Abstract

Introduction:

Acute myeloid leukemia (AML) is one of the most frequent forms of leukemia in children younger than 15 years. The detection of several mutations in a blast population of pediatric AML (pAML) is supposed to be caused by a clonal evolution from a leukemic stem cell (LSC) to leukemic blasts. LSC are believed to be more resistant to chemotherapy, to be able to survive during treatment and to be responsible for the emergence of a relapse due to the persistence in the bone marrow (BM) niche. Since LSC and potential leukemic subclones are only present in small subpopulations, it has been a major technical challenge to particular analyse only the specific population. To acquire a better understanding of the underlying mechanisms of mutagenesis, clonal evolution and leukemogenesis, the aim of this study was to establish methods that allow the analysis and detection of mutations in single cells of a subpopulation known to contain HSC as well as LSC (CD34+CD38-). We especially focused on a pAML subgroup with mutations in Nucleophosmin (NPM1) and/or fms related tyrosine kinase 3 (Flt3).

Methods and Results:

We established methods to perform single cell sorting, whole genome amplification (WGA) using multiple displacement amplification (MDA) technology (Qiagen) and subsequent whole exome sequencing. The sorting efficiency was checked as Hoechst stained cells were sorted onto glas slides with 48 defined spots and the presence of single cells was checked under an inverse fluorescent microscope. Subsequently, single CD34+CD38- patient derived cells were sorted into 0,5ml low binding tubes containing 4µl PBS followed by WGA and whole exome sequencing. The mutational status of the sorted single cells from three patients suffering from pAML was analysed and compared to mutations detected at initial diagnosis in DNA from a bulk of BM cells. WGA from single CD34+CD38-PI- cells resulted in an amount of 29 to 31.7µg DNA from each of five single cells. The quality of the amplified DNA was sufficient for whole exome sequencing. A 4bp insertion in exon 12 of NPM1 reflecting a common NPM1 mutation (MutA) initially detected from a bulk of cells was identified in amplified DNA from single cells using whole exome sequencing in 2/3 patients. Internal tandem duplications in Flt3 indicated by mismatches in the alignment could be detected in amplified DNA from single cells of two patients. The detected ITD resemble those initially detected in DNA from a bulk of BM cells.

Discussion and Conclusion:

Single cell sequencing provides a useful tool to amend the detection of genetic aberrations from a bulk of cells and to confirm the presence of specific mutations in single cells from small subpopulations. It therefore helps to get further insights into the clonal evolution in pAML.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.