Small EVs-Associated DNA as Complementary Biomarker to Circulating Tumor DNA in Plasma of Metastatic Colorectal Cancer Patients

It is widely accepted that assessing circular tumor DNA (ctDNA) in the plasma of cancerpatients is a promising practice to evaluate somatic mutations from solid tumors noninvasively.Recently, it was reported that isolation of extracellular vesicles improves the detection of mutantDNA from plasma in metastatic patients; however, no consensus on the presence of dsDNA inexosomes has been reached yet. We analyzed small extracellular vesicle (sEV)-associated DNA ofeleven metastatic colorectal cancer (mCRC) patients and compared the results obtained by microarrayand droplet digital PCR (ddPCR) to those reported on the ctDNA fraction. We detected the samemutations found in tissue biopsies and ctDNA in all samples but, unexpectedly, in one sample, wefound a KRAS mutation that was not identified either in ctDNA or tissue biopsy. Furthermore, toassess the exact location of sEV-associated DNA (outside or inside the vesicle), we treated with DNaseI sEVs isolated with three different methodologies. We found that the DNA inside the vesicles is onlya small fraction of that surrounding the vesicles. Its amount seems to correlate with the total amountof circulating tumor DNA. The results obtained in our experimental setting suggest that integratingctDNA and sEV-associated DNA in mCRC patient management could provide a complete real-timeassessment of the cancer mutation status.