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KRAS mutation analysis in genomic DNA isolated from formalin-fixed paraffin-embedded ovarian tissue: evaluation of a strip-based reverse-hybridisation assay
  1. Gernot Kriegshäuser1,
  2. Veronika Auner2,
  3. Eva Schuster3,
  4. Barbara Holzer3,
  5. Christian Oberkanins1,
  6. Reinhard Horvat4,
  7. Paul Speiser3,
  8. Robert Zeillinger2,3
  1. 1ViennaLab Diagnostics GmbH, Vienna, Austria
  2. 2Ludwig-Boltzmann Institute for Gynecological Oncology, Cluster Translational Oncology, Vienna, Austria
  3. 3Department of Obstetrics and Gynecology, Division of Gynecology, Molecular Oncology Group, Medical University of Vienna, Vienna, Austria
  4. 4Department of Clinical Pathology, Medical University of Vienna, Vienna, Austria
  1. Correspondence to Gernot Kriegshäuser, ViennaLab Diagnostics GmbH, Gaudenzdorfer Gürtel 43–45, 1120 Vienna, Austria; kriegshauser{at}viennalab.com.

Abstract

Aims To evaluate a reverse-hybridisation assay (strip assay) designed for the sensitive detection of 10 mutations in codons 12 and 13 of the KRAS gene. The strip assay relies on mutant-enriched PCR followed by reverse-hybridisation of biotinylated amplification products to oligonucleotide probes immobilised as an array of parallel lines on nitrocellulose test strips.

Methods The strip assay was used to analyse genomic DNA isolated from 120 formalin-fixed paraffin-embedded (FFPE) ovarian tissue samples. The samples were analysed in parallel using a biochip-based protocol (biochip assay) covering the same mutation spectrum, and results were compared with respect to sensitivity, specificity and operational input.

Results The strip assay identified 19 (16%) of 120 FFPE samples to carry a KRAS mutation; results were in agreement with those obtained by biochip hybridisation. Both assays had an analytical sensitivity of 1% when performed on FFPE-extracted DNA with approximately the same operational input needed for post-PCR processing. In contrast to the biochip assay, strip assay hybridisation may be automated to a large extent.

Conclusions The strip assay is an accurate and sensitive tool for the low to medium throughput detection of KRAS mutation in genomic DNA isolated from FFPE tissue.

  • Cancer
  • diagnostics
  • laboratory tests
  • molecular pathology
  • ovary

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Footnotes

  • GK and VA contributed equally to this study.

  • Competing interests GK and CO are employees, and RZ is shareholder of ViennaLab Diagnostics GmbH.

  • Ethics approval This study was conducted with the approval of the local institutional review board, General Hospital, Vienna, Austria.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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