Abstract

A Development and Analysis Pipeline for microRNA Biomarker Signatures in Clinical and Molecular Diagnostics Based on Circulating EVs

Michael Pfaffl, Professor, Technical University of Munich

Extracellular vesicles (EVs) circulate in body liquids and are involved in the intercellular communication. They have significant regulative functions in almost any physiological or pathological process. Especially exosome-like small EVs have gained huge scientific and clinical interests because of their specific microRNA biomarker signature and hence future potential in molecular diagnostics. The past decade has brought about the development and commercialization of a multitude of extraction methods to isolate EVs, primarily from blood compartments. The EV purity and which EV subpopulations are captured strongly depend on the applied isolation method, which in turn determines how suitable resulting samples are for potential downstream applications and microRNA biomarker discovery. Hence, a development and analysis pipeline was developed, optimized and validated on various clinical cohorts. Applied isolation methods were benchmarked regarding their suitability for microRNA biomarker discovery as well as biological characteristics of captured vesicles, according to the latest MISEV 2018 guidelines. To deeply analyze the small-RNA deep sequencing results various self-established bioinformatical tools were used: microRNA analysis pipeline (based on R), analysis of microRNA isoforms (via isomiRROR), identification of stable microRNA references (via miREV). Differential expressed microRNAs candidates were identified by multivariate statistics (HCA, PCA, PLS-DA) to find reliable biomarkers. Final goal was the development of microRNA biomarker signatures for an early diagnosis and a valid classification of critical ill patients. Various independent 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 microRNA biomarkers have high future potential for early diagnosis of pneumonia and to indicate disease progression towards severe inflammation events. Furthermore, the methodological findings provides guidance for navigating the multitude of EV isolation methods available, and helps researchers and clinicians in the field of molecular diagnostics to make the right choice about the optimal isolation strategy to get the most valid EV biomarker signature.