Sensitive, Easy And Fast Monitoring Of Treatment Efficiency, Resistance Development And Disease Progression Using Open Platform QPCR On Liquid Biopsies From Cancer Patients

Ulf Bech Christensen, Rasmus Koefoed Petersen
PentaBase, Denmark

Personalized administration of present and future targeted cancer therapies demands fast, low cost and easy-to-use companion diagnostics (CDx) providing doctors and patients with the answers they need to maximize treatment efficiency and minimize treatment costs. We have developed and clinically validated several real-time PCR based assay for liquid biopsies. Sensitivity studies show that our SensiScreen® Liquid assays can detect down to a single copy of mutated DNA in a background of wild type plasma cell-free DNA (cfDNA) in a simple and robust workflow.
The extreme sensitivity of our assays is obtained by use of BaseBlockers™, that suppress amplification of wild type DNA while allowing amplification of mutated DNA, modified primers and sensitive dual-labelled Probes. BaseBlockers™, primers and probes are all based on the DNA platform technology – Intercalating Nucleic Acid, INA®.
We will present a case study using our SensiScreen® Liquid CE IVD Assay for monitoring disease progression and treatment efficiency in a clinical setting, in a BRAF V600E mutated metastatic colorectal cancer patient. After initial diagnosis and genotyping, we have monitored the patient’s cfDNA levels and the cfDNA amount of BRAF V600E in plasma for more than 700 days during three different regimes of treatment and compared with carcinoembryonic antigen and CA 19-9 levels.
We believe that sensitive monitoring of liquid-based biopsies present unique opportunities for healthcare providers, targeted therapy developers, and eventually cancer patients. The information and additional knowledge gained on patient response and efficacy of treatment can be used as a tool to reduce use of inefficient treatment and improve clinical output.

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Exosome-derived Epigenetic Biomarkers for Saliva Diagnostics

Christa Nöhammer1,
Ulrike Kegler1, Manuela Hofner1, Anja Buhmann1, Helene Scharkosi1, Walter Pulverer1, Michael Leutner2, Klemens Vierlinger1, Alexandra Kautzky-Willer2, Christa Nöhammer1
1) AIT Austrian Institute of Technology GmbH, Molecular Diagnostics, Vienna, Austria;
2) Medical University Vienna, Division for Endocrinology and Metabolism, Vienna, Austria;

The aim of our research activities at AIT, the Austrian Institute of Technology, is to define reliable biomarkers suitable for early and non-invasive disease diagnosis and prognosis. To this end we have been establishing and optimizing a whole range of multiplex capability technologies (e.g. microarrays, quantitative PCR, Luminex bead technology) to meet the special demands and challenges of diagnostic biomarker discovery – and validation in body fluids. Using this specific technology expertise we e.g. successfully discovered autoantibody- as well as DNA methylation -based diagnostic marker panels for the big 4 cancer entities (breast, colon, prostate, lung) in serum or plasma. Based on these success stories and the evident advantages of saliva as a diagnostic matrix our recent special interest is to go for saliva diagnostics and to evaluate saliva for its suitability for circulating biomarker-based diagnostics. Along these lines we will show proof of concept studies for autoantibody- and DNA-methylation based salivary diagnostics and report on the evaluation of different commercially available strategies for isolation of exosomes from human serum and saliva. We will further present data from comparative profiling studies in salivary – and serum-derived exosomes including targeted protein-, genome-wide microRNA – as well as DNA-methylation profiling. Last but not least we will report on first results of a research project where we are looking for salivary and plasma exosome-derived epigenetic biomarkers for early type 2 diabetes diagnosis.

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New Developments in Sample Quality Control – Focus on Cell-free DNA

Bettina Strauch, Elisa Viering
Agilent Technologies, Germany

Quality control of nucleic acid starting material is essential to ensure the success of downstream experiments. Especially, Next Generation Sequencing (NGS) developed to a powerful tool in almost all genetic research and diagnostic areas. Due to the establishment of low input library protocols for NGS workflows sequencing of cell-free DNA (cfDNA) became possible. Since the downstream applications are often time-consuming and expensive, tight QC steps are required to avoid a “garbage in-garbage out” situation. This talk focuses on standardized nucleic acid quality assessment using different automated electrophoresis platforms to ensure that samples are “fit for purpose”. Accurate quantification of starting material (DNA, RNA, cfDNA, FFPE samples) is essential to determine suitable input amounts for library preparation prior to sequencing. Depending on preanalytical sample treatment or extraction methods the quality of e.g. cfDNA can vary. This results in various electropherogram patterns after electrophoretic separation as presented in this talk. Moreover, the electrophoretic separation enables to qualify cfDNA samples according to their contamination level with high molecular weight material. Likewise, quality scores for gDNA, RNA as well as FFPE RNA can be assessed using automated electrophoresis systems, which allow defining a quality threshold for specific types of samples or preparation. This allows defining thresholds for objective sample qualification prior to library preparation.

