title: Mutation detection in formalin-fixed prostate cancer biopsies taken at the time of diagnosis using next-generation DNA sequencing.
abstract: OBJECTIVE
  Assessing whether next-generation DNA sequencing (NGS) can be used to screen prostate cancer for multiple gene alterations in men routinely diagnosed with this disease and/or who are entered into clinical trials. Previous studies are limited and have reported only low success rates.
  
  
  METHODS
  We marked areas of cancer on H&E-stained sections from formalin-fixed needle biopsies, and used these as templates to dissect cancer-rich tissue from adjacent unstained sections. DNA was prepared using a Qiagen protocol modified to maximise DNA yield. The DNA was screened simultaneously for mutations in 365 cancer-related genes using an Illumina HiSeq 2000 NGS platform.
  
  
  RESULTS
  From 63 prostate cancers examined, 59(94%) of the samples yielded at least 30 ng of DNA, the minimum amount of DNA considered suitable for NGS analysis. Patients in the D'Amico high-risk group yielded an average of 1033 ng, intermediate-risk patients 401 ng, and low-risk patients 97 ng. NGS of eight samples selected from high-risk and intermediate-risk groups gave a median exon read depth of 962 and detected TMPRRS2-ERG fusions, as well as a variety of mutations including those in the SPOP, TP53, ATM, MEN1, NBPF10, NCOR2, PIK3CB and MAP2K5 (MEK5) genes.
  
  
  CONCLUSIONS
  Using the methods presented here, NGS technologies can be used to screen a high proportion of patients with prostate cancer for mutations in cancer-related genes in tissue samples opening up its general use in the context of clinical trials or routine diagnosis.
authors.affiliation: Department of Urology, Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, UK.
authors.forename: David
authors.initials: D
authors.lastname: Manson-Bahr
authors.affiliation: Department of Histopathology, Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Ball
authors.affiliation: Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
authors.forename: Gunes
authors.initials: G
authors.lastname: Gundem
authors.affiliation: Department of Urology, Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, UK.
authors.forename: Krishna
authors.initials: K
authors.lastname: Sethia
authors.affiliation: Department of Urology, Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Mills
authors.affiliation: Department of Urology, Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Rochester
authors.affiliation: Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
authors.forename: Victoria
authors.initials: V
authors.lastname: Goody
authors.affiliation: Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Anderson
authors.affiliation: Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: O'Meara
authors.affiliation: Norwich Medical School and School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, UK.
authors.forename: Marcus
authors.initials: M
authors.lastname: Flather
authors.affiliation: Department of Information Services, Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Keeling
authors.affiliation: Department of Urology, Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, UK.
authors.forename: Marcelino
authors.initials: M
authors.lastname: Yazbek-Hanna
authors.affiliation: Norwich Medical School and School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, UK.
authors.forename: Rachel
authors.initials: R
authors.lastname: Hurst
authors.affiliation: Norwich Medical School and School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Curley
authors.affiliation: Norwich Medical School and School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, UK.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Clark
authors.affiliation: Norwich Medical School and School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, UK.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Brewer
authors.affiliation: Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
authors.forename: Ultan
authors.initials: U
authors.lastname: McDermott
authors.affiliation: Norwich Medical School and School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, UK.
authors.forename: Colin
authors.initials: C
authors.lastname: Cooper
mesh_terms: D000368:Aged; D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics*; D044963:Biopsy, Fine-Needle; D004252:DNA Mutational Analysis / Q000379:methods*; D005404:Fixatives; D005557:Formaldehyde; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007610:Kallikreins / Q000097:blood; D008297:Male; D008875:Middle Aged; D009154:Mutation; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D010641:Phenotype; D011237:Predictive Value of Tests; D017430:Prostate-Specific Antigen / Q000097:blood; D011471:Prostatic Neoplasms / Q000097:blood / Q000235:genetics* / Q000473:pathology; D018570:Risk Assessment; D012307:Risk Factors; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor; D005404:Fixatives; D005557:Formaldehyde; C549870:KLK3 protein, human; D007610:Kallikreins; D017430:Prostate-Specific Antigen
keywords: CANCER; CANCER GENETICS; GENETICS; MOLECULAR GENETICS; PROSTATE