Abstract

Liquid Biopsy -- Exosomal microRNA Biomarker Signatures in Clinical Diagnostics

Michael Pfaffl, Professor, Technical University of Munich

Extracellular vesicles (EVs) are circulating in body liquids and are involve in the intercellular communication with key functions in physiological and pathological processes. In recent time especially the exosomes have gained huge interest because of their molecular diagnostic potential, mainly based on the containing microRNAs.

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. The 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 performance of various optimized isolation principles for serum EVs/exosomes in healthy individuals and critically ill patients, suffering pneumonia and/or various stages of sepsis. The isolation methods were benchmarked regarding their suitability for biomarker discovery as well as biological characteristics of captured vesicles. Isolated vesicles were phenotypic and molecular characterized by Nano Tracking Analysis, (NTA) (EV concentration, EV mean size, EV size distribution), surface marker proteins (positive and negative markers via western blotting), and containing small-RNA families (small-RNA and isomiRs via small-RNA NGS). To analyze the deep sequencing results, a self-established bioinformatics pipeline for microRNA (based on R) and a deeper analysis of their isoforms (via isomiRROR) was applied.

First goal was the development of microRNA/isomiR biomarker signature in a learning cohort with over 116 patients for an early diagnosis and for a valid classification of critical ill patients. Various patient groups were investigated: healthy volunteers, sepsis (referred to mild or severe pneumonia), acute pulmonary failure (ARDS) and septic shock. 21 miRNAs were significantly regulated in all patient groups compared to healthy controls, and different disorders showed unique miRNA expression profiles. Distinct miRNA subsets were identified, which are applicable to indicate disease progression from limited inflammation present in pneumonia to severe inflammatory changes as seen in ARDS and sepsis shock. The found biomarker signatures are now verified in a second and independent confirmation cohort with over 100 new patients.

To conclude, this study results indicate that EV miRNA biomarkers have a great potential for diagnosis of pneumonia and to indicate disease progression towards severe inflammation events. Our findings are of clinical relevance, as the timely diagnosis of pneumonia can be challenging, and secondary complications such as ARDS and sepsis might be prevented by early intervention and fast treatment. Further 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.