title: Enriched Bone Marrow Derived Disseminated Neuroblastoma Cells Can Be a Reliable Source for Gene Expression Studies-A Validation Study.
abstract: BACKGROUND
  Metastases in the bone marrow (BM) in form of disseminated tumor cells (DTCs) are frequent events at diagnosis and also at relapse in high-risk neuroblastoma patients. The frequently highly diluted occurrence of DTCs requires adequate enrichment strategies to enable their detailed characterization. However, to avoid methodical artifacts we tested whether pre-analytical processing steps-including transport duration, temperature and, importantly, tumor cell enrichment techniques-are confounding factors for gene expression analysis in DTCs.
  
  
  METHODS
  LAN-1 neuroblastoma cells were spiked into tumor free BM and/or peripheral blood and: i) kept at room temperature or at 4°C for 24, 48 and 72 hours; ii) frozen down at -80°C and thawed; iii) enriched via magnetic beads. The effect on the gene expression signature of LAN-1 cells was analyzed by qPCR arrays and gene expression microarrays.
  
  
  RESULTS
  Neither storage at -80°C in DMSO and subsequent thawing nor enrichment of spiked-in neuroblastoma cells changed the expression of the analyzed genes significantly. Whereas storage at 4°C altered the expression of analyzed genes (14.3%) only at the 72h-timepoint in comparison to the 0h-timepoint, storage at room temperature had a much more profound effect on gene expression by affecting 20% at 24h, 26% at 48h and 43% at 72h of the analyzed genes.
  
  
  CONCLUSIONS
  Using neuroblastoma as a model, we show that tumor cell enrichment by magnetic bead separation has virtually no effect on gene expression in DTCs. However, transport time and temperature can influence the expression profile remarkably. Thus, the expression profile of routinely collected BM samples can be analyzed without concern as long as the transport conditions are monitored.
authors.affiliation: CCRI, Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: Fikret
authors.initials: F
authors.lastname: Rifatbegovic
authors.affiliation: CCRI, Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: M Reza
authors.initials: MR
authors.lastname: Abbasi
authors.affiliation: CCRI, Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: Sabine
authors.initials: S
authors.lastname: Taschner-Mandl
authors.affiliation: CCRI, Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Kauer
authors.affiliation: Molecular Diagnostics, Health & Environment Department, AIT Austrian Institute of Technology GmbH, Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Weinhäusel
authors.affiliation: Children's University Hospital, University of Tuebingen, Tuebingen, Germany.
authors.forename: Rupert
authors.initials: R
authors.lastname: Handgretinger
authors.affiliation: CCRI, Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria; Department of Pediatrics, Medical University of Vienna, Vienna, Austria.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Ambros
mesh_terms: D014408:Biomarkers, Tumor; D019046:Bone Marrow Neoplasms / Q000235:genetics* / Q000556:secondary*; D045744:Cell Line, Tumor; D016000:Cluster Analysis; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009447:Neuroblastoma / Q000235:genetics* / Q000473:pathology*; D013696:Temperature; D059467:Transcriptome
chemical_list: D014408:Biomarkers, Tumor