November 9, 2021
Circulating Biomarkers & Extracellular Vesicles
Chairmen : Michael Pfaffl & Patrice Martin
EV associated Biomarkers in Liquid Biopsies -- How to identify valid ‘Biomarker Signatures’ in blood and circulating extracellular vesicles
|15.40-16.15|| Jo Vandesompele ||Extracellular RNA profiling in human biofluids: about standardization and beyond microRNAs and plasma|
EV in liquid biopsies: from technological considerations towards biomarker discovery
|17.00-17.35|| Mikael Kubista ||Two-tailed PCR for Precision Diagnostics|
|A round-table discussion will conclude the webinar|
Patrice Martin (INRAE, France): Introduction
Michael Pfaffl (Technical University of Munich, Germany): "EV associated Biomarkers in Liquid Biopsies -- How to identify valid ‘Biomarker Signatures’ in blood and circulating extracellular vesicle"
Extracellular vesicles (EVs) circulate in body liquids and are involved in the intercellular communication, and they have important regulative functions in almost any physiological or pathological process. In recent time, especially the exosomes or exosome-like small EV have gained huge scientific interest because of their molecular diagnostic potential, mainly based on their nucleic acid and microRNA content.
Herein we compared the overall performance of various optimized isolation principles for serum EVs/exosomes, primarily from blood compartments, in healthy individuals and various cohorts of critically ill patients. Furthermore, we investigated combined EV isolation methods and compared cell-free and vesicular microRNAs from matched arterial and venous sera. Optimized isolation methods were benchmarked regarding their suitability for microRNA biomarker discovery as well as biological characteristics of captured EVs, according to the latest MISEV 2018 quality control guidelines. To analyze the small-RNA deep sequencing results, a self-established bioinformatics R pipeline for microRNA was applied and isomiRROR for the deep analysis of their isoforms. Differential expressed microRNAs candidates were analyzed by multivariate statistics (HCA, PCA, PLS-DA) to find reliable biomarker signatures. Final goal was the development of a valid microRNA biomarker signature for an early diagnosis and a valid classification of critical ill patients. Various patient cohorts were investigated: healthy volunteers, mild- or severe pneumonia, acute pulmonary failure (ARDS), septic shock, and recently Covid-19 patients. Distinct miRNA signatures could be identified, which are applicable to indicate disease progression from limited inflammation present in pneumonia to severe inflammatory changes as seen in ARDS, septic shock or Covid-19.
The study results indicate that EV associated miRNA biomarkers have high potential for early diagnosis of pneumonia and to indicate disease progression towards severe inflammation events towards Covid-19. Furthermore the methodological findings provides guidance for navigating the multitude of EV/exosome isolation methods available, and helps researchers and clinicians in the field of molecular diagnostics to make the right choice about the EV/exosome isolation strategy and data analysis pipeline.
Jo Vandesompele (Ghent University, Belgium): Extracellular RNA profiling in human biofluids: about standardization and beyond microRNAs and plasma
Various human biofluids are rich sources of messenger RNAs, circular RNAs and microRNAs(1). The fluids display a 10,000 fold difference in RNA concentration, and the extracellular transcriptomes reflects the tissues that produce or transport the fluids. Circular RNAs are significantly enriched in fluids compared to tissues. Pre-analytical standardization (RNA extraction, blood collection tube, time points) is key to achieve reliable extracellular transcriptome sequencing results(2). Large performance differences among RNA extraction kits and blood collection tubes are demonstrated. Selected case studies show applicability of liquid biopsy-based RNA profiling using 2 different RNA-seq methods(3,4).
An Hendrix (Ghent University, Belgium): EV in liquid biopsies: from technological considerations towards biomarker discovery
Separating extracellular vesicles (EVs) from blood plasma is challenging and complicates their biological understanding and biomarker development (PMID: 33568799). We fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EVs (PMID: 31776460; PMID: 34429857) . Using blood plasma samples from cancer patients and reference materials to ensure standardized EV measurements (PMID: 31337761; PMID: 33452501), we demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from cancer patients we show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides 1) a catalogue of putative EV corona proteins, 2) a list of proteins not associated with systemically circulating EV, and 3) reveals the presence of systemically circulating bacterials EVs (PMID: 30518529). In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV.
Mikael Kubista (Institute for Biotechnology, Republic Czech): Two-tailed PCR for Precision Diagnostics
We present a highly specific, sensitive and cost-effective system to quantify miRNA and cell-free DNA in liquid biopsies based on novel chemistry called Two-tailed PCR. Two-tailed PCR takes advantage of target-specific primers composed of two hemiprobes complementary to two different parts of the target molecule connected by a hairpin structure. The introduction of a short hemiprobe that senses the variable sequences confers exceeding sequence specificity while maintaining the very high sensitivity of PCR. Highly similar targets can be distinguished with superior precision irrespectively of the position of the mismatched nucleotide. Further, the target molecule can be very short, making Two-tailed PCR the preferred method for microRNA profiling as well as analysis of rare sequence variants in cell-free DNA or RNA and formalin fixed paraffine embedded (FFPE) tissue. Two-tailed RT-qPCR has a dynamic range of 7 logs and a sensitivity to detect less than ten target miRNA molecules. Two-tailed PCR is readily multiplexed.
(1) Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. P Androvic, L Valihrach, J Elling, R Sjoback, M Kubista. Nucleic acids research 45 (15), e144-e144
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