Automated large-volume extraction of circulating, cell-free DNA to improve the sensitivity of tumor biomarker detection.

Abstract

19 Background: Because of its low concentration and high degree of fragmentation, the extraction and detection of tumor-derived circulating cell-free DNA (ccfDNA) is technically challenging. An optimized ccfDNA extraction method was developed and evaluated by comparison to an existing extraction protocol running on the QIAsymphony instrument and to a manual reference. Methods: 5mlEDTA plasma from healthy donors (with IRB approval) was processed. ccfDNA was bound to magnetic particles with novel surface chemistry and recovered in 150µl. As an alternative method, the “Virus cell-free 1000 protocol” using the QIAsymphony Virus/Pathogen Midi Kit was modified for the processing of higher sample volumes. ccfDNA was extracted from 4ml plasma and eluted in 90µl. The QIAamp Circulating Nucleic Acid (CNA) Kit served as a reference. ccfDNA yield was quantified by qPCR (66bp within the 18S rDNA). To determine the DNA fragment size-dependent recovery, another qPCR assay was run, quantifying four target sequences of different lengths within the APP gene. 5ml plasma was processed and eluted in 150µl. Results: The mean ccfDNA recovery (18S 66bp target; compared to the QIAamp CNA Kit) was 87% (N=6; +/-46%) for the newly developed automated extraction chemistry and 85% (N=6; +/-8%) for the modified extraction protocol. For the APP assay, ratios between the copy numbers of different target sizes were calculated. The mean ratios were: 67/476bp = 11 (N=12; +/-6.2), 180/476bp = 8.1 (N=12; +/-3.6) and 67/180bp = 1.4 (N=12; +/-0.3). Conclusions: The automated protocol versions led to an overall similar ccfDNA recovery compared to the QIAamp CNA Kit. Using the new extraction chemistry a generally improved recovery of tumor-derived circulating DNA is possible. This improvement leads to a higher sensitivity of tumor biomarker detection, which is, besides a high specificity, very important for the use of tumor biomarkers as non-invasive tool in cancer diagnosis and prognosis. The applications presented here are for research use only. Not for use in diagnostic procedures. This work has received funding from the European Union FP7 Programme under grant agreement no. 222916, SPIDIA project ( www.spidia.eu ).