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Single-Cell Genotyping Study Underscores Clonal Diversity in AML

NEW YORK (GenomeWeb) – In a study appearing online today in Science Translational Medicine, researchers from the Fred Hutchinson Cancer Research Center and elsewhere described the extensive clonal diversity that they detected in acute myeloid leukemia samples using a new single-cell genotyping method.

The team used a single-cell, multiplexed PCR-based approach to assess mutation patterns in individual cells from seven AML samples, focusing on alterations in the frequently mutated genes FLT3 and NPM1. Each of the samples contained nine or more different clonal populations comprised of cells with glitches in one or both copies of FLT3 and NPM1, pointing to pronounced diversity in the tumors.

"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," Fred Hutchinson Cancer Research Center researcher Jerald Radich, the study's senior author, and his co-authors wrote.

Prior research has pointed to a role for clonal diversity in everything from tumor development to outcomes and treatment resistance. For the current study, Radich and his colleagues reasoned that they might get a more refined understanding of AML evolution by carefully tracking key mutations in individual cells rather than trying to piece together patterns from bulk DNA sequence data.

With that in mind, the researchers first validated their single-cell, multiplexed PCR mutation assay in cell lines and in nearly 600 sorted cells from the same AML patient samples, demonstrating that the approach could accurately detect FLT3 internal tandem duplications (FLT3-ITDs) and insertions in the NPM1 gene.

From there, the team turned its attention to cells from seven AML tumors that were previously shown to carry at least one FLT3-ITD and NPM1 mutation apiece.

The researchers then tallied up the types of mutations in each cell, considering distinguishing features such as varying internal tandem duplication lengths in FLT3, differences in NPM1 insertion sequences, and the presence of such alterations in one or both copies of the genes.

Although the general mutation patterns they found matched those expected from bulk sample genotyping, the single cell data revealed that these alterations exist in a staggering number of combinations that likely stem from convergent evolution in different tumor sub-clones.

"Our data suggest that clonal structure based on concurrent mutations can be more complex than is perceived by bulk allelic frequencies," the study's authors wrote, "and that the assumption that mutations occur only in the heterozygous state may lead to underestimation of the clonal diversity. 

By following the FLT3 and NPM1 mutations found in cells from two patients' tumors at the time of diagnosis and at relapse, meanwhile, the investigators got a glimpse at the shifting clonal complexity that can accompany relapse.

The representation of FLT3-ITD alterations — particularly those affecting both copies of the gene — appeared to rise in recurring AML tumors. But their results suggest that the other mutational sub-clones also tend to stick around over the course of disease to one degree or another.