Determination of Exosome Mitochondrial DNA as a Biomarker of Renal Cancer Aggressiveness

SIMPLE SUMMARY: Components of liquid biopsy are potential non-invasive biomarkers for monitoring renal cell carcinoma (RCC) status. The aim of our study was to examine mitochondrial genes (such as HV1 and CYB) included in exosomal fractions as promising and innovative biomarkers in RCC. We found that phase C containing different types of vesicles and phase F rich in exosomes with a high mitochondrial DNA (mtDNA) content could be considered as powerful biomarkers for susceptibility to RCC. Interestingly, mtDNA was a good genetic marker when aggressiveness was evaluated. ABSTRACT: Here, the role of non-invasive biomarkers in liquid biopsy was evaluated, mainly in exosomes and mitochondrial DNA (mtDNA) as promising, novel, and stable biomarkers for renal cell carcinoma (RCC). A total of 140 fractions (named from B to F) obtained by ultracentrifugations of whole blood samples from 28 individuals (13 patients and 15 controls) were included. Nanoparticle Tracking Analysis (NTA) was conducted to characterized exosomal fraction. Subsequently, an analysis of digital PCR (dPCR) using the QuantStudio™ 3D Digital PCR platform was performed and the quantification of mtDNA copy number by QuantStudioTM 12K Flex Real-Time PCR System (qPCR) was developed. Moreover, Next Generation Sequencing (NGS) analyses were included using MiSeq system (Illumina, San Diego, CA, USA). An F fraction, which contains all exosome data and all mitochondrial markers, was identified in dPCR and qPCR with statistically significant power (adjusted p values ≤ 0.03) when comparing cases and controls. Moreover, present analysis in mtDNA showed a relevant significance in RCC aggressiveness. To sum up, this is the first time a relation between exosomal mtDNA markers and clinical management of RCC is analyzed. We suggest a promising strategy for future liquid biopsy RCC analysis, although more analysis should be performed prior to application in routine clinical practice.