in chemotherapy treated mCRC patients and we hypothesized that such biomarker could be used to identify cases most likely to benefit from regorafenib (i.e. patients with PFS longer than 4 months).
• Assessment of pre and on treatment blood samples confirmed the prognostic value of circulating methylated DNA and suggest its use as biomarker for regorafenib since it may predict unresponsiveness to the treatment.
Possibilities of Nanopore Long Read Sequencing in Liquid Biopsy
ANDREAS HAUSER, Staff Scientist, Ludwig-Maximilians-Universität (LMU), Munich and the Laboratory for Functional Genome Analysis (LAFUGA), Germany
As sequencing continues to become cheaper and more realtime, more opportunities arise in liquid biopsies. Long Read Nanopore Sequencing offers especially interesting possibilities. In addition to common short reads benefits, like SNP calling, long reads enable cheap assembly of host or pathogen genomes, identification of structural variation, repeat array resolution. Nanopore sequencing specifically also allows for unamplified sequencing, direct RNA sequencing and methylation calling on DNA and RNA. The currently available devices also allow analysis while running making it possible to continuously monitor or stop until detection of specific sequences.
Chair’s Closing Remarks / Conference Close
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