title: The In Vitro Stability of Circulating Tumour DNA.
abstract: OBJECTIVE
  DNA from apoptotic cancer cells, present in the circulation, has the potential to facilitate genomic profiling and disease monitoring. However, only low fractions of total cell-free DNA originates from cancer cells, limiting the applicability of circulating tumour DNA (ctDNA). Optimal sample processing is consequently of uttermost importance. Therefore, we evaluated the in vitro stability of ctDNA.
  
  
  METHODS
  Blood was collected in 10 ml EDTA or Streck tubes. Three conditions (EDTA and Streck tubes in room temperature, EDTA tubes at five degrees) and four time points (plasma harvested from blood aliquots of each 10 ml tube in a time series up to 24 h) were investigated. Each condition was evaluated in five metastatic prostate cancer patients. Subsequently, three additional patients were collected enabling investigation of the in vitro stability in EDTA tubes up to 48 h.
  
  
  METHODS
  The in vitro stability of ctDNA was interrogated by low-pass whole genome sequencing which allows for the identification of somatic copy-number alterations (CNAs). In silico simulations demonstrated that non-parametric testing could detect a 1% contamination by white blood cell DNA. Mutational profiling was performed by targeted, in-solution based hybridization capture and subsequent sequencing. The allelic fraction of individual mutations was used as an estimate of the in vitro stability.
  
  
  RESULTS
  Somatic CNAs were detected in all patients. Surprisingly, the ctDNA levels at zero hours were not significantly different to 24 or 48 hour in vitro incubation in any investigated condition. Subsequently, mutational profiling corroborated the conclusions from the CNA analysis.
  
  
  CONCLUSIONS
  The stability of ctDNA simplifies logistics without the requirement of immediate processing or applying fixatives to prevent white blood cell lysis.
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Henao Diaz
authors.affiliation: Department of Oncology-Pathology, Karolinska Institutet and University Hospital, Stockholm, Sweden.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Yachnin
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Grönberg
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Lindberg
mesh_terms: D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000097:blood* / Q000235:genetics; D059018:DNA Contamination; D056915:DNA Copy Number Variations; D004273:DNA, Neoplasm / Q000097:blood* / Q000235:genetics; D055088:Early Detection of Cancer / Q000379:methods / Q000592:standards; D006801:Humans; D008297:Male; D009154:Mutation; D009362:Neoplasm Metastasis; D011471:Prostatic Neoplasms / Q000097:blood* / Q000235:genetics / Q000473:pathology
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm