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Low frequency clonal mutations recoverable by deep sequencing in patients with aplastic anemia

Leukemia volume 27, pages 968–971 (2013)Cite this article

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Up to 20% of patients with aplastic anemia (AA) who do not undergo allogeneic hematopoietic stem cell transplantation (HSCT) will ultimately develop myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML).1 Distinguishing which patients will evolve to MDS could help guide decision-making between immunosuppresive therapy (IST) and HSCT, but risk factors for malignant evolution remain poorly defined. Recent studies utilizing single nucleotide polymorphism (SNP) arrays have identified somatic alterations in 15–20% of patients with AA.2, 3 However, the unbiased identification of variants by either SNP arrays or traditional Sanger sequencing typically requires an allele fraction >25% of the involved specimen. Because the progression from AA to MDS may occur over years or even decades, subclones that ultimately evolve into MDS could be present at very low fractions at the time of AA diagnosis.

Sanger sequencing of specimens from patients with AA has not identified recurrent alterations, suggesting that they are either not present or exist within subclones below the level of detection with this method. Next-generation sequencing to a high-depth of coverage offers the potential to identify subclonal mutations with sensitivity proportional to the depth of sequencing. We developed a platform for exon capture followed by deep sequencing4 of 219 genes (Supplementary Table 1) that are recurrently mutated in hematologic malignancies (see Supplementary Methods). Patients with AA and available blood or bone marrow were identified by searching databases of stored specimens at the Dana-Farber Cancer Institute and Brigham and Women’s Hospital. All studies were performed under the approval of the Institutional Review Board of the Dana-Farber Cancer Institute/Harvard Cancer Center.

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Acknowledgements

The authors thank Paul van Hummelen for assistance with sequencing. This research was supported by the Edward P Evans Foundation (AY), the Conquer Cancer Foundation (AAL), Lauri Strauss Leukemia Foundation (AAL), and Leukemia and Lymphoma Society (AAL), the Jock and Bunny Adams Education and Research Fund (JHA), the Stellato Fund (DMW) and a Stand Up To Cancer Innovative Research Grant (DMW).

Author contributions

AAL designed and performed research, collected and interpreted data, and wrote the manuscript. OO designed and performed research, interpreted data and performed statistical analysis. NK and SK performed research, collected and analyzed data. AY designed research. RE designed research and interpreted data. NW contributed analytical tools. GAA performed statistical analysis and interpreted data. SJR performed research, contributed analytical tools and interpreted data. JHA designed research and wrote the manuscript. DMW designed research, interpreted data and wrote the manuscript.

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Authors and Affiliations

  1. Department of Medical Oncology, Boston, MA, USA

    A A Lane, O Odejide, N Kopp, S Kim, A Yoda, N Wagle, G A Abel, J H Antin & D M Weinstock

  2. Harvard Medical School, Boston, MA, USA

    A A Lane, N Wagle, G A Abel, S J Rodig, J H Antin & D M Weinstock

  3. Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, MA, USA

    R Erlich

  4. Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA

    S J Rodig

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  1. A A Lane
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Correspondence to D M Weinstock.

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Supplementary Information accompanies this paper on the Leukemia website

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Lane, A., Odejide, O., Kopp, N. et al. Low frequency clonal mutations recoverable by deep sequencing in patients with aplastic anemia. Leukemia 27, 968–971 (2013). https://doi.org/10.1038/leu.2013.30

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