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Research ArticleCancer Diagnostics

Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing

Sonia Mansukhani, Louise J. Barber, Dimitrios Kleftogiannis, Sing Yu Moorcraft, Michael Davidson, Andrew Woolston, Paula Zuzanna Proszek, Beatrice Griffiths, Kerry Fenwick, Bram Herman, Nik Matthews, Ben O'Leary, Sanna Hulkki, David Gonzalez De Castro, Anisha Patel, Andrew Wotherspoon, Aleruchi Okachi, Isma Rana, Ruwaida Begum, Matthew N. Davies, Thomas Powles, Katharina von Loga, Michael Hubank, Nick Turner, David Watkins, Ian Chau, David Cunningham, Stefano Lise, Naureen Starling, Marco Gerlinger
DOI: 10.1373/clinchem.2018.289629 Published September 2018
Sonia Mansukhani
Centre for Evolution and Cancer, Division of Molecular Pathology, The Institute of Cancer Research, London, UK;
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Louise J. Barber
Centre for Evolution and Cancer, Division of Molecular Pathology, The Institute of Cancer Research, London, UK;
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Dimitrios Kleftogiannis
Centre for Evolution and Cancer, Division of Molecular Pathology, The Institute of Cancer Research, London, UK;
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Sing Yu Moorcraft
Gastrointestinal Cancer Unit, The Royal Marsden NHS Foundation Trust, London and Sutton, UK;
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Michael Davidson
Gastrointestinal Cancer Unit, The Royal Marsden NHS Foundation Trust, London and Sutton, UK;
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Andrew Woolston
Centre for Evolution and Cancer, Division of Molecular Pathology, The Institute of Cancer Research, London, UK;
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Paula Zuzanna Proszek
Centre for Molecular Pathology, The Royal Marsden NHS Foundation Trust, Sutton, UK;
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Beatrice Griffiths
Centre for Evolution and Cancer, Division of Molecular Pathology, The Institute of Cancer Research, London, UK;
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Kerry Fenwick
Tumour Profiling Unit, The Institute of Cancer Research, London, UK;
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Bram Herman
Diagnostics and Genomics Group, Agilent Technologies Inc., Santa Clara, CA;
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Nik Matthews
Tumour Profiling Unit, The Institute of Cancer Research, London, UK;
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Ben O'Leary
Breast Cancer Now Research Centre, The Institute of Cancer Research, London, UK;
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Sanna Hulkki
Centre for Molecular Pathology, The Royal Marsden NHS Foundation Trust, Sutton, UK;
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David Gonzalez De Castro
Centre for Cancer Research and Cell Biology, Belfast, UK;
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Anisha Patel
Department for Radiology, The Royal Marsden NHS Foundation Trust, London and Sutton, UK;
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Andrew Wotherspoon
Department of Histopathology, The Royal Marsden NHS Foundation Trust, London and Sutton, UK;
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Aleruchi Okachi
Gastrointestinal Cancer Unit, The Royal Marsden NHS Foundation Trust, London and Sutton, UK;
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Isma Rana
Gastrointestinal Cancer Unit, The Royal Marsden NHS Foundation Trust, London and Sutton, UK;
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Ruwaida Begum
Gastrointestinal Cancer Unit, The Royal Marsden NHS Foundation Trust, London and Sutton, UK;
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Matthew N. Davies
Centre for Evolution and Cancer, Division of Molecular Pathology, The Institute of Cancer Research, London, UK;
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Thomas Powles
Barts Cancer Institute, Queen Mary University of London, London, UK;
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Katharina von Loga
Centre for Evolution and Cancer, Division of Molecular Pathology, The Institute of Cancer Research, London, UK;
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Michael Hubank
Centre for Molecular Pathology, The Royal Marsden NHS Foundation Trust, Sutton, UK;
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Nick Turner
Breast Cancer Now Research Centre, The Institute of Cancer Research, London, UK;Breast Cancer Unit, The Royal Marsden NHS Foundation Trust, London and Sutton, UK.
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David Watkins
Gastrointestinal Cancer Unit, The Royal Marsden NHS Foundation Trust, London and Sutton, UK;
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Ian Chau
Gastrointestinal Cancer Unit, The Royal Marsden NHS Foundation Trust, London and Sutton, UK;
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David Cunningham
Gastrointestinal Cancer Unit, The Royal Marsden NHS Foundation Trust, London and Sutton, UK;
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Stefano Lise
Centre for Evolution and Cancer, Division of Molecular Pathology, The Institute of Cancer Research, London, UK;
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Naureen Starling
Gastrointestinal Cancer Unit, The Royal Marsden NHS Foundation Trust, London and Sutton, UK;
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Marco Gerlinger
Centre for Evolution and Cancer, Division of Molecular Pathology, The Institute of Cancer Research, London, UK;Gastrointestinal Cancer Unit, The Royal Marsden NHS Foundation Trust, London and Sutton, UK;
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  • For correspondence: marco.gerlinger@icr.ac.uk
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Abstract

Background: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies.

Methods: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction.

Results: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to >95% of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100% of single-nucleotide variants, with 0.15% variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS, parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing.

Conclusions: This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution.

  • Received for publication March 21, 2018.
  • Accepted for publication July 17, 2018.
  • © 2018 American Association for Clinical Chemistry
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Clinical Chemistry: 65 (2)
Vol. 65, Issue 2
February 2019
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Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing
Sonia Mansukhani, Louise J. Barber, Dimitrios Kleftogiannis, Sing Yu Moorcraft, Michael Davidson, Andrew Woolston, Paula Zuzanna Proszek, Beatrice Griffiths, Kerry Fenwick, Bram Herman, Nik Matthews, Ben O'Leary, Sanna Hulkki, David Gonzalez De Castro, Anisha Patel, Andrew Wotherspoon, Aleruchi Okachi, Isma Rana, Ruwaida Begum, Matthew N. Davies, Thomas Powles, Katharina von Loga, Michael Hubank, Nick Turner, David Watkins, Ian Chau, David Cunningham, Stefano Lise, Naureen Starling, Marco Gerlinger
Clinical Chemistry Jan 2018, clinchem.2018.289629; DOI: 10.1373/clinchem.2018.289629
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Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing
Sonia Mansukhani, Louise J. Barber, Dimitrios Kleftogiannis, Sing Yu Moorcraft, Michael Davidson, Andrew Woolston, Paula Zuzanna Proszek, Beatrice Griffiths, Kerry Fenwick, Bram Herman, Nik Matthews, Ben O'Leary, Sanna Hulkki, David Gonzalez De Castro, Anisha Patel, Andrew Wotherspoon, Aleruchi Okachi, Isma Rana, Ruwaida Begum, Matthew N. Davies, Thomas Powles, Katharina von Loga, Michael Hubank, Nick Turner, David Watkins, Ian Chau, David Cunningham, Stefano Lise, Naureen Starling, Marco Gerlinger
Clinical Chemistry Jan 2018, clinchem.2018.289629; DOI: 10.1373/clinchem.2018.289629

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