Comparison of different semi-automated cfDNA extraction methods in combination with UMI-based targeted sequencing
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Anna Streubel1, Albrecht Stenzinger2,3, Susann Stephan-Falkenau1, Jens Kollmeier4, Daniel Misch4, Torsten Gerriet Blum4, Torsten Bauer4, Olfert Landt5, Alexander Am Ende5, Peter Schirmacher2, Thomas Mairinger1 and Volker Endris2
1 Department of Pathology, Helios Klinikum Emil von Behring, Berlin, Germany
2 Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany
3 German Cancer Consortium (DKTK), Heidelberg, Germany
4 Department of Pneumology, Helios Klinikum Emil von Behring, Berlin, Germany
5 TIB Molbiol Syntheselabor GmbH, Berlin, Germany
Keywords: lung cancer; liquid biopsy; cfDNA; molecular diagnostics; T790M
Received: May 04, 2019 Accepted: July 17, 2019 Published: October 1, 2019
The analysis of circulating cell-free DNA (cfDNA) extracted from peripheral blood can serve as a minimally invasive alternative to tumor tissue biopsies in cases with impaired access to tissue. Its clinical utility has been well demonstrated for EGFR T790M testing in lung cancer patients suffering progress after tyrosine kinase inhibitor treatment. At present, highly sensitive unique molecular identifiers (UMI)-based NGS for liquid biopsy testing is less established compared to single gene assays. However, the critical bottleneck are sufficient cfDNA yields, which are essentially required to obtain meaningful test results.
We compared four different cfDNA extraction methods (Qiagen, Promega, Thermo and Stratec) using the same plasma samples in order to evaluate their suitability for further NGS analysis. We managed to draw 60 ml blood from 12 patients each and equally collected 30ml in PAXgene and EDTA tubes at the same time point, sufficient for total of 96 cfDNA extractions. CfDNA concentrations and total amounts were highest for Qiagen and Promega protocols, showing the best read length profiles after sequencing.
Known oncogenic driver mutations were identified in 9 out of 12 patients with at least one of the cfDNA extraction methods, again favoring the extraction protocols from Qiagen and Promega. We also uncovered putative sequencing artefacts including known driver genes pointing to a careful consideration for the limit of detection of this methodology. Our study shows that pre-analytical optimization is necessary to achieve the maximum sensitivity of UMI-based sequencing but also highlights the low abundance of tumor-derived cfDNA in lung cancer samples.
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