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. 2013 Dec;23(12):2115-25.
doi: 10.1101/gr.159913.113. Epub 2013 Sep 20.

Single-cell mutational profiling and clonal phylogeny in cancer

Nicola E Potter  1 , Luca Ermini, Elli Papaemmanuil, Giovanni Cazzaniga, Gowri Vijayaraghavan, Ian Titley, Anthony Ford, Peter Campbell, Lyndal Kearney, Mel Greaves
Affiliations

Single-cell mutational profiling and clonal phylogeny in cancer

Nicola E Potter et al. Genome Res. 2013 Dec.

Abstract

The development of cancer is a dynamic evolutionary process in which intraclonal, genetic diversity provides a substrate for clonal selection and a source of therapeutic escape. The complexity and topography of intraclonal genetic architectures have major implications for biopsy-based prognosis and for targeted therapy. High-depth, next-generation sequencing (NGS) efficiently captures the mutational load of individual tumors or biopsies. But, being a snapshot portrait of total DNA, it disguises the fundamental features of subclonal variegation of genetic lesions and of clonal phylogeny. Single-cell genetic profiling provides a potential resolution to this problem, but methods developed to date all have limitations. We present a novel solution to this challenge using leukemic cells with known mutational spectra as a tractable model. DNA from flow-sorted single cells is screened using multiplex targeted Q-PCR within a microfluidic platform allowing unbiased single-cell selection, high-throughput, and comprehensive analysis for all main varieties of genetic abnormalities: chimeric gene fusions, copy number alterations, and single-nucleotide variants. We show, in this proof-of-principle study, that the method has a low error rate and can provide detailed subclonal genetic architectures and phylogenies.

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Figures

Figure 1.
Figure 1.
Schematic workflow of the multiplex targeted Q-PCR approach for the simultaneous detection of gene fusions, DNA copy number alterations, and mutations in single cells. Initially, CFSE-labeled single cells are sorted into each well of a 96-well plate and then lysed. Multiplex DNA specific target amplification is then completed to amplify a DNA region of interest (fusion gene, copy number alteration, or SNV) from the two copies found in a single cell to an amount that can be detected by Q-PCR using the BioMark HD. Amplified samples and assays are then loaded into a 96.96 dynamic array that utilizes a valve-controlled capillary network to bring these two mixes together at nanoliter volumes (completed in the IFC controller) for the Q-PCR reaction to take place. This final Q-PCR step determines the gene fusion, mutation, or copy number alteration status for each single cell.
Figure 2.
Figure 2.
Single-cell genetic analysis of leukemic cells using the multiplex targeted Q-PCR approach and the BioMark HD platform. (A) Heatmap depicting an example of raw Q-PCR data from the BioMark HD. The rows represent single cells including six cord blood cells and seven REH cells. The columns represent assays, each completed in quadruplicate including B2M (one of three assays), MX1 (two of three assays), CDKN2A (two of three assays), the ETV6-RUNX1 fusion gene assay, and the EPOR SNP (rs318720) assay containing two Taqman probes, one complementary to the wild-type sequence (labeled with FAM) and the other complementary to the SNP sequence (labeled with VIC). The colored boxes at the junction of a row and column indicate the raw CT value (according to the key on the right) obtained for a Q-PCR reaction involving the indicated cell and assay. Assays targeting a mutation or the fusion gene provide a definitive positive or negative result indicating the presence or absence, respectively, of an alteration. The DNA copy number assays provide a raw CT value that requires further analysis (standard ΔΔCT method, Applied Biosystems) to attribute a DNA copy number to the target gene of interest for a single cell; an example can be found in B (refer to the Single-Cell Analysis section in the Methods, and Supplemental Material). (Green arrow) The Q-PCR amplification curves generated from each copy number assay for a single cord blood cell. (Black cross in a colored box) An inadequate amplification curve. (B) Graph depicting the estimated DNA copy number of MX1 attributed reliably to 89 single cells given the assay results from B2M (assay 3) and MX1 (assay 3); one of the nine estimated copy number results used to confidently attribute a DNA copy number to the gene of interest for a single cell. The height of the bar indicates the estimated DNA copy number, and the color of the bar indicates the integer; (light blue) one copy; (dark blue) two copies; (green) three copies; and (yellow) four copies. (CB) Cord blood. (C) Subclonal genetic architecture of the REH cell line inferred by multiplex targeted Q-PCR and confirmed by FISH analysis (126 and 100 cells, respectively); the percentages in parentheses are those obtained by FISH analysis. All cells harbored the ETV6-RUNX1 fusion (F) and the EPOR SNP compared with cord blood cells. DNA copy number is indicated for each gene and subclonal population. Representative FISH images are shown next to their respective subclone. (D) Subclonal genetic architecture of leukemic cells from a child with Down’s syndrome and acute lymphoblastic leukemia (DS-ALL) generated by multiplex targeted Q-PCR and FISH analysis (115 and 100 cells, respectively); 98% of cells harbored the P2RY8-CRLF2 fusion and the IL7R mutation (IL7Rm) by multiplex targeted Q-PCR. Of these, the majority were heterozygous mutations (IL7Rm hete). A minor subclone (2%) had a homozygous IL7R mutation (IL7Rm homo). Loss of CDKN2A was subclonal to the IL7R mutation and proceeded to homozygous loss in 11% of cells. FISH for the P2RY8-CRLF2 fusion and CDKN2A copy number confirmed these results (percentages in parentheses); the IL7R mutation cannot be detected by FISH.
Figure 3.
Figure 3.
Phylogenetic analysis results for Cases A and B. In each case the observed clone is indicated by a circle. Yellow circles indicate tumor clones, and black circles indicate the normal cell population. The alterations are listed below each subclone; excluding ETV6-RUNX1, those without a number indicate the presence of a mutation and those with a number indicate DNA copies accordingly. The boxed subclone in gray is inferred; a group of cells that has died out or been outcompeted, or if still present, exists at a low frequency that cannot be reliably detected by this approach. The number in italics at each node indicates the jackknifing value. The distance unit is indicated. (A) One parsimonious tree was found for Case A consisting of four subclones with modest heterogeneity. The major clone (A4) represents 87.4% of the population. The size of each circle is proportional to the number of single cells included in the subclone except A4, which has been reduced by a third in this tree. (B) In Case B, there are two equally parsimonious trees composed of seven subclones. These two trees differ by the position of subclones B4 and B5, which are equal parsimonious ancestors to subclones B3, B7, and B8. This case shows increased heterogeneity with the major clone representing 54.9% of the population (B3). The major clone B3 is reduced by half in this tree.
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