Single-cell genotyping demonstrates complex clonal diversity in acute myeloid leukemia

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Science Translational Medicine  01 Apr 2015:
Vol. 7, Issue 281, pp. 281re2
DOI: 10.1126/scitranslmed.aaa0763
  • Fig. 1. Underlying diversity obscured by describing clonal evolution in bulk AML samples.

    (A) Large NGS efforts identify the allele frequency (AF) in bulk samples for multiple genes simultaneously (genes A to H). These allele frequencies are then used to calculate the population frequency of the clones that contain each mutation by assuming all mutations are heterozygous and are added sequentially (a clone containing the cluster of mutations including A/B would be the “founder” clone, and the subclones must contain A/B/C/D and would then be assumed to be subsequent to the founder clone). Mutant alleles below the threshold of detection, which depends on the type of NGS used, the sample quality, and the coverage/filtering, cannot be attributed to a cancer clone mathematically. (B) Examples of clonal distributions for gene A that would produce the same allele frequency when analyzed in the bulk sample. If the mutation is not constrained to be heterozygous, then the same allele frequency observed in a bulk sample can be obtained in many different distributions of zygosity. When combining multiple mutations, this allows for an exponentially increasing number of possible genotype combinations that would each produce the observed overall allele frequencies for each mutant allele. (C) Many combinations of genotypes exist even when multiple mutations occur in the same sample. In the case of two mutations, there are nine possible mutation/zygosity combinations that could occur in individual cells, which, depending on their relative proportions, could reconstitute the bulk allele frequencies.

  • Fig. 2. Validation of single-cell genotyping via a comparison to the bulk data.

    (A to D) A reference patient sample containing two different FLT3-ITDs of different lengths was analyzed by single-cell genotyping for FLT3 during technique validation. The approach used for bulk analyses clinically is to determine the allele ratio of mutant (ITD-1 or ITD-2) alleles to that of the wild type (WT) as demonstrated in (A). Adaptation of this technique to single cells was applied to the reference sample, with a total sample size of 593 cells. Error bars in (B) are the 99% confidence intervals for the population frequencies of each FLT3-ITD zygosity. Het, heterozygous; Mut, homozygous mutant. Bulk allelic ratio was calculated via bulk fragment analysis with the same primers used for single-cell genotyping, converted to an allele frequency [AF = AR/(1 − AR)], and compared to the calculated numbers obtained by single-cell genotyping to ensure accuracy in the overall data set (C). These values were then tracked for the seven patient samples analyzed for FLT3 and NPM1 (D).

  • Fig. 3. Genetically distinct clonal frequencies in AML with respect to FLT3-ITD and NPM1.

    Bubble plot of the clonal distribution of six AML patient samples at diagnosis (and one analyzed at relapse, Pt4-R) showed the presence of all possible combined genotypes for the mutant genes present in each sample, although at a variable frequency. Pt1 to Pt4, and Pt4-R contained one each of FLT3-ITD and NPM1 mutations, and Pt5 and Pt6 had two FLT3-ITDs and an NPM1 mutation. Note that none of the samples with two ITD lengths had clones containing both ITDs simultaneously.

  • Fig. 4. Comparison of observed clonal distributions to those calculated with bulk data.

    Observed cell numbers scored with each genetic identity in the single cells of each patient sample are shown in shaded bars. Null distributions generated by predicting clonal distributions based on technical uncertainty and existing assumptions of heterozygosity and concurrence of mutations to link mutant allele frequency and clonal frequency are shown as wireframe. This distribution is based on the allelic frequencies in the bulk samples (Fig. 2D).

Supplementary Materials

  • Supplementary Material for:

    Single-cell genotyping demonstrates complex clonal diversity in acute myeloid leukemia

    Amy L. Paguirigan,* Jordan Smith, Soheil Meshinchi, Martin Carroll, Carlo Maley, Jerald P. Radich

    *Corresponding author. E-mail: apaguiri{at}

    Published 1 April 2015, Sci. Transl. Med. 7, 281re2 (2015)
    DOI: 10.1126/scitranslmed.aaa0763

    This PDF file includes:

    • Table S1. Efficiency data calculated for multigene single-cell genotyping.
    • Table S2. Primer sequences used for single-cell genotyping.
    • Table S3. Plasmid insert sequences for FLT3-ITD and NPM1.

    [Download PDF]

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