Abstract
It is widely accepted that assessing circular tumor DNA (ctDNA) in the plasma of cancer patients 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 mutant DNA from plasma in metastatic patients; however, no consensus on the presence of dsDNA in exosomes has been reached yet. We analyzed small extracellular vesicle (sEV)-associated DNA of eleven metastatic colorectal cancer (mCRC) patients and compared the results obtained by microarray and droplet digital PCR (ddPCR) to those reported on the ctDNA fraction. We detected the same mutations found in tissue biopsies and ctDNA in all samples but, unexpectedly, in one sample, we found a KRAS mutation that was not identified either in ctDNA or tissue biopsy. Furthermore, to assess the exact location of sEV-associated DNA (outside or inside the vesicle), we treated with DNase I sEVs isolated with three different methodologies. We found that the DNA inside the vesicles is only a small fraction of that surrounding the vesicles. Its amount seems to correlate with the total amount of circulating tumor DNA. The results obtained in our experimental setting suggest that integrating ctDNA and sEV-associated DNA in mCRC patient management could provide a complete real-time assessment of the cancer mutation status.
Highlights
To shed light on this topic, we investigated the presence of DNA in small extracellular vesicle (sEV) fractions of eleven metastatic colorectal cancer patients with two different methodologies and compared the results with those previously obtained analyzing circular tumor DNA (ctDNA) in the same samples [14]
Our data confirm the presence of KRAS and BRAF mutations in cell-free circulating and sEV-associated DNA, in particular a higher concentration and fractional abundance is detected in ctDNA
Using eleven samples of the cohort reported in reference [14], we investigate which of the different tumor-driven component—ctDNA, DNA internally or externally associated with exosomes—should be used to determine tumor-specific molecular alterations and the differences in the information provided
Summary
In 2014, Thakur et al demonstrated for the first time that the majority of DNA associated with tumor exosomes is double-stranded (dsDNA) and represents the whole genomic DNA [4]. Zocco et al [13], in disagreement with Jeppesen, suggested that about 90% of the isolated mutant DNA is out of the sEVs (associated with the outer surface of the EV membrane or independently co-purified within protein aggregates) They found that only about 10% of DNA was detected inside the exosomes, after treatment with. Our data confirm the presence of KRAS and BRAF mutations in cell-free circulating and sEV-associated DNA, in particular a higher concentration and fractional abundance is detected in ctDNA. Our results suggest that besides ctDNA, the tumor-derived fragmented DNA in the bloodstream that is not associated with cells, sEV-associated DNA fractions can help to identify and monitor mutations in mCRC patients
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