@article{3077470,
    title = "Multicenter evaluation of circulating cell-free DNA extraction and downstream analyses for the development of standardized (Pre)analytical work flows",
    author = "Lampignano, R. and Neumann, M.H.D. and Weber, S. and Kloten, V. and Herdean, A. and Voss, T. and Groelz, D. and Babayan, A. and Tibbesma, M. and Schlumpberger, M. and Chemi, F. and Rothwell, D.G. and Wikman, H. and Galizzi, J.-P. and Bergheim, I.R. and Russnes, H. and Mussolin, B. and Bonin, S. and Voigt, C. and Musa, H. and Pinzani, P. and Lianidou, E. and Brady, G. and Speicher, M.R. and Pantel, K. and Betsou, F. and Schuuring, E. and Kubista, M. and Ammerlaan, W. and Sprenger-Haussels, M. and Schlange, T. and Heitzer, E.",
    journal = "Advances in Clinical Chemistry",
    year = "2020",
    volume = "66",
    number = "1",
    pages = "149-160",
    publisher = "American Association for Clinical Chemistry Inc.",
    doi = "10.1373/clinchem.2019.306837",
    keywords = "DNA;  protein p53;  cell free nucleic acid;  circulating tumor DNA;  protein p53;  TP53 protein, human, Article;  controlled study;  DNA extraction;  DNA purification;  downstream processing;  droplet digital polymerase chain reaction;  gene mutation;  human;  lung cancer cell line;  multicenter study;  quantitative analysis;  real time polymerase chain reaction;  workflow;  blood;  blood sampling;  chemistry;  dna mutational analysis;  genetics;  high throughput sequencing;  metabolism;  neoplasm;  nucleosome;  pathology;  pre-analytical phase;  procedures;  single nucleotide polymorphism;  standard;  tumor cell line, Blood Specimen Collection;  Cell Line, Tumor;  Cell-Free Nucleic Acids;  Circulating Tumor DNA;  DNA Mutational Analysis;  High-Throughput Nucleotide Sequencing;  Humans;  Neoplasms;  Nucleosomes;  Polymorphism, Single Nucleotide;  Pre-Analytical Phase;  Real-Time Polymerase Chain Reaction;  Reference Standards;  Tumor Suppressor Protein p53",
    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. © 2019 American Association for Clinical Chemistry."
}