title: Impact of biospecimens handling on biomarker research in breast cancer.
abstract: BACKGROUND
  Gene expression profiling is moving from the research setting to the practical clinical use. Gene signatures able to correctly identify high risk breast cancer patients as well as to predict response to treatment are currently under intense investigation. While technical issues dealing with RNA preparation, choice of array platforms, statistical analytical tools are taken into account, the tissue collection process is seldom considered. The time elapsed between surgical tissue removal and freezing of samples for biological characterizations is rarely well defined and/or recorded even for recently stored samples, despite the publications of standard operating procedures for biological sample collection for tissue banks.
  
  
  METHODS
  Breast cancer samples from 11 patients were collected immediately after surgical removal and subdivided into aliquots. One was immediately frozen and the others were maintained at room temperature for respectively 2, 6 and 24 hrs. RNA was extracted and gene expression profile was determined using cDNA arrays. Phosphoprotein profiles were studied in parallel.
  
  
  RESULTS
  Delayed freezing affected the RNA quality only in 3 samples, which were not subjected to gene profiling. In the 8 breast cancer cases with apparently intact RNA also in sample aliquots frozen at delayed times, 461 genes were modulated simply as a function of freezing timing. Some of these genes were included in gene signatures biologically and clinically relevant for breast cancer. Delayed freezing also affected detection of phosphoproteins, whose pattern may be crucial for clinical decision on target-directed drugs.
  
  
  CONCLUSIONS
  Time elapsed between surgery and freezing of samples appears to have a strong impact and should be considered as a mandatory variable to control for clinical implications of inadequate tissue handling.
authors.affiliation: Fondazione Istituto FIRC di Oncologia Molecolare, Via Adamello16, 20139 Milano, Italy. loris.dececco@ifom-ieo-campus.it
authors.forename: Loris
authors.initials: L
authors.lastname: De Cecco
authors.forename: Valeria
authors.initials: V
authors.lastname: Musella
authors.forename: Silvia
authors.initials: S
authors.lastname: Veneroni
authors.forename: Vera
authors.initials: V
authors.lastname: Cappelletti
authors.forename: Italia
authors.initials: I
authors.lastname: Bongarzone
authors.forename: Maurizio
authors.initials: M
authors.lastname: Callari
authors.forename: Barbara
authors.initials: B
authors.lastname: Valeri
authors.forename: Marco A
authors.initials: MA
authors.lastname: Pierotti
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Daidone
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis*; D035843:Biomedical Research / Q000379:methods* / Q000592:standards; D001943:Breast Neoplasms / Q000235:genetics*; D005260:Female; D005615:Freezing; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013048:Specimen Handling / Q000379:methods* / Q000592:standards; D013995:Time
chemical_list: D014408:Biomarkers, Tumor; D012333:RNA, Messenger