title: Development of a semi-conductor sequencing-based panel for genotyping of colon and lung cancer by the Onconetwork consortium.
abstract: BACKGROUND
  The number of predictive biomarkers that will be necessary to assess in clinical practice will increase with the availability of drugs that target specific molecular alterations. Therefore, diagnostic laboratories are confronted with new challenges: costs, turn-around-time and the amount of material required for testing will increase with the number of tests performed on a sample. Our consortium of European clinical research laboratories set out to test if semi-conductor sequencing provides a solution for these challenges.
  
  
  METHODS
  We designed a multiplex PCR targeting 87 hotspot regions in 22 genes that are of clinical interest for lung and/or colorectal cancer. The gene-panel was tested by 7 different labs in their own clinical setting using ion-semiconductor sequencing.
  
  
  RESULTS
  We analyzed 155 samples containing 112 previously identified mutations in the KRAS, EGFR en BRAF genes. Only 1 sample failed analysis due to poor quality of the DNA. All other samples were correctly genotyped for the known mutations, even as low as 2%, but also revealed other mutations. Optimization of the primers used in the multiplex PCR resulted in a uniform coverage distribution over the amplicons that allows for efficient pooling of samples in a sequencing run.
  
  
  CONCLUSIONS
  We show that a semi-conductor based sequencing approach to stratify colon and lung cancer patients is feasible in a clinical setting.
authors: Department of Pathology, Radboud University Medical Centre (Radboudumc), PO box 9101, 6500 HB, Nijmegen, The Netherlands. bastiaan.tops@radboudumc.nl.
authors: Bastiaan B J
authors: BB
authors: Tops
authors: Cell Biology and Biotherapy Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale", IRCCS, 80131, Naples, Italy. nicnorm@yahoo.com.|Pharmacogenomic Laboratory, CROM - Centro Ricerche Oncologiche di Mercogliano, Mercogliano, 83013, Avellino, Italy. nicnorm@yahoo.com.
authors: Nicola
authors: N
authors: Normanno
authors: VIOLLIER, Department of Genetics/Molecular Biology, Basle, Switzerland. henriette.kurth@viollier.ch.
authors: Henriette
authors: H
authors: Kurth
authors: ARC-NET Miriam Cherubini Research Centre, University of Verona, Verona, Italy. eliana.amato@univr.it.
authors: Eliana
authors: E
authors: Amato
authors: ARC-NET Miriam Cherubini Research Centre, University of Verona, Verona, Italy. andrea.mafficini@univr.it.
authors: Andrea
authors: A
authors: Mafficini
authors: Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany. n.rieber@dkfz-heidelberg.de.
authors: Nora
authors: N
authors: Rieber
authors: Paris Sorbonne Cité; INSERM UMR-S775, Bases moléculaires des la réponses aux xénobiotiques, Paris, France. delphine.lecorre@parisdescartes.fr.
authors: Delphine
authors: D
authors: Le Corre
authors: Cell Biology and Biotherapy Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale", IRCCS, 80131, Naples, Italy. anmarachiglio@yahoo.it.
authors: Anna Maria
authors: AM
authors: Rachiglio
authors: Department of Pathology, Warwick Medical School, University Hospitals Coventry and Warwickshire, Coventry, CV2 2DX, UK. a.reiman@warwick.ac.uk.
authors: Anne
authors: A
authors: Reiman
authors: Department of Histopathology, The University of Dublin, Trinity College, Dublin, Ireland. osheils@tcd.ie.
authors: Orla
authors: O
authors: Sheils
authors: VIOLLIER, Department of Genetics/Molecular Biology, Basle, Switzerland. christoph.noppen@viollier.ch.
authors: Christoph
authors: C
authors: Noppen
authors: Department of Medical Biology and Pathology, Translational research Laboratory and Biobank (UMS3655 CNRS/US23 INSERM), INSERM Unit U981, Gustave Roussy, Villejuif, France. ludovic.lacroix@igr.fr.
authors: Ludovic
authors: L
authors: Lacroix
authors: Department of Pathology, Warwick Medical School, University Hospitals Coventry and Warwickshire, Coventry, CV2 2DX, UK. i.a.cree@warwick.ac.uk.
authors: Ian A
authors: IA
authors: Cree
authors: ARC-NET Miriam Cherubini Research Centre, University of Verona, Verona, Italy. aldo.scarpa@univr.it.|Department of Pathology and Diagnostics, University of Verona, Verona, Italy. aldo.scarpa@univr.it.
authors: Aldo
authors: A
authors: Scarpa
authors: Department of Pathology, Radboud University Medical Centre (Radboudumc), PO box 9101, 6500 HB, Nijmegen, The Netherlands. marjolijn.ligtenberg@radboudumc.nl.|Department of Human Genetics, Radboud University Medical Centre (Radboudumc), PO box 9101, 6500 HB, Nijmegen, The Netherlands. marjolijn.ligtenberg@radboudumc.nl.
authors: Marjolijn J L
authors: MJ
authors: Ligtenberg
authors: Paris Sorbonne Cité; INSERM UMR-S775, Bases moléculaires des la réponses aux xénobiotiques, Paris, France. pierre.laurent-puig@parisdescartes.fr.
authors: Pierre
authors: P
authors: Laurent-Puig
mesh_terms: D003110:Colonic Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D059645:Mutation Rate; D015203:Reproducibility of Results
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