Single Cell Molecular Genotyping Reveals Significant Molecular Heterogeneity at Diagnosis and Emergence of Clonal Dominance at Relapse

Abstract

Acute myeloid leukemia is a heterogeneous disease with significant variation in morphologic, cytogenetic, and molecular characteristics. We used single cell molecular genotyping to investigate the extent of molecular heterogeneity in individual patients with FLT3/ITD, WT1 and c-KIT mutations. Diagnostic leukemic samples from patients with FLT3/ITD (N=10) with varying FLT3/ITD allelic ratios (proportion of mutant to wild type product, ITD-AR) were flow sorted to immunophenotypic homogeneity (CD45dim/CD34+/CD33+) and individual leukemic blasts were isolated and directly genotyped. Single cell signal recovery as evident by the detection of FLT3 signal ranged from 39% to 88% (median 58%). Single cells from FLT3/ITD-positive samples demonstrated significant molecular heterogeneity with the presence of 3 distinct FLT3 genotypes: heterozygous FLT3/ITD, homozygous FLT3/ITD and wild type FLT3 (FLT3/WT). Proportion of cells with homozygous FLT3/ITD varied from 3% to 80% and directly correlated with the diagnostic ITD-AR, where those with high allelic ratios had a higher proportion of homozygous ITD cells and in those with the highest ITD-AR, with demonstrated 13q uniparental disomy (13q UPD), cells with homozygous ITD were the dominant clone. We further evaluated relapse specimens from 3 patients with FLT3/ITD, where cells with homozygous ITD was a minor clone at diagnosis (15%–30%). Single cell FLT3 genotyping of these specimens revealed significant expansion and clonal dominance of the homozygous ITD at relapse, where >85% of leukemic cells harbored homozygous ITD (and presumed 13q UPD). We demonstrated similar molecular heterogeneity in patients with WT1 and c-KIT mutations, with detection of homozygous mutations in 20–60% of the cells tested. Single cell genotyoing demonstrates significant molecular heterogeneity in AML that may not be evident by conventional methods. This data provides direct evidence that cells with homozygous FLT3/ITD and associated 13q UPD may be present as a minor population at the time of diagnosis and emerge as the dominant clone at relapse. Such a molecular heterogeneity and emergence of clonal dominance by an individual clone may provide insight into mechanism of disease resistance. Extent of molecular heterogeneity may also be important in our interpretation of data obtained from biologic specimens.