An Improved Detection of Circulating Tumor DNA in Extracellular Vesicles-Depleted Plasma

Abstract

Circulating tumor DNA (ctDNA) in plasma has been used as a biomarker for cancer detection and outcome prediction. In this study, we collected the five precipitates (fractions 1–5) and leftover supernatant plasma component (fraction 6) by a sequential centrifugation in plasma samples from nine small cell lung cancer (SCLC) patients. The fractions 3, 5 and 6 were large vesicles, exosomes and extracellular vesicles (EVs)-depleted plasma, respectively. Fragment size analysis using DNAs from these fractions showed dramatical differences from a peak of 7–10 kb in fraction 1 to 140–160 bp in fraction 6. To determine ctDNA content, we performed whole genome sequencing and applied copy number-based algorithm to calculate ctDNA percentage. This analysis showed the highest ctDNA content in EV-depleted plasma (average = 27.22%), followed by exosomes (average = 22.09%) and large vesicles (average = 19.70%). Comparatively, whole plasma, which has been used in most ctDNA studies, showed an average of 23.84% ctDNA content in the same group of patients. To further demonstrate higher ctDNA content in fraction 6, we performed mutational analysis in the plasma samples from 22 non-small cell lung cancer (NSCLC) patients with known EGFR mutations. This analysis confirmed higher mutation detection rates in fraction 6 (14/22) than whole plasma (10/22). This study provides a new insight into potential application of using fractionated plasma for an improved ctDNA detection.

EV‐ADD, a database for EV‐associated DNA in human liquid biopsy samples

“…However, other studies reported the presence of shorter DNA fragments (152.4 bp, 160 bp) within EVs, with larger DNA fragments possibly resulting from contamination with apoptotic bodies (Sun et al., 2021 ; Zhang et al., 2019 ). The comparison between the above‐mentioned studies is difficult due to different EV isolation methods resulting in purification of various EV subtypes.…”

“…It has been reported that longer EV-DNA fragments may improve the detection of single nucleotide variations, copy number variations (San Lucas et al, 2016) and insertions/deletions during NGS sequencing performance and bioinformatic analysis (Waldenmaier et al, 2022). However, other studies reported the presence of shorter DNA fragments (152.4 bp, 160 bp) within EVs, with larger DNA fragments possibly resulting from contamination with apoptotic bodies (Sun et al, 2021;. The comparison between the above-mentioned studies is difficult due to different EV isolation methods resulting in purification of various EV subtypes.…”

Genetic Characteristics Associated With Drug Resistance in Lung Cancer and Colorectal Cancer Using Whole Exome Sequencing of Cell-Free DNA

“…ctDNA fraction was estimated by FACETS from data of the WES cfDNA sample and absolute copy number (ABCN) were called depends on tumor fraction estimation from cfDNA as previously described ( 24 ), and mean tcn.em values were used. To estimate ctDNA amount, mean tcn.em values were used to calculate ctDNA content of total cfDNA ( 25 ).…”

Identification of Circulating Exosomal miR-101 and miR-125b Panel Act as a Potential Biomarker for Hepatocellular Carcinoma

“…Exosomes were isolated using the Total Exosome Isolation Kit (Invitrogen, USA) according to the manufacturer's instruction as previously described [ 21 – 23 ]. The serum was thawed in a 25°C water bath and then subjected to centrifugation at 2,000 × g for 30 min to remove possible residual cells debris.…”