Abstract 1355: Comparison of five commercial kits on extraction of cfDNA from human plasma containing defined amount of genomic DNA contamination: The usage of DNA-depleted plasma as a control matrix for monitoring ctDNA assay
Authors: Lu Yabin , Polonskaia Alina , Shi Keegan , Fu Thomas 
Published: August 2019

The use of PCR-based or NGS-based liquid biopsy assays detecting circulating tumor DNA (ctDNA) is rapidly increasing in clinical labs. However, there are still uncertainties around assay performance and consistency among assays from different developers. One lacking area of research is on the understanding of how plasma factors affect the cell-free DNA (cfDNA) extraction. For certain applications, the contamination of large molecular weight genomic DNA (gDNA) from cell lysis can cause interference to the quantitation of ctDNA. The ability to minimize this contamination by commercial extraction kits was examined.

Anchor Molecular Inc. has developed a proprietary technology that specifically removes the DNA from plasma without affecting other composition of the plasma. The effect of plasma factors on extraction and ctDNA quantitation was studied by quantitatively spiking low concentration of DNA into the DNA-depleted plasma. The result indicated that, while the recovery from DNA extraction was comparable for the 170bp DNA fragment, there was 2.3 fold more gDNA extracted from plasma than from buffer (quantitated by calculating the area from Bioanalyzer). This suggested plasma factors affected the large molecular weight gDNA recovery more significantly. The ability of extracting gDNA from plasma sample was compared among five commercial cfDNA extraction kits. The plasma contained a mixture of synthetic 170bp ctDNA fragment and large molecular weight undigested gDNA. After extraction, the number of copies of the recovered ctDNA and gDNA (represented by RNasP) was assayed by qPCR. The calculated copy numbers were represented as a percentage of the originally spiked copy numbers. From Kit1 to Kit5, the recovery was 74, 60, 27, 34 and 68%, respectively for the 170 bp DNA, and 75, 24, 0, 42 and 29%, respectively for the gDNA. Kit3 was able to completely eliminate the gDNA (0%) while others showed varying degree of contamination. The relative capacity to retain gDNA contamination was 101, 40, 0, 124 and 42%, respectively. This variation could cause large differences in Allele Frequency determination if the relative amount of gDNA contamination were significant. The results from plasma-based sample and from buffer-based (or synthetic plasma) sample was also compared.

The data demonstrated that the DNA profile from plasma is different from buffer. Different extraction kits showed different ability in eliminating contaminating gDNA. Quality Controls samples based on DNA-free Plasma are good tools for ctDNA assay validation and extraction monitoring.

Citation Format: Yabin Lu, Alina Polonskaia, Keegan Shi, Thomas Fu. Comparison of five commercial kits on extraction of cfDNA from human plasma containing defined amount of genomic DNA contamination: The usage of DNA-depleted plasma as a control matrix for monitoring ctDNA assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1355.