Physical activity as pre-analytical parameter for liquid biopsies

Background: Liquid biopsies often aim to compare the amount of nucleic acids of tumour origin with nucleic acids that are shed from healthy cells. While it is understood that the amount of tumour material in circulation strongly depends on tumour stage, the conditions influencing the amount of wildtype nucleic acids are less well described. The amount of RNA contained in exosomes and other extracellular vesicles (exoRNA), as well as the amount of cell-free DNA (cfDNA), may be influenced by preanalytical parameters - potentially changing the interpretation of a liquid biopsy result. Here, we analyse the release of exoRNA and cfDNA by physical activity. Methods: We sampled the blood of four healthy individuals by venipuncture before and after 30 min of continuous physical exercise and used a combined co-extraction of exoRNA and cfDNA to analyse their relative abundance in plasma. Size distribution of the total nucleic acids was accessed using a BioAnalyzer 6000 Pico Kit. The content of exoRNA was quantified using RT-qPCR for specific mRNAs (GAPDH and RPL0) and the content of cfDNA was quantified using primers for the loci of often used oncogenes (KRAS, BRAF and PIK3CA). Results: All four individuals showed increased amount of total nucleic acids after physical exercise, mainly corresponding to the size of monoand dinucleosomal DNA. Comparing the pre- and post-exercise datapoints of each patient, the increase in cfDNA was measured consistently between 10 and 30-fold, while the increase in exosomal mRNAs was only two-fold on average. Summary/Conclusion: While both exoRNA and cfDNA increased already after 30 min of exercise, the increase in cfDNA consistently exceeded the increase of exosomal RNA by an order of magnitude. This effect of physical activity has to be taken into account when interpreting datasets that use the absolute or relative amount of nucleic acids in liquid biopsies.