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An ultra-deep sequencing strategy to detect sub-clonal TP53 mutations in presentation chronic lymphocytic leukaemia cases using multiple polymerases

Oncogene volume 35, pages 5328–5336 (2016)Cite this article

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Abstract

Chronic lymphocytic leukaemia (CLL) is the most common clonal B-cell disorder characterized by clonal diversity, a relapsing and remitting course, and in its aggressive forms remains largely incurable. Current front-line regimes include agents such as fludarabine, which act primarily via the DNA damage response pathway. Key to this is the transcription factor p53. Mutations in the TP53 gene, altering p53 functionality, are associated with genetic instability, and are present in aggressive CLL. Furthermore, the emergence of clonal TP53 mutations in relapsed CLL, refractory to DNA-damaging therapy, suggests that accurate detection of sub-clonal TP53 mutations prior to and during treatment may be indicative of early relapse. In this study, we describe a novel deep sequencing workflow using multiple polymerases to generate sequencing libraries (MuPol-Seq), facilitating accurate detection of TP53 mutations at a frequency as low as 0.3%, in presentation CLL cases tested. As these mutations were mostly clustered within the regions of TP53 encoding DNA-binding domains, essential for DNA contact and structural architecture, they are likely to be of prognostic relevance in disease progression. The workflow described here has the potential to be implemented routinely to identify rare mutations across a range of diseases.

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Acknowledgements

This project was funded by the Kay Kendall Leukaemia Fund (www.kklf.org.uk). We would also like to thank the staff in the Haematological Malignancy Diagnostic Services Unit at Leeds Teaching Hospital who provided information on diagnostic patient data and samples for the study, and the sequencing facility at the University of Leeds who provided a technical service running the Illumina next-generation sequencing systems.

Author contributions

LW prepared the manuscript and performed the validation experiments. PB, MAC and LH carried out the bioinformatics, and PB and LH co-wrote the bioinformatics sections. AV and TM obtained and prepared patient samples. PAE performed mutational analysis of IGHV and SJO carried out FISH cytogenetic analysis. AR performed flow analysis and assisted in study design. RMT helped design the study and reviewed the article. PH and DJN conceived and designed the study, and DJN wrote part of the manuscript and carried out the laboratory work.

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Authors and Affiliations

  1. Section of Experimental Haematology, Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, UK

    L Worrillow, M A Care, R M Tooze, P Hillmen & D J Newton

  2. Department of Evolutionary Biology, Max Planck Institute for Developmental Biology, Tuebingen, Germany

    P Baskaran

  3. Haematological Malignancy Diagnostic Services, Leeds Teaching Hospitals National Health Services Trust, Leeds, UK

    A Varghese, T Munir, P A Evans, S J O'Connor & A Rawstron

  4. Cancer Research UK, Ling Ka Shing Centre, University of Cambridge, Cambridge, UK

    L Hazelwood

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Correspondence to D J Newton.

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Worrillow, L., Baskaran, P., Care, M. et al. An ultra-deep sequencing strategy to detect sub-clonal TP53 mutations in presentation chronic lymphocytic leukaemia cases using multiple polymerases. Oncogene 35, 5328–5336 (2016). https://doi.org/10.1038/onc.2016.73

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