title: Simultaneous detection of clinically relevant mutations and amplifications for routine cancer pathology.
abstract: In routine cancer molecular pathology, various independent experiments are required to determine mutation and amplification status of clinically relevant genes. Most of these tests are designed to identify a limited number of genetic aberrations, most likely in a given tumor type. We present a modified version of a multiplexed PCR and IonTorrent-based sequencing approach that can replace a large number of existing assays. The test allows for the simultaneous detection of point mutations and gene amplifications in 40 genes, including known hotspot regions in oncogenes (KRAS, BRAF), inactivating mutations in tumor suppressors (TP53, PTEN), and oncogene amplifications (ERBB2, EGFR). All point mutations were confirmed using certified diagnostic assays, and a sensitivity and specificity of 100% (95% CI, 0.875-1.0) and 99% (95% CI, 0.960-0.999), respectively, were determined for amplifications in FFPE material. Implementation of a single assay to effectively detect mutations and amplifications in clinically relevant genes not only improves the efficiency of the workflow within diagnostic laboratories but also increases the chance of detecting (rare) actionable variants for a given tumor type that are typically missed in routine pathology. The ability to obtain comprehensive and rapid mutational overviews is key for improving the efficiency of cancer patient care through tailoring treatments based on the genetic characteristics of individual tumors.
authors.affiliation: Department of Medical Oncology, University Medical Center Utrecht, Heidelberglaan Utrecht, Utrecht, the Netherlands; Netherlands Center for Personalized Cancer Treatment, Universiteitsweg Utrecht, Utrecht, the Netherlands.
authors.forename: Marlous
authors.initials: M
authors.lastname: Hoogstraat
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Universiteitsweg Utrecht, Utrecht, the Netherlands.
authors.forename: John W J
authors.initials: JW
authors.lastname: Hinrichs
authors.affiliation: Department of Medical Oncology, University Medical Center Utrecht, Heidelberglaan Utrecht, Utrecht, the Netherlands; Netherlands Center for Personalized Cancer Treatment, Universiteitsweg Utrecht, Utrecht, the Netherlands.
authors.forename: Nicolle J M
authors.initials: NJ
authors.lastname: Besselink
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Universiteitsweg Utrecht, Utrecht, the Netherlands.
authors.forename: Joyce H
authors.initials: JH
authors.lastname: Radersma-van Loon
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Universiteitsweg Utrecht, Utrecht, the Netherlands.
authors.forename: Carmen M A
authors.initials: CM
authors.lastname: de Voijs
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Universiteitsweg Utrecht, Utrecht, the Netherlands.
authors.forename: Ton
authors.initials: T
authors.lastname: Peeters
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Universiteitsweg Utrecht, Utrecht, the Netherlands; Center for Molecular Medicine, University Medical Center Utrecht, Universiteitsweg Utrecht, Utrecht, the Netherlands.
authors.forename: Isaac J
authors.initials: IJ
authors.lastname: Nijman
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Universiteitsweg Utrecht, Utrecht, the Netherlands.
authors.forename: Roel A
authors.initials: RA
authors.lastname: de Weger
authors.affiliation: Department of Medical Oncology, University Medical Center Utrecht, Heidelberglaan Utrecht, Utrecht, the Netherlands; Netherlands Center for Personalized Cancer Treatment, Universiteitsweg Utrecht, Utrecht, the Netherlands.
authors.forename: Emile E
authors.initials: EE
authors.lastname: Voest
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Universiteitsweg Utrecht, Utrecht, the Netherlands; Department of Pathology, University Medical Center Utrecht, Universiteitsweg Utrecht, Utrecht, the Netherlands. Electronic address: s.m.willems-4@umcutrecht.nl.
authors.forename: Stefan M
authors.initials: SM
authors.lastname: Willems
authors.affiliation: Netherlands Center for Personalized Cancer Treatment, Universiteitsweg Utrecht, Utrecht, the Netherlands; Center for Molecular Medicine, University Medical Center Utrecht, Universiteitsweg Utrecht, Utrecht, the Netherlands; Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center Utrecht, Utrecht, the Netherlands. Electronic address: e.cuppen@hubrecht.eu.
authors.forename: Edwin
authors.initials: E
authors.lastname: Cuppen
authors.affiliation: Department of Medical Oncology, University Medical Center Utrecht, Heidelberglaan Utrecht, Utrecht, the Netherlands; Netherlands Center for Personalized Cancer Treatment, Universiteitsweg Utrecht, Utrecht, the Netherlands.
authors.forename: Marco J
authors.initials: MJ
authors.lastname: Koudijs
mesh_terms: D005557:Formaldehyde; D005784:Gene Amplification; D015870:Gene Expression; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D009363:Neoplasm Proteins / Q000235:genetics*; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D010232:Paraffin; D016612:Paraffin Embedding; D017354:Point Mutation; D057285:Precision Medicine; D012680:Sensitivity and Specificity; D016707:Tissue Fixation
chemical_list: D009363:Neoplasm Proteins; D005557:Formaldehyde; D010232:Paraffin