title: Next-generation RNA sequencing of archival formalin-fixed paraffin-embedded urothelial bladder cancer.
abstract: Molecular profiling of individual cancers is key to personalised medicine. While sequencing technologies have required stringent sample collection and handling, recent technical advances offer sequencing from tissues collected in routine practice and tissues already stored in archives. In this paper, we establish methods for whole-transcriptome RNA sequencing (RNA-seq) from formalin-fixed paraffin-embedded tissues. We obtain average RNA-seq reads of >100 million per sample using the Illumina HiSeq2000 platform. We find high concordance with results from matching fresh frozen samples (>0.8 Spearman correlation). For validation, we compared low- and high-grade bladder cancer transcriptomes in 49 tumour samples after transurethral resection of bladder tumour. We found 947 differentially expressed protein-coding genes. While high-grade lesions exhibited distinct intertumour transcriptome heterogeneity, the transcriptome of low-grade tumours was homogeneous.
  
  
  RESULTS
  In this report, we show that it is now possible to use universally available bladder cancer samples that have been fixed in formalin to perform high-quality transcriptome analysis. This ability will facilitate the development of transcriptome-wide tests based on gene expression correlated with clinical outcome.
authors.affiliation: The Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Surgery, Division of Urology, University of Toronto, Mount Sinai Hospital and University Health Network, Toronto, Ontario, Canada.
authors.forename: Aidan P
authors.initials: AP
authors.lastname: Noon
authors.affiliation: The Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Aguiar Cabeza
authors.affiliation: The Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Jess
authors.initials: J
authors.lastname: Shen
authors.affiliation: Department of Surgery, Division of Urology, University of Toronto, Mount Sinai Hospital and University Health Network, Toronto, Ontario, Canada.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Kuk
authors.affiliation: Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Christine
authors.initials: C
authors.lastname: Ilczynski
authors.affiliation: Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Ruoyu
authors.initials: R
authors.lastname: Ni
authors.affiliation: Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Balram
authors.initials: B
authors.lastname: Sukhu
authors.affiliation: The Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Kin
authors.initials: K
authors.lastname: Chan
authors.affiliation: Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, Toronto, Ontario, Canada; Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Nuno L
authors.initials: NL
authors.lastname: Barbosa-Morais
authors.affiliation: Department of Surgery, Division of Urology, University of Toronto, Mount Sinai Hospital and University Health Network, Toronto, Ontario, Canada.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hermanns
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada; Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Blencowe
authors.affiliation: Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Azar
authors.initials: A
authors.lastname: Azad
authors.affiliation: Department of Pathology, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada.
authors.forename: Theodorus H
authors.initials: TH
authors.lastname: van der Kwast
authors.affiliation: The Academic Urology Unit and Academic Unit of Molecular Oncology, University of Sheffield, Sheffield, UK.
authors.forename: James W F
authors.initials: JW
authors.lastname: Catto
authors.affiliation: Department of Surgery, Division of Urology, University of Toronto, Mount Sinai Hospital and University Health Network, Toronto, Ontario, Canada.
authors.forename: Alexandre R
authors.initials: AR
authors.lastname: Zlotta
authors.affiliation: The Centre for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. Electronic address: wrana@lunenfeld.ca.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Wrana
mesh_terms: D005404:Fixatives; D005557:Formaldehyde; D020869:Gene Expression Profiling; D006801:Humans; D060787:Neoplasm Grading; D016612:Paraffin Embedding; D017423:Sequence Analysis, RNA / Q000379:methods*; D013048:Specimen Handling; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000473:pathology
chemical_list: D005404:Fixatives; D005557:Formaldehyde
keywords: Formalin-fixed paraffin-embedded; Grade; Personalised medicine; Protein–protein interaction; RNA sequencing; Transcriptome; Urinary bladder neoplasms