title: Reliable gene expression profiling of formalin-fixed paraffin-embedded breast cancer tissue (FFPE) using cDNA-mediated annealing, extension, selection, and ligation whole-genome (DASL WG) assay.
abstract: The difficulties in using formalin-fixed and paraffin-embedded (FFPE) tumour specimens for molecular marker studies have hampered progress in translational cancer research. The cDNA-mediated, annealing, selection, extension, and ligation (DASL) assay is a platform for gene expression profiling from FFPE tissue and hence could allow analysis of large collections of tissue with associated clinical data from existing archives, therefore facilitating the development of novel biomarkers.
  
  
  
  RNA isolated from matched fresh frozen (FF) and FFPE cancer specimens was profiled using both the DASL whole-genome (WG) platform, and Illumina BeadArray's, and results were compared. Samples utilized were obtained from the breast cancer tumour bank held at the Cambridge University Hospitals NHS Foundation Trust.
  
  
  
  The number of reliably detected probes was comparable between the DASL and BeadArray platforms, indicating that the source of RNA did not result in a significant difference in the detection rates (Mean probes- 17114 in FFPE & 17400 in FF). There was a significant degree of correlation between replicates within the FF and FFPE sample sets (r (2) = 0.96-0.98) as well as between the two platforms (DASL vs. BeadArray r (2) = range 0.83-0.89). Hierarchical clustering using the most informative probes showed that replicate and matched samples were grouped into the same sub-cluster, regardless of whether RNA was derived from FF or FFPE tissue.
  
  
  
  Both FF and FFPE material generated reproducible gene expression profiles, although there was more noise in profiles from FFPE specimens. We have shown that the DASL WG platform is suitable for profiling formalin-fixed paraffin-embedded samples, but robust bioinformatics analysis is required.
authors.affiliation: Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK. mahesh.iddawela@monash.edu.|Department of Oncology, Addenbrooke's Hospital, University of Cambridge, Hills Road, Cambridge, CB1 9RN, UK. mahesh.iddawela@monash.edu.|Cambridge Breast Unit, Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK. mahesh.iddawela@monash.edu.|NIHR Cambridge Biomedical Research Centre and Cambridge Experimental Cancer Medicine Centre, Cambridge, UK. mahesh.iddawela@monash.edu.|Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, 3800, Australia. mahesh.iddawela@monash.edu.|School of Clinical Sciences, Monash University, Clayton, Victoria, Australia. mahesh.iddawela@monash.edu.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Iddawela
authors.affiliation: Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK.
authors.forename: Oscar M
authors.initials: OM
authors.lastname: Rueda
authors.affiliation: Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK.
authors.forename: Marcus
authors.initials: M
authors.lastname: Klarqvist
authors.affiliation: Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK.
authors.forename: Stefan
authors.initials: S
authors.lastname: Graf
authors.affiliation: Department of Oncology, Addenbrooke's Hospital, University of Cambridge, Hills Road, Cambridge, CB1 9RN, UK.|Cambridge Breast Unit, Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.|NIHR Cambridge Biomedical Research Centre and Cambridge Experimental Cancer Medicine Centre, Cambridge, UK.
authors.forename: Helena M
authors.initials: HM
authors.lastname: Earl
authors.affiliation: Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK.|Department of Oncology, Addenbrooke's Hospital, University of Cambridge, Hills Road, Cambridge, CB1 9RN, UK.|Cambridge Breast Unit, Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.|NIHR Cambridge Biomedical Research Centre and Cambridge Experimental Cancer Medicine Centre, Cambridge, UK.
authors.forename: Carlos
authors.initials: C
authors.lastname: Caldas
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology*; D018076:DNA, Complementary / Q000235:genetics; D005557:Formaldehyde; D020869:Gene Expression Profiling / Q000379:methods*; D023281:Genomics / Q000379:methods*; D016612:Paraffin Embedding; D016707:Tissue Fixation
chemical_list: D018076:DNA, Complementary; D005557:Formaldehyde
keywords: Breast Cancer; FFPE; Molecular Markers; WG DASL