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

Abstract

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 highthroughput 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. (Blood. 2012;120(3):603-612)

A generic, cost-effective, and scalable cell lineage analysis platform

“…The previous platform (Reizel et al , 2012Shlush et al 2012) was based on small multiplex groups (4×) using fluorophores and different MS lengths in CE, similarly to the protocol used for forensic analysis. The platform presented here demonstrates an immense advancement over our previous lineage analysis platform:…”

“…Most of the MS panel is designed for (A) MSs of type AC on the X Chromosome to allow for monoallelic MS calling Wasserstrom et al 2008;Reizel et al 2011;Segev et al 2011;Shlush et al 2012) and for (B) the longest MS loci possible, which exhibit a higher mutation rate in vivo (Ellegren 2004;Supplemental Table S2). Notably, primers were designed such that the entire MS will be covered within a 150-bp Examples of single-cell analyses to reconstruct clonal/lineage analysis Wang et al 2012;Xu et al 2012;Gawad et al 2014;Lohr et al 2014;Lodato et al 2015) (Navin et al 2011;Cai et al 2014;Wang et al 2014) ) Salipante et al 2010;Reizel et al 2011Reizel et al , 2012Shlush et al 2012;Evrony et al 2015) read (see Methods). (2) We modified the second PCR to apply a sample barcode by utilizing combinations of forward and reverse PCR primer combinations, resulting in a dual indexed NGS library (Supplemental Fig.…”

“…Our laboratory has demonstrated (Frumkin et al 2005Wasserstrom et al 2008;Reizel et al 2011Reizel et al , 2012Segev et al 2011;Shlush et al 2012) the feasibility of cell lineage analysis utilizing a low-resolution capillary electrophoresis (CE)-based system (∼100 loci per cell). We analyzed microsatellite (MS) loci, which have high mutation rates in vivo (Table 1) and are considered neutral (Ellegren 2004).…”

Change comes like a little wind: tales in MDS evolution

“…The resistant clones were only a minor fraction at the time of diagnosis and were the result of branching evolution. Accordingly, studies in T-cell acute lymphoblastic leukemia, 3 acute myeloid leukemia, 4 and chronic lymphocytic leukemia 5 all suggested that resistant clones originated from ancestral minor populations. This is of no surprise at all, as most of the current treatment protocols aim at eliminating the major population observed at diagnosis.…”

Microbial Community Structure and Functionality in Ruminants Fed the Probiotic Bacillus amyloliquefaciens H57

“…In humans, analysing the genetic variation in individual tumor cells has provided insights into the evolutionary development of cancer cells, thus identifying mechanisms causing relapse (Shlush et al, 2012). …”