Single cell analysis exposes intratumor heterogeneity and suggests that FLT3-ITD is a late event in leukemogenesis.

FMS-like tyrosine kinase 3 receptor-internal tandem duplication (FLT3-ITD) commonly occurs in acute myeloid leukemia and is considered rare in acute lymphocytic leukemia. Acute leukemia has poor prognosis, mainly due to relapse. Standard FLT3-ITD diagnostic techniques are based on genomic polymerase chain reaction and have recently incorporated GeneScan (Applied Biosystems, Foster City, CA) to identify variations of the FLT3 gene. As this is an average-based assay utilized in a heterogeneous leukemic cell population, we hypothesized that cells of acute leukemia, considered FLT3-ITD-negative by standard methods, could possess a fraction of FLT3-ITD-positive cells. The present study employed single cell mutation analysis to evaluate the FLT3-ITD status in newly diagnosed acute myeloid leukemia (n = 5) and acute lymphocytic leukemia (n = 3) patients. A total of 541 single leukemic cells and 36 mononuclear cells from healthy volunteers were analyzed. Seven patients, considered FLT3-ITD-negative according to bulk DNA analysis, appeared to possess a small fraction of FLT3-ITD-positive cells based on single cell analysis. Moreover, this approach revealed the heterogeneity of the tumor as evident by different FLT3-ITD mutations present in the same patient. The presence of a minor clone carrying FLT3-ITD in almost all patients tested provides evidence that this lesion is a common late event in leukemogenesis. Additionally, 3 relapsed patients demonstrated loss of heterozygosity of the normal allele, affecting 25%-100% of the cells found to be FLT3-ITD-positive. Though further clinical testing is warranted, these findings may have implications on the prognostic significance of FLT3-ITD and the use of targeted therapy.

Cell lineage analysis of acute leukemia relapse uncovers the role of replication-rate heterogeneity and microsatellite instability.

Human cancers display substantial intratumoral genetic heterogeneity, which facilitates tumor survival under changing microenvironmental conditions. Tumor substructure and its effect on disease progression and relapse are incompletely understood. In the present study, a high-throughput method that uses neutral somatic mutations accumulated in individual cells to reconstruct cell lineage trees was applied to hundreds of cells of human acute leukemia harvested from multiple patients at diagnosis and at relapse. The reconstructed cell lineage trees of patients with acute myeloid leukemia showed that leukemia cells at relapse were shallow (divide rarely) compared with cells at diagnosis and were closely related to their stem cell subpopulation, implying that in these instances relapse might have originated from rarely dividing stem cells. In contrast, among patients with acute lymphoid leukemia, no differences in cell depth were observed between diagnosis and relapse. In one case of chronic myeloid leukemia, at blast crisis, most of the cells at relapse were mismatch-repair deficient. In almost all leukemia cases, > 1 lineage was observed at relapse, indicating that diverse mechanisms can promote relapse in the same patient. In conclusion, diverse relapse mechanisms can be observed by systematic reconstruction of cell lineage trees of patients with leukemia.