Publication

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

Innovative Medicines Initiative CANCER-ID Consortium, Lampignano, R., Neumann, M. H. D., Weber, S., Kloten, V., Herdean, A., Voss, T., Groelz, D., Babayan, A., Tibbesma, M., Schlumpberger, M., Chemi, F., Rothwell, D. G., Wikman, H., Galizzi, J-P., Bergheim, I. R., Russnes, H., Mussolin, B., Bonin, S., Voigt, C., Musa, H., Pinzani, P., Lianidou, E., Brady, G., Speicher, M. R., Pantel, K., Betsou, F., Schuuring, E., Kubista, M., Ammerlaan, W., Sprenger-Haussels, M., Schlange, T. & Heitzer, E., 2019, In : Clinical Chemistry. 12 p.

Research output: Contribution to journalArticleAcademicpeer-review

Copy link to clipboard

Documents

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

    Final publisher's version, 2 MB, PDF document

    Request copy

DOI

  • Innovative Medicines Initiative CANCER-ID Consortium
  • 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 Musa
  • Pamela 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

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.

Original languageEnglish
Number of pages12
JournalClinical Chemistry
Publication statusE-pub ahead of print - 2019

ID: 107647091