Simultaneous Detection of Clinically Relevant Mutations and Amplifications for Routine Cancer Pathology

Marlous Hoogstraat, John W. J. Hinrichs, Nicolle J. M. Besselink, Joyce H. Radersma-van Loon, Carmen M. A. de Voijs, Ton Peeters, Isaac J. Nijman, Roel A. de Weger, Emile E. Voest, Stefan M. Willems, Edwin Cuppen*, Marco J. Koudijs

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


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.

Original languageEnglish
Pages (from-to)10-18
Number of pages9
JournalJournal of Molecular Diagnostics
Issue number1
Publication statusPublished - Jan 2015


  • Formaldehyde
  • Gene Amplification
  • Gene Expression
  • High-Throughput Nucleotide Sequencing
  • Humans
  • In Situ Hybridization, Fluorescence
  • Multiplex Polymerase Chain Reaction
  • Neoplasm Proteins
  • Neoplasms
  • Paraffin
  • Paraffin Embedding
  • Point Mutation
  • Precision Medicine
  • Sensitivity and Specificity
  • Tissue Fixation
  • Journal Article
  • Research Support, Non-U.S. Gov't


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