Single‐cell DNA and surface protein characterization of high hyperdiploid acute lymphoblastic leukemia at diagnosis and during treatment

High hyperdiploid (HeH) B-cell acute lymphoblastic leukemia (B-ALL) is the most prevalent subtype of childhood ALL. This leukemia is characterized by trisomies and tetrasomies of specific chromosomes and additional point mutations. Here, we used single-cell targeted DNA and antibody sequencing to determine the clonal evolution of HeH B-ALL during development and chemotherapy treatment. Chromosomal copy number changes were mostly stable over all the leukemia cells, while mutations were typically subclonal. Within all 13 cases, at least one <i>RAS</i> mutant (<i>KRAS</i> or <i>NRAS</i>) subclone was detected (range: 1 to 4 subclones with <i>RAS</i> mutations), indicating the importance of <i>RAS</i> signaling in HeH B-ALL development. <i>NSD2</i> mutations were detected in 4 out of 13 cases and always in a subclone with <i>RAS</i> signaling mutations. Single-cell DNA sequencing detected residual leukemia cells during chemotherapy treatment, and analysis of chromosomal copy number changes aided in the accurate detection of these cells. Our single-cell data demonstrate that chromosomal changes are acquired prior to additional mutations and that RAS signaling mutations are present in all HeH cases, often as subclonal mutations. This single-cell multi-omics study enabled us to extensively characterize the genetic and surface protein heterogeneity in patients with HeH B-ALL.