Login to Access Video or Poster Abstract: MP78-07
Sources of Funding: Freunde des Universitätsklinikums e.V. Homburg

Introduction

In previous studies we identified specific miRNA alterations in tumor tissues of clear cell renal cell cancer (ccRCC) with diagnostic and prognostic value relating to the presence of metastasis. For few years it has been known, that miRNAs are actively packed in exosomes which can be released into body fluids. Therefore, we hypothesize that in a simple blood based test we are able to use specific miRNA signatures as minimal invasive biomarkers for confirmation of the diagnosis and evaluation of the metastatic risk in ccRCC.

Methods

Initially, exosomes were isolated from 1 ml serum of 10 ccRCC patients (metastatic and non metastatic tumors) and 10 healthy volunteers using appropriated exosome isolation kit protocol (Thermo Fisher Scientific). Quality of exosomes was proven using exosomal markers (CD63, CD81, Alix, Synthenin) and Nano Particle Tracking Analysis. Exosomal totalRNA was isolated using miRNeasy Mini Kit (Qiagen). For miRNA analyses total RNA was reverse transcribed using TaqMan Reverse Transcription Kit (Thermo Fisher Scientific), cDNA was preamplified using TaqMan® PreAmp Master Mix (Thermo Fisher Scientific) and real time PCR was performed using Gene Expression master mix (Thermo Fisher Scientific). Expression differences were calculated using REST Software and SPSS.

Results

We have shown that 1 ml serum is sufficient to analyze miRNA expression in exosomes. Exosomes isolated from serum exhibit high amount of exosomal markers and possess the typical exosomal size (30-120 nm). CcRCC serum samples are characterized by downregulation of several miRNAs (including miR-10b and miR451). Furthermore, specific miRNAs (including miR-30c) differentiate patients with or without metastases as well as healthy volunteers.

Conclusions

These initial data confirm our hypothesis that the tissue based miRNA signature could be used as biomarkers for detection of aggressive ccRCC analyzing exosomes from liquid biopsy. This minimal invasive blood based test is easy to handle in clinical practice and could be a powerful tool for early detection and monitoring of metastatic disease. To validate these results the expansion of the sample set is ongoing.