Methods for the purification and detection of single nucleotide KRAS mutations on extrachromosomal circular DNA in human plasma

Abstract

Backgrounds: Despite recent advances, many cancers are still detected too late for curative treatment. There is, therefore, a need for the development of new diagnostic methods and biomarkers. One approach may arise from the detection of extrachromosomal circular DNA (eccDNA), which is part of cell-free DNA in human plasma.

Aims: First, we assessed and compared two methods for the purification of eccDNA from plasma. Second, we tested for an easy diagnostic application of eccDNA liquid biopsy-based assays.

Materials & methods: For the comparison we tested a solid-phase silica purification method and a phenol/chloroform method with salt precipitation. For the diagnostic application of eccDNA we developed and tested a qPCR primer-based SNP detection system, for the detection of two well-established cancer-causing KRAS mutations (G12V and G12R) on circular DNA. This investigation was supported by purifying, sequencing, and analysing clinical plasma samples for eccDNAs containing KRAS mutant alleles in 0.5 mL plasma from 16 pancreatic ductal adenocarcinoma patients and 19 healthy controls.

Results: In our method comparison we observed, that following exonuclease treatment a lower eccDNA yield was found for the phenol/chloroform method (15.7%-26.7%) compared with the solid-phase purification approach (47.8%-65.9%). For the diagnostic application of eccDNA tests, the sensitivity of the tested qPCR assay only reached ~10^-3 in a background of 10⁵ wild type (wt) KRAS circular entities, which was not improved by general amplification or primer-based inhibition of wt KRAS amplification. Furthermore, we did not detect eccDNA containing KRAS in any of the clinical samples.

Discussion: A potential explanation for our inability to detect any KRAS mutations in the clinical samples may be related to the general low abundance of eccDNA in plasma.

Conclusion: Taken together our results provide a benchmark for eccDNA purification methods while raising the question of what is required for the optimal fast and sensitive detection of SNP mutations on eccDNA with greater sensitivity than primer-based qPCR detection.

Keywords: KRAS mutations; circular DNA; eccDNA; liquid biopsy; phenol/chloroform DNA extraction; plasmids.

FIGURE 1

DNA purification yields from plasma using the solid‐phase purification method (QIAamp, Qiagen). DNA was detected by qPCR, and the first five bars represent measured spike‐in baseline controls, which have been set to 100%. Three independent purifications from plasma were conducted and measured in triplicates by qPCR, and the purified DNA yields were calculated from the ct values relative to each sample control (Bars 1–5). Bars 1–5 (positive baseline controls, nine data points/bar), Bars 6–15 (samples purified from plasma, 27 data points/bar), and Bars 16–20 (negative controls, nine data points/bar). Circular DNA is presented using orange bars, and linear DNA fragments are presented using blue bars. All data are presented as mean ± standard deviation.

FIGURE 2

DNA purification yield from plasma using phenol/chloroform purification and salt precipitation. DNA was detected by qPCR, and the first five bars represent measured spike‐in baseline controls, which have been set to 100%. Three independent purifications from plasma were conducted and measured in triplicates by qPCR, and the purified DNA yields were calculated from the ct values relative to each sample control (Bars 1–5). Bars 1–5 (positive baseline controls, nine data points/bar), Bars 6–15 (samples purified from plasma, 27 data points/bar), and Bars 16–20 (negative controls, nine data points/bar). Circular DNA is presented using orange bars, and linear DNA fragments are presented using blue bars. All data are presented as mean ± standard deviation.

FIGURE 3

Violin plot presents the total DNA purification yields from plasma for the solid‐phase and phenol/chloroform purification methods. The total DNA was detected using Qubit. Six to fifteen independent measurements were conducted. All data are presented as mean ± standard deviation.

FIGURE 4

Detection of KRAS SNPs in a wt KRAS background and the effect of Φ29 amplification on the sensitivity. Three independent purifications were conducted and measured in triplicates by qPCR. (A) detection of the G12V KRAS mutant and (B) detection of the G12R KRAS mutant. Each bar includes nine data points. Significance was assessed by comparing each detection with the 10⁵ wt background level using a t‐test. ns denotes “not significant” (p > 0.05), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. All data are presented as mean ± standard deviation.

FIGURE 5

Detection of KRAS SNPs in a background of KRAS wt with and without dd‐primer KRAS wt primer. Three independent purifications were conducted and measured in triplicates by qPCR. (A) shows the G12V KRAS mutant and (B) shows the G12R KRAS mutant. Each bar represents nine data points. Data were analyzed by t‐test. ns denotes “not significant” (p > 0.05), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. All data are presented as mean ± standard deviation.