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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

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1437-4331
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Volume 52, Issue 5 (May 2014)

Issues

Routine use of the Ion Torrent AmpliSeq™ Cancer Hotspot Panel for identification of clinically actionable somatic mutations

Gregory J. Tsongalis
  • Corresponding author
  • Department of Pathology, Geisel School of Medicine at Dartmouth and Dartmouth Hitchcock Medical Center, Lebanon, NH, USA
  • Email:
/ Jason D. Peterson
  • Department of Pathology, Geisel School of Medicine at Dartmouth and Dartmouth Hitchcock Medical Center, Lebanon, NH, USA
/ Francine B. de Abreu
  • Department of Pathology, Geisel School of Medicine at Dartmouth and Dartmouth Hitchcock Medical Center, Lebanon, NH, USA
/ Christopher D. Tunkey
  • Department of Pathology, Geisel School of Medicine at Dartmouth and Dartmouth Hitchcock Medical Center, Lebanon, NH, USA
/ Torrey L. Gallagher
  • Department of Pathology, Geisel School of Medicine at Dartmouth and Dartmouth Hitchcock Medical Center, Lebanon, NH, USA
/ Linda D. Strausbaugh
  • Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA
/ Wendy A. Wells
  • Department of Pathology, Geisel School of Medicine at Dartmouth and Dartmouth Hitchcock Medical Center, Lebanon, NH, USA
/ Christopher I. Amos
  • Departments of Medicine and Community and Family Medicine, Geisel School of Medicine at Dartmouth and Dartmouth Hitchcock Medical Center, Lebanon, NH, USA
Published Online: 2013-12-13 | DOI: https://doi.org/10.1515/cclm-2013-0883

Abstract

Background: Somatic mutation analysis is standard of practice for solid tumors in order to identify therapeutic sensitizing and resistance mutations. Our laboratory routinely performed standalone PCR-based methods for mutations in several genes. Rapid discovery and introduction of new therapeutics has demanded additional genomic information for adequate management of the cancer patient. We evaluated a next generation sequencing assay, the Ion Torrent AmpliSeq Cancer Hotspot Panelv2 (CHPv2), capable of identifying multiple somatic mutations in 50 genes in a single assay.

Methods: Accuracy, precision, limit of detection, and specificity were evaluated using DNA from well-characterized cell lines, genetically engineered cell lines fixed and embedded in paraffin, and previously tested mutation positive or negative, formalin-fixed, paraffin-embedded (FFPE) tissues. Normal kidney, tonsil and colon FFPE tissues were used as controls.

Results: Accuracy studies showed 100% concordance in each patient sample between previous PCR results and the corresponding variants identified using the Ion Torrent panel. Precision studies gave consistent results when libraries were prepared from the same original DNA and were run on multiple 316 chips. The limit of detection was determined to be 5% for single nucleotide variants (SNVs) and 20% for insertions and deletions (indels). Specificity studies using normal FFPE tissue previously tested by PCR methods were also 100%.

Conclusions: We have evaluated the performance of the AmpliSeq Cancer Panel Hotspotv2 and show that it is suitable for clinical testing. This next generation sequencing panel has allowed the laboratory to consolidate a broader range of molecular oncology testing to a single platform and single assay.

Keywords: cancer; colon cancer; glioma; lung cancer; melanoma; next generation sequencing; somatic mutations

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About the article

Corresponding author: Gregory J. Tsongalis, Department of Pathology, Geisel School of Medicine at Dartmouth and Dartmouth Hitchcock Medical Center, 1 Medical Center Drive, Lebanon, NH 03756, USA, Phone: +1 603 650 5498, E-mail:


Received: 2013-10-14

Accepted: 2013-11-23

Published Online: 2013-12-13

Published in Print: 2014-05-01


Citation Information: Clinical Chemistry and Laboratory Medicine, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2013-0883.

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