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Comparative Study
, 13 (2), 392-402

High-throughput Isolation of Circulating Tumor DNA: A Comparison of Automated Platforms

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Comparative Study

High-throughput Isolation of Circulating Tumor DNA: A Comparison of Automated Platforms

Lisanne F van Dessel et al. Mol Oncol.

Abstract

The emerging interest in circulating tumor DNA (ctDNA) analyses for clinical trials has necessitated the development of a high-throughput method for fast, reproducible, and efficient isolation of ctDNA. Currently, the majority of ctDNA studies use the manual QIAamp (QA) platform to isolate DNA from blood. The purpose of this study was to compare two competing automated DNA isolation platforms [Maxwell (MX) and QIAsymphony (QS)] to the current 'gold standard' QA to facilitate high-throughput processing of samples in prospective trials. We obtained blood samples from healthy blood donors and metastatic cancer patients for plasma isolation. Total cell-free DNA (cfDNA) quantity was assessed by TERT quantitative PCR. Recovery efficiency was investigated by quantitative PCR analysis of spiked-in synthetic plant DNA. In addition, a β-actin fragmentation assay was performed to determine the amount of contamination by genomic DNA from lysed leukocytes. ctDNA quality was assessed by digital PCR for somatic variant detection. cfDNA quantity and recovery efficiency were lowest using the MX platform, whereas QA and QS showed a comparable performance. All platforms preferentially isolated small (136 bp) DNA fragments over large (420 and 2000 bp) DNA fragments. Detection of the number variant and wild-type molecules was most comparable between QA and QS. However, there was no significant difference in variant allele frequency comparing QS and MX to QA. In summary, we show that the QS platform has comparable performance to QA, the 'gold standard', and outperformed the MX platform depending on the readout used. We conclude that the QS can replace the more laborious QA platform, especially when high-throughput cfDNA isolation is needed.

Keywords: automation; cell-free DNA; circulating tumor DNA; isolation.

Figures

Figure 1
Figure 1
Effect of increasing cRNA input (0–4 μg) on cfDNA quantity and quality using the Maxwell and QIAsymphony platforms. The effect on cfDNA concentration (ng·mL −1 plasma) was measured by Qubit (A) and TERT qPCR (B). The recovery efficiency of each platform was analyzed by qPCR using spiked‐in synthetic plant DNA (C). Differences in cfDNA fragment size, expressed as number of β‐actin fragments for each fragment size (136, 420 and 2000 bp), were analyzed by dPCR (D). Boxes (interquartile ranges; IQR) and whiskers (1.5× IQR) are shown together with the median (black horizontal line). Outliers are indicated as single black points. Symbols ● and ▲ are mean values shown with whiskers (standard deviation). The Friedman test was used to test the group difference between Maxwell and QIAsymphony samples. Significant differences were post hoc analyzed using the Wilcoxon signed‐rank test. N = 5.
Figure 2
Figure 2
Compatibility of EDTA and CellSave blood collection tubes with the Maxwell and QIAsymphony platforms. The effects on cfDNA concentration (ng·mL −1 plasma) measured by TERT qPCR (A), recovery efficiency measured by plant DNA qPCR (B), and β‐actin fragmentation assay analyzed with dPCR are shown (C). Boxes (interquartile ranges; IQR) and whiskers (1.5× IQR) are shown together with the median (black horizontal line). Outliers are indicated as single black points. Symbols ● and ▲ are mean values shown with whiskers (standard deviation). The Wilcoxon signed‐rank test was used to test the difference between blood collection tubes for each platform. N = 9.
Figure 3
Figure 3
Effect of the different isolation platforms (QIAamp, Maxwell, and QIAsymphony) on downstream cfDNA analysis. cfDNA was isolated from 2 mL matched plasma samples of HBDs (N = 10) and patients with metastatic cancer (N = 10) and analyzed by TERT qPCR assay for cfDNA concentration (ng·mL −1 plasma) (A), plant DNA qPCR assay to determine recovery efficiency (B), and dPCR β‐actin fragmentation assay to evaluate cfDNA fragment sizes (C). Boxes (interquartile ranges; IQR) and whiskers (1.5× IQR) are shown together with the median (black horizontal line). Outliers are indicated as single black points. Symbols ■, ●, and ▲ are mean values shown with whiskers (standard deviation). The Friedman test was used to test the group difference between matched samples processed by the three platforms. Significant differences were post hoc analyzed using the Wilcoxon signed‐rank test.
Figure 4
Figure 4
Somatic variant detection in patients with metastatic cancer on samples isolated with the three different isolation platforms (QIAamp, Maxwell, and QIAsymphony). Somatic variant status had been assessed in patients’ primary and/or metastatic lesion as part of the standard of care. In all patients (N = 10), the known somatic variant was detected in plasma isolated from the three platforms. The ratios of the mutant copy number (A), wild‐type copy number (B), and variant allele frequency (VAF) measured in the Maxwell and QIAsymphony vs. QIAamp are shown (C). The dashed line (ratio of 1) resembles the situation when platforms have similar results. The Wilcoxon signed‐rank test was used to test the difference between the platforms.

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