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

Rita Lampignano, Martin H D Neumann, Sabrina Weber, Vera Kloten, Andrei Herdean, Thorsten Voss, Daniel Groelz, Anna Babayan, Marco Tibbesma, Martin Schlumpberger, Francesca Chemi, Dominic G Rothwell, Harriet Wikman, Jean-Pierre Galizzi, Inger Riise Bergheim, Hege Russnes, Benedetta Mussolin, Serena Bonin, Christine Voigt, Hanny MusaPamela Pinzani, Evi Lianidou, Ged Brady, Michael R Speicher, Klaus Pantel, Fay Betsou, Ed Schuuring, Mikael Kubista, Wim Ammerlaan, Markus Sprenger-Haussels, Thomas Schlange, Ellen Heitzer

Research output: Contribution to journalArticlepeer-review

Abstract

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. 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. 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. 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.
Original languageUndefined/Unknown
Pages (from-to)149-160
Number of pages12
JournalClinical Chemistry
Volume66
DOIs
Publication statusPublished - Jan 1 2020

Keywords

  • Blood Specimen Collection
  • Cell Line
  • Tumor
  • Cell-Free Nucleic Acids
  • chemistry
  • metabolism
  • standards
  • Circulating Tumor DNA
  • blood
  • DNA Mutational Analysis
  • High-Throughput Nucleotide Sequencing
  • methods
  • Humans
  • Neoplasms
  • genetics
  • pathology
  • Nucleosomes
  • Polymorphism
  • Single Nucleotide
  • Pre-Analytical Phase
  • Real-Time Polymerase Chain Reaction
  • Reference Standards
  • Tumor Suppressor Protein p53

Cite this