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Clinical Chemistry and Laboratory Medicine (CCLM)

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Volume 52, Issue 5 (May 2014)

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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

References

  • 1.

    Tuononen K, Mäki-Nevala S, Sarhadi VK, Wirtanen A, Rönty M, Salmenkivi K, et al. Comparison of targeted next-generation sequencing (NGS) and real-time PCR in the detection of EGFR, KRAS, and BRAF mutations on formalin-fixed, paraffin-embedded tumor material of non-small cell lung carcinoma-superiority of NGS. Genes Chromosomes Cancer 2013;5:503–11. [Web of Science] [Crossref]

  • 2.

    Peeters M, Oliner KS, Parker A, Siena S, Van Cutsem E, Huang J, et al. Massively parallel tumor multigene sequencing to evaluate response to panitumumab in a randomized phase III study of metastatic colorectal cancer. Clin Cancer Res 2013;19:1902–12. [Crossref]

  • 3.

    Amado RG, Wolf M, Peeters M, Van Cutsem E, Siena S, Freeman DJ, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008;26:1626–34. [PubMed] [Crossref]

  • 4.

    Tol J, Koopman M, Cats A, Rodenburg CJ, Creemers GJ, Schrama JG, et al. Chemotherapy, bevacizumab, and cetuximab in metastatic colorectal cancer. N Engl J Med 2009;360:563–72. [Web of Science]

  • 5.

    Di Nicolantonio F, Martini M, Molinari F, Sartore-Bianchi A, Arena S, Saletti P, et al. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol 2008;26:5705–12. [Crossref] [PubMed]

  • 6.

    Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497–500.

  • 7.

    Hudis CA. Trastuzumab – mechanism of action and use in clinical practice. N Engl J Med 2007;357:39–51.

  • 8.

    Walther A, Johnstone E, Swanton C, Midgley R, Tomlinson I, Kerr D. Genetic prognostic and predictive markers in colorectal cancer. Nat Rev Cancer 2009;9:489–99. [Crossref] [PubMed] [Web of Science]

  • 9.

    Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:949–54.

  • 10.

    Eberhard DA, Johnson BE, Amler LC, Goddard AD, Heldens SL, Herbst RS, et al. Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol 2005;23:5900–9. [PubMed] [Crossref]

  • 11.

    Pao W, Miller VA. Epidermal growth factor receptor mutations, small-molecule kinase inhibitors, and non-small-cell lung cancer: current knowledge and future directions. J Clin Oncol 2005;23:2556–68. [Crossref] [PubMed]

  • 12.

    Sequist LV, Joshi VA, Jänne PA, Muzikansky A, Fidias P, Meyerson M, et al. Response to treatment and survival of patients with non-small cell lung cancer undergoing somatic EGFR mutation testing. Oncologist 2007;12:90–8. [Crossref] [Web of Science] [PubMed]

  • 13.

    Marks JL, Broderick S, Zhou Q, Chitale D, Li AR, Zakowski MF, et al. Prognostic and therapeutic implications of EGFR and KRAS mutations in resected lung adenocarcinoma. J Thorac Oncol 2008;3:111–6. [Crossref] [PubMed]

  • 14.

    Siegfried JM, Gillespie AT, Mera R, Casey TJ, Keohavong P, Testa JR, et al. Prognostic value of specific KRAS mutations in lung adenocarcinomas. Cancer Epidemiol Biomarkers Prev 1997;6:841–7. [PubMed]

  • 15.

    Wertheim GB, Daber R, Bagg A. Molecular diagnostics of acute myeloid leukemia: it’s a (next) generational thing. J Mol Diagn 2013;1:27–30. [Crossref]

  • 16.

    Spencer DH, Abel HJ, Lockwood CM, Payton JE, Szankasi P, Kelley TW, et al. Detection of FLT3 internal tandem duplication in targeted, short-read-length, next-generation sequencing data. J Mol Diagn 2013;1:81–93.

  • 17.

    Metzker ML. Sequencing technologies – the next generation. Nat Rev Genet 2010;11:31–46. [Crossref] [PubMed]

  • 18.

    Quail MA, Smith M, Coupland P, Otto TD, Harris SR, Connor TR, et al. A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics 2012;13:341. [Crossref] [PubMed]

  • 19.

    Hadd AG, Houghton J, Choudhary A, Sah S, Chen L, Marko AC, et al. Targeted, high-depth, next-generation sequencing of cancer genes in formalin-fixed, paraffin-embedded and fine-needle aspiration tumor specimens. J Mol Diagn 2013;2:234–47. [Crossref]

  • 20.

    Beadling C, Neff TL, Heinrich MC, Rhodes K, Thornton M, Leamon J, et al. Combining highly multiplexed PCR with semiconductor-based sequencing for rapid cancer genotyping. J Molec Diagn 2013;15:171–6. [Crossref]

  • 21.

    Singh RR, Patel KP, Routbort MJ, Reddy NG, Barkoh BA, Handal B, et al. Clinical validation of a next generation sequencing screen for mutational hotspots in 46 cancer related genes. J Molec Diagn 2013;15:607–22. [Crossref]

  • 22.

    Merriman B, Ion Torrent R&D Team, Rothberg JM. Progress in ion torrent semiconductor chip based sequencing. Electrophoresis 2012;33:3397–417. [Crossref] [Web of Science]

  • 23.

    Rothberg JM, Hinz W, Rearick TM, Schultz J, Mileski W, Davey M, et al. An integrated semiconductor device enabling non-optical genome sequencing. Nature 2011;475:348–52. [Web of Science]

  • 24.

    Yousem SA, Dacic S, Nikiforov YE, Nikiforova M. Pulmonary Langerhans cell histiocytosis: profiling of multifocal tumors using next-generation sequencing identifies concordant occurrence of BRAF V600E mutations. Chest 2013;143:1679–84. [Web of Science]

  • 25.

    Lièvre A, Bachet JB, Boige V, Cayre A, Le Corre D, Buc E, et al. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol 2008;26:374–9. [PubMed] [Crossref]

  • 26.

    Jarry A, Masson D, Cassagnau E, Parois S, Laboisse C, Denis MG. Real-time allele-specific amplification for sensitive detection of the BRAF mutation V600E. Mol Cell Probes 2004;18:349–52.

  • 27.

    Pan Q, Pao W, Ladanyi M. Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas. J Mol Diagn 2005;7:396–403. [Crossref] [PubMed]

  • 28.

    Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol 2011;29:24–6. [Web of Science] [PubMed] [Crossref]

  • 29.

    Kaushal D, Naeve CW. An overview of Spotfire for gene-expression studies. Curr Protoc Bioinformatics 2004;7:7.7. [PubMed] [Crossref]

  • 30.

    Forbes SA, Bindal N, Bamford S, Cole C, Kok CY, Beare D, et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res 2011;39:D945–50. [Web of Science] [Crossref]

  • 31.

    Worthey EA, Mayer AN, Syverson GD, Helbling D, Bonacci BB, Decker B, et al. Making a definitive diagnosis: successful clinical application of whole exome sequencing in a child with intractable inflammatory bowel disease. Genet Med 2011;3:255–62. [Crossref] [Web of Science]

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. Export Citation

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