Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows.

Lampignano, R; Neumann, MHD; Weber, S; Kloten, V; Herdean, A; Voss, T; Groelz, D; Babayan, A; Tibbesma, M; Schlumpberger, M; Chemi, F; Rothwell, DG; Wikman, H; Galizzi, J-P; Riise Bergheim, I; Russnes, H; Mussolin, B; Bonin, S; Voigt, C; Musa, H; Pinzani, P; Lianidou, E; Brady, G; Speicher, MR; Pantel, K; Betsou, F; Schuuring, E; Kubista, M; Ammerlaan, W; Sprenger-Haussels, M; Schlange, T; Heitzer, E

Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows.

Lampignano, R Neumann, MHD Weber, S Kloten, V Herdean, A

Voss, T Groelz, D Babayan, A Tibbesma, M Schlumpberger, M Chemi, F Rothwell, DG Wikman, H Galizzi, J-P Riise Bergheim, I Russnes, H Mussolin, B Bonin, S Voigt, C Musa, H Pinzani, P Lianidou, E Brady, G Speicher, MR Pantel, K Betsou, F Schuuring, E Kubista, M Ammerlaan, W Sprenger-Haussels, M Schlange, T Heitzer, E

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Publisher:: OXFORD UNIV PRESS INC
Publication Type:: Journal Article
Citation:: Clinical chemistry, 2020, 66, (1), pp. 149-160
Issue Date:: 2020-01

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Field	Value	Language
dc.contributor.author	Lampignano, R
dc.contributor.author	Neumann, MHD
dc.contributor.author	Weber, S
dc.contributor.author	Kloten, V
dc.contributor.author	Herdean, A https://orcid.org/0000-0003-2143-0213
dc.contributor.author	Voss, T
dc.contributor.author	Groelz, D
dc.contributor.author	Babayan, A
dc.contributor.author	Tibbesma, M
dc.contributor.author	Schlumpberger, M
dc.contributor.author	Chemi, F
dc.contributor.author	Rothwell, DG
dc.contributor.author	Wikman, H
dc.contributor.author	Galizzi, J-P
dc.contributor.author	Riise Bergheim, I
dc.contributor.author	Russnes, H
dc.contributor.author	Mussolin, B
dc.contributor.author	Bonin, S
dc.contributor.author	Voigt, C
dc.contributor.author	Musa, H
dc.contributor.author	Pinzani, P
dc.contributor.author	Lianidou, E
dc.contributor.author	Brady, G
dc.contributor.author	Speicher, MR
dc.contributor.author	Pantel, K
dc.contributor.author	Betsou, F
dc.contributor.author	Schuuring, E
dc.contributor.author	Kubista, M
dc.contributor.author	Ammerlaan, W
dc.contributor.author	Sprenger-Haussels, M
dc.contributor.author	Schlange, T
dc.contributor.author	Heitzer, E
dc.date.accessioned	2021-01-08T00:06:02Z
dc.date.available	2019-08-05
dc.date.available	2021-01-08T00:06:02Z
dc.date.issued	2020-01
dc.identifier.citation	Clinical chemistry, 2020, 66, (1), pp. 149-160
dc.identifier.issn	0009-9147
dc.identifier.issn	1530-8561
dc.identifier.uri	http://hdl.handle.net/10453/145203
dc.description.abstract	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.
dc.format	Print
dc.language	eng
dc.publisher	OXFORD UNIV PRESS INC
dc.relation.ispartof	Clinical chemistry
dc.relation.isbasedon	10.1373/clinchem.2019.306837
dc.rights	info:eu-repo/semantics/closedAccess
dc.subject	1004 Medical Biotechnology, 1101 Medical Biochemistry and Metabolomics, 1103 Clinical Sciences
dc.subject.classification	General Clinical Medicine
dc.subject.mesh	Cell Line, Tumor
dc.subject.mesh	Nucleosomes
dc.subject.mesh	Humans
dc.subject.mesh	Neoplasms
dc.subject.mesh	Blood Specimen Collection
dc.subject.mesh	DNA Mutational Analysis
dc.subject.mesh	Polymorphism, Single Nucleotide
dc.subject.mesh	Reference Standards
dc.subject.mesh	Tumor Suppressor Protein p53
dc.subject.mesh	High-Throughput Nucleotide Sequencing
dc.subject.mesh	Real-Time Polymerase Chain Reaction
dc.subject.mesh	Pre-Analytical Phase
dc.subject.mesh	Cell-Free Nucleic Acids
dc.subject.mesh	Circulating Tumor DNA
dc.subject.mesh	Cell Line, Tumor
dc.subject.mesh	Nucleosomes
dc.subject.mesh	Humans
dc.subject.mesh	Neoplasms
dc.subject.mesh	Blood Specimen Collection
dc.subject.mesh	DNA Mutational Analysis
dc.subject.mesh	Polymorphism, Single Nucleotide
dc.subject.mesh	Reference Standards
dc.subject.mesh	Tumor Suppressor Protein p53
dc.subject.mesh	High-Throughput Nucleotide Sequencing
dc.subject.mesh	Real-Time Polymerase Chain Reaction
dc.subject.mesh	Pre-Analytical Phase
dc.subject.mesh	Cell-Free Nucleic Acids
dc.subject.mesh	Circulating Tumor DNA
dc.subject.mesh	Blood Specimen Collection
dc.subject.mesh	Cell Line, Tumor
dc.subject.mesh	Cell-Free Nucleic Acids
dc.subject.mesh	Circulating Tumor DNA
dc.subject.mesh	DNA Mutational Analysis
dc.subject.mesh	High-Throughput Nucleotide Sequencing
dc.subject.mesh	Humans
dc.subject.mesh	Neoplasms
dc.subject.mesh	Nucleosomes
dc.subject.mesh	Polymorphism, Single Nucleotide
dc.subject.mesh	Pre-Analytical Phase
dc.subject.mesh	Real-Time Polymerase Chain Reaction
dc.subject.mesh	Reference Standards
dc.subject.mesh	Tumor Suppressor Protein p53
dc.title	Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows.
dc.type	Journal Article
utslib.citation.volume	66
utslib.location.activity	England
utslib.for	1004 Medical Biotechnology
utslib.for	1101 Medical Biochemistry and Metabolomics
utslib.for	1103 Clinical Sciences
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - C3 - Climate Change Cluster
pubs.organisational-group	/University of Technology Sydney
utslib.copyright.status	closed_access	*
pubs.consider-herdc	false
dc.date.updated	2021-01-08T00:05:48Z
pubs.issue	1
pubs.publication-status	Published
pubs.volume	66
utslib.citation.issue	1

Abstract:

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.

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http://hdl.handle.net/10453/145203