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Single‐cell whole exome and targeted sequencing in NPM1/FLT3 positive pediatric acute myeloid leukemia
Author(s) -
Walter Christiane,
Pozzorini Christian,
Reinhardt Katarina,
Geffers Robert,
Xu Zhenyu,
Reinhardt Dirk,
Neuhoff Nils,
Hanenberg Helmut
Publication year - 2018
Publication title -
pediatric blood and cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.116
H-Index - 105
eISSN - 1545-5017
pISSN - 1545-5009
DOI - 10.1002/pbc.26848
Subject(s) - exome sequencing , npm1 , myeloid leukemia , medicine , myeloid , exome , cancer research , gene , microbiology and biotechnology , biology , genetics , mutation , karyotype , chromosome
Background The small portion of leukemic stem cells (LSCs) in acute myeloid leukemia (AML) present in children and adolescents is often masked by the high background of AML blasts and normal hematopoietic cells. The aim of the current study was to establish a simple workflow for reliable genetic analysis of single LSC‐enriched blasts from pediatric patients. Procedure For three AMLs with mutations in nucleophosmin 1 and/or fms‐like tyrosine kinase 3, we performed whole genome amplification on sorted single‐cell DNA followed by whole exome sequencing (WES). The corresponding bulk bone marrow DNAs were also analyzed by WES and by targeted sequencing (TS) that included 54 genes associated with myeloid malignancies. Results Analysis revealed that read coverage statistics were comparable between single‐cell and bulk WES data, indicating high‐quality whole genome amplification. From 102 single‐cell variants, 72 single nucleotide variants and insertions or deletions (70%) were consistently found in the two bulk DNA analyses. Variants reliably detected in single cells were also present in TS. However, initial screening by WES with read counts between 50–72× failed to detect rare AML subclones in the bulk DNAs. Conclusions In summary, our study demonstrated that single‐cell WES combined with bulk DNA TS is a promising tool set for detecting AML subclones and possibly LSCs.