title: Global gene expression changes induced by prolonged cold ischemic stress and preservation method of breast cancer tissue.
abstract: BACKGROUND
  Tissue handling can alter global gene expression potentially affecting the analytical performance of genomic signatures, but such effects have not been systematically evaluated.
  
  
  METHODS
  Tissue samples from 11 previously untreated breast tumors were minced and aliquots were either snap frozen or placed in RNAlater immediately or after 20, 40, 60, 120 or 180 min at room temperature. RNA was profiled on Affymetrix HG-U133A arrays. We used probe-set-wise hierarchical models to evaluate the effect of preservation method on transcript expression and linear mixed effects models to assess the effect of cold ischemic delay on the expression of individual probe sets. Gene set enrichment analysis identified pathways overrepresented in the affected transcripts. We combined the levels of 41 most sensitive transcripts to develop an index of ischemic stress.
  
  
  RESULTS
  Concordance in global gene expression between the baseline and 40 min delay was higher for samples preserved in RNAlater (average concordance correlation coefficient CCC = 0.92 compared to 0.88 for snap frozen). Overall, 481 transcripts (3%) were significantly affected by the preservation method, most of them involved in processes important in cancer. Prolonged cold ischemic delay of up to 3 h induced marginal global gene expression changes (average CCC = 0.90 between baseline and 3 h delay). However 41 transcripts were significantly affected by cold ischemic delay. Among the induced transcripts were stress response genes, apoptotic response genes; among the downregulated were genes involved in metabolism, protein processing and cell cycle regulation. An index combining the expression levels of these genes was proportional to the cold ischemic delay.
  
  
  CONCLUSIONS
  Prolonged cold ischemia induces significant transcriptional changes in a small subset of transcripts in the tissue. Furthermore, the expression level of about 3% of the transcripts is affected by the preservation method. These sensitive transcripts should not be included in genomic signatures for more reliable analytical performance.
authors.affiliation: Section of Medical Oncology, Yale Cancer Center, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
authors.forename: Bilge
authors.initials: B
authors.lastname: Aktas
authors.affiliation: Department of Pathology, UT M.D. Anderson Cancer Center, P.O. Box 85, 1515 Holcombe Blvd, Houston, TX 77030, USA.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Sun
authors.affiliation: Department of Bioinformatics and Computational Biology, UT M.D. Anderson Cancer Center, P.O. Box 301402, Houston, TX 77230, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Yao
authors.affiliation: Section of Medical Oncology, Yale Cancer Center, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Shi
authors.affiliation: Department of Pathology, UT M.D. Anderson Cancer Center, P.O. Box 85, 1515 Holcombe Blvd, Houston, TX 77030, USA.
authors.forename: Rebekah
authors.initials: R
authors.lastname: Hubbard
authors.affiliation: Department of Pathology, UT M.D. Anderson Cancer Center, P.O. Box 85, 1515 Holcombe Blvd, Houston, TX 77030, USA.
authors.forename: Ya
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Section of Medical Oncology, Yale Cancer Center, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
authors.forename: Tingting
authors.initials: T
authors.lastname: Jiang
authors.affiliation: Section of Medical Oncology, Yale Cancer Center, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
authors.forename: Sophia N
authors.initials: SN
authors.lastname: Ononye
authors.affiliation: Section of Medical Oncology, Yale Cancer Center, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
authors.forename: Vikram B
authors.initials: VB
authors.lastname: Wali
authors.affiliation: Section of Medical Oncology, Yale Cancer Center, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
authors.forename: Lajos
authors.initials: L
authors.lastname: Pusztai
authors.affiliation: Department of Pathology, UT M.D. Anderson Cancer Center, P.O. Box 85, 1515 Holcombe Blvd, Houston, TX 77030, USA.
authors.forename: W Fraser
authors.initials: WF
authors.lastname: Symmans
authors.affiliation: Section of Medical Oncology, Yale Cancer Center, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. Electronic address: christos.hatzis@yale.edu.
authors.forename: Christos
authors.initials: C
authors.lastname: Hatzis
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001943:Breast Neoplasms / Q000150:complications / Q000235:genetics*; D015687:Cell Hypoxia; D003080:Cold Temperature; D015925:Cryopreservation / Q000379:methods; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D006801:Humans; D007511:Ischemia / Q000150:complications / Q000235:genetics; D008875:Middle Aged; D012333:RNA, Messenger / Q000235:genetics*; D014021:Tissue Preservation / Q000379:methods
chemical_list: D012333:RNA, Messenger
keywords: Cold ischemia; Genomic index; Hypoxia; Ischemic stress index; Microarrays; RNAlater; Snap freezing