Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing

BACKGROUND: The implementation of liquid biopsy for biomarker testing and response to treatment monitoring in cancer patients would presumable increase laboratory throughput, requiring the development of automated methods for circulating free DNA (cfDNA) isolation. METHODS: RSC (MR) ccfDNA Plasma Kit and the later with QIAamp Circulating Nucleid Acid (QCNA) Kit using 57 plasma samples from cancer patients. cfDNA concentration was measured using the Qubit fluorometer. DNA fragments lengt were assessed using the Agilent 2100 Bioanalyzer. Circulating tumor DNA (ctDNA) was quantified by digital PCR (dPCR). RESULTS: Firstly, we observed that MPC method significantly extracted less cfDNA than MR (P<0.0001). However, there were no significant differences in extraction yields of QCNA and MR kits. cfDNA isolation yield was also associated with tumor stage but not with tumor location. Secondly, an oligonucleosomal DNA ladder pattern was observed in 88% of the samples and significant differences in the recovery of mono-, di- and tri-nucleosomes DNA fragments were observed between MPC and MR methodologies. Finally, tumor mutation quantification on cfDNA was performed on 38 paired samples using digital PCR. Mutant allele fractions (MAFs) between paired samples were not significantly different. CONCLUSIONS: Methods for isolation of cfDNA can affect DNA yield and molecular weight fractions recovery. These observations should be taken into account for cfDNA analysis in routine clinical practice.