title: Single-cell genotyping demonstrates complex clonal diversity in acute myeloid leukemia.
abstract: Clonal evolution in cancer-the selection for and emergence of increasingly malignant clones during progression and therapy, resulting in cancer metastasis and relapse-has been highlighted as an important phenomenon in the biology of leukemia and other cancers. Tracking mutant alleles to determine clonality from diagnosis to relapse or from primary site to metastases in a sensitive and quantitative manner is most often performed using next-generation sequencing. Such methods determine clonal frequencies by extrapolation of allele frequencies in sequencing data of DNA from the metagenome of bulk tumor samples using a set of assumptions. The computational framework that is usually used assumes specific patterns in the order of acquisition of unique mutational events and heterozygosity of mutations in single cells. However, these assumptions are not accurate for all mutant loci in acute myeloid leukemia (AML) samples. To assess whether current models of clonal diversity within individual AML samples are appropriate for common mutations, we developed protocols to directly genotype AML single cells. Single-cell analysis demonstrates that mutations of FLT3 and NPM1 occur in both homozygous and heterozygous states, distributed among at least nine distinct clonal populations in all samples analyzed. There appears to be convergent evolution and differential evolutionary trajectories for cells containing mutations at different loci. This work suggests an underlying tumor heterogeneity beyond what is currently understood in AML, which may be important in the development of therapeutic approaches to eliminate leukemic cell burden and control clonal evolution-induced relapse.
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98117, USA. apaguiri@fredhutch.org.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Paguirigan
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98117, USA.
authors.forename: Jordan
authors.initials: J
authors.lastname: Smith
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98117, USA.
authors.forename: Soheil
authors.initials: S
authors.lastname: Meshinchi
authors.affiliation: University of Pennsylvania, Philadelphia, PA, 19104, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Carroll
authors.affiliation: Center for Evolution and Cancer, Helen Diller Family Comprehensive Cancer Center and Department of Surgery, University of California, San Francisco, San Francisco, CA 94158, USA. School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA.
authors.forename: Carlo
authors.initials: C
authors.lastname: Maley
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98117, USA.
authors.forename: Jerald P
authors.initials: JP
authors.lastname: Radich
mesh_terms: D016477:Artifacts; D060965:Clonal Evolution / Q000235:genetics*; D005787:Gene Frequency / Q000235:genetics; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology*; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D000090243:Nucleophosmin; D012008:Recurrence; D015203:Reproducibility of Results; D059010:Single-Cell Analysis / Q000379:methods*; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics
chemical_list: C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D051941:fms-Like Tyrosine Kinase 3
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