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Liquid Biopsy — Exosomal microRNA Biomarker Signatures in Clinical Diagnostics

Michael W Pfaffl
Division of Animal Physiology & Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Germany

Extracellular vesicles (EVs) circulate in body liquids and are involved in intercellular communication. They have important regulative functions in almost any physiological or pathological process. In recent time especially exosomes have gained huge scientific interest because of their molecular diagnostic potential, mainly based on the contained microRNA pattern. The past decade has brought about the development and commercialization of a multitude of extraction methods to isolate EVs and exosomes, primarily from blood compartments. Exosome purity and which subpopulations of EVs are captured strongly depend on the applied isolation method, which in turn determines how suitable resulting samples are for potential downstream applications and biomarker discovery. Herein we compared the overall performance of various optimized isolation principles for serum EVs/exosomes in healthy individuals and critically ill patients suffering mainly from pneumonia and sepsis. The isolation methods were benchmarked regarding their suitability for biomarker discovery as well as biological characteristics of captured vesicles, according to the latest MISEV 2018 guidelines. To analyze the small-RNA deep sequencing results, a self-established bioinformatics pipeline for microRNA (based on R) and a deeper analysis of their isoforms (via isomiRROR) was applied. Final goal was the development of a microRNA/isomiR biomarker signature for an early diagnosis and a valid classification of critical ill patients. Various patient cohorts were investigated: healthy volunteers, sepsis (referred to mild or severe pneumonia), acute pulmonary failure (ARDS) and septic shock. Distinct miRNA signatures were identified, which are applicable to indicate disease progression from limited inflammation present in pneumonia to severe inflammatory changes as seen in ARDS or sepsis shock. The study results indicate that EV miRNA biomarkers have future potential for early diagnosis of pneumonia and to indicate disease progression towards severe inflammation events. Further the methodological findings provide guidance for navigating the multitude of EV/exosome isolation methods available, and help researchers and clinicians in the field of molecular diagnostics to make the right choice about the EV/exosome isolation strategy.

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Exploiting RNA in Liquid Biopsies for Precision Medicine Purposes

Jo Vandesompele1,2
1) Ghent University, Belgium;
2) Biogazelle, Ghent, Belgium;

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 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. Apart from RNA abundance profiling, this type of data can also be used to detect structural RNA variants, such as somatic mutations, fusion genes and RNA editing events, all known to play an important role in disease, including cancer. The resulting RNA profiles can be deconvoluted to enumerate the cells, tissues and organs 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 the first insights of the Human Biofluid RNA Atlas, in which we have deeply probed into the extracellular transcriptome of 22 human biofluids, providing a solid foundation for exploiting biofluids for diagnostic purposes. (on behalf of Extracellular RNA Quality Control consortium, Human Biofluid RNA Atlas consortium).

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Clinical Application of Liquid Profiling for Precision Medicine

Stefan Holdenrieder
German Heart Center Munich, TUM, Germany

The concept of “precision medicine” in the treatment of cancer patients aims to specifically target deregulated molecular cancer pathways that are involved in cancer cell proliferation, angiogenesis, metastasis and evasion of the immune control. Classical ways are extracellular blockage of membrane-bound growth receptors by specific antibodies or intracellular inhibition of cancer pathways by small molecules like tyrosine kinase inhibitors that have shown promising therapeutic results. New approaches aim to restore and reactivate immune cell functions to attack the tumor in a more sustainable way. Precondition for precisely acting drugs is the presence of the respective molecular changes that have to be detected in tumor tissue or in the blood plasma – also known as “liquid biopsy” or “liquid profiling” – prior to therapy. Sensitive blood-based diagnostics are able to identify druggable mutations in cell-free plasma tumor DNA (ctDNA) and circulating tumor cells (CTC). Current diagnostic strategies include single-, multi-gene and whole exome / genome approaches that have recently become more sensitive and specific by the introduction of tumor-enrichment and error-reducing techniques. As liquid profiling is only minimally invasive it can be used to complement tissue biopsy for patient stratification and for the serial monitoring of successfully treated and newly occurring resistant cell clones at an individual level. With only few exceptions, plasma ctDNA is measurable in patients with most tumor types, particularly at advanced stage of disease. Concordance with tumor tissue is around 90% if highly sensitive methods are used. ctDNA diagnostics support therapy stratification if tumor biopsy is not available or insufficient, has predictive and prognostic power and can be used as modern, quantifiable tumor marker for monitoring mutation-positive patients. Finally, multiplexing and sequencing enables the detection of new mutations. Preanalytics and rigorous quality control have turned out to be critical for reproducible results. Today, the use of blood conserving tubes, double centrifugation, standardized DNA extraction, enrichment, quantification and sequencing techniques are basic requirements for ctDNA diagnostics. Plasma-based ctDNA “companion diagnostics” has developed to a valuable new tool for precision medicine. First assays are available as IVD-CE labelled methods to be applied in routine diagnostics. High grades of technological and quality standards as well as future combination with protein and metabolome markers will help to improve the diagnostic accuracy and facilitate new applications in other fields of precision medicine such as in immune checkpoint therapies.

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Cell-Free Tumor DNA Analysis In Liquid Biopsy Using Ultrasensitive Sequencing

Anders Ståhlberg1,2
1) University of Gothenburg, Sweden;
2) Sahlgrenska University Hospital, Sweden;

Liquid biopsy and detection of tumor associated-mutations in cell-free circulating DNA (ctDNA) often requires the ability to identify single nucleotide variants at allele frequencies below 0.1%. Standard sequencing protocols cannot achieve this level of sensitivity due to background noise from DNA damage, polymerase induced errors. Addition of unique molecular identifiers allows identification and removal of errors responsible for this background noise. In addition, the entire liquid biopsy workflow needs to be carefully optimized to enable reliable ctDNA analysis. Here, we discuss important considerations for ctDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition. Furthermore, ctDNA assay performance is also demonstrated to be affected by both DNA fragmentation and target sequence. We show that quantitative PCR is useful to estimate the required sequencing depth and to monitor DNA losses throughout the workflow. Theoretically, high fidelity enzymes will reduce error rates in barcoded NGS but this has not been thoroughly explored. We evaluated the impact of polymerase fidelity on the magnitude of error reduction at different steps of barcoded NGS library construction. The use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine.

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