TY - JOUR
T1 - Multicenter evaluation of circulating cell-free DNA extraction and downstream analyses for the development of standardized (Pre)analytical work flows
AU - Lampignano, Rita
AU - Neumann, Martin H.D.
AU - Weber, Sabrina
AU - Kloten, Vera
AU - Herdean, Andrei
AU - Voss, Thorsten
AU - Groelz, Daniel
AU - Babayan, Anna
AU - Tibbesma, Marco
AU - Schlumpberger, Martin
AU - Chemi, Francesca
AU - Rothwell, Dominic G.
AU - Wikman, Harriet
AU - Galizzi, Jean Pierre
AU - Bergheim, Inger Riise
AU - Russnes, Hege
AU - Mussolin, Benedetta
AU - Bonin, Serena
AU - Voigt, Christine
AU - Musa, Hanny
AU - Pinzani, Pamela
AU - Lianidou, Evi
AU - Brady, Ged
AU - Speicher, Michael R.
AU - Pantel, Klaus
AU - Betsou, Fay
AU - Schuuring, Ed
AU - Kubista, Mikael
AU - Ammerlaan, Wim
AU - Sprenger-Haussels, Markus
AU - Schlange, Thomas
AU - Heitzer, Ellen
N1 - Funding Information:
Employment or Leadership: R. Lampignano, Bayer AG; M.H.D. Neumann, Bayer AG, QIAGEN; V. Kloten, Lab GBU Research; A. Herdean, Tataa Biocenter; T. Voss, QIAGEN GmbH; D. Groelz, QIAGEN GmbH; M. Schlumpberger, QIAGEN GmbH; G. Brady, University of Manchester; K. Pantel, guest editor, Clinical Chemistry, AACC; M. Kubista, TATAA Biocenter AB; M. Sprenger-Haussels, QIAGEN GmbH; T. Schlange, Bayer AG. Consultant or Advisory Role: T. Schlange, DeciBio. Stock Ownership: D. Groelz, QIAGEN GmbH; M. Schlumpberger, QIAGEN; M. Kubista, TATAA Biocenter AB; M. Sprenger-Haussels, QIAGEN GmbH; T. Schlange, Bayer AG. Honoraria: None declared. Research Funding: The authors are participants in the Innovative Medicines Initiative consortium CANCER-ID. CANCER-ID is supported by the Innovative Medicines Initiative (IMI) Joint Undertaking under Grant Agreement no. 115749, resources of which are composed of financial contribution from the European Union’s Seventh Frame-work Programme (FP7/2007–2013) and European Federation of Pharmaceutical Industries and Associations companies’ in-kind contributions. Several authors are participants in the European Union’s Horizon 2020 SPIDIA4P project (Grant agreement no. 733112) or members of the Christian Doppler Research Laboratory for Liquid Biopsies for Early Detection of Cancer led by E. Heitzer funded by the Austrian Federal Ministry for Digital and Economic Affairs. H. Russnes, Health Region South East; M. Kubista, RVO: 86652036 and BIOCEV (CZ.1.05/ 1.1.00/02.0109) from the ERDF. Expert Testimony: None declared. Patents: D. Groelz, WO2015140218 (A1); M. Sprenger-Haussels, WO2012EP68847, WO2012EP68892, WO2009EP03364, WO2013EP70016, WO2012EP68893.
Publisher Copyright:
© 2019 American Association for Clinical Chemistry.
PY - 2020
Y1 - 2020
N2 - BACKGROUND: In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making. METHODS: We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http:// www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites. RESULTS: We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT. CONCLUSIONS: This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.
AB - BACKGROUND: In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making. METHODS: We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http:// www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites. RESULTS: We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT. CONCLUSIONS: This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.
UR - http://www.scopus.com/inward/record.url?scp=85077657390&partnerID=8YFLogxK
U2 - 10.1373/clinchem.2019.306837
DO - 10.1373/clinchem.2019.306837
M3 - Article
C2 - 31628139
AN - SCOPUS:85077657390
SN - 0009-9147
VL - 66
SP - 149
EP - 160
JO - Clinical Chemistry
JF - Clinical Chemistry
IS - 1
ER -