Single-cell sequencing reveals the origin and the order of mutation acquisition in T-cell acute lymphoblastic leukemia

Jolien De Bie,Heidi Segers,Jan Cools,Peter Vandenberghe,Ellen Geerdens,Llucia Alberti-Servera,Lucienne Michaux,Kim De Keersmaecker,Nancy Boeckx,Michaël Broux,Sofie Demeyer,Thierry Voet,Anne Uyttebroeck

doi:10.1038/s41375-018-0127-8

Abstract

Next-generation sequencing has provided a detailed overview of the various genomic lesions implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL). Typically, 10–20 protein-altering lesions are found in T-ALL cells at diagnosis. However, it is currently unclear in which order these mutations are acquired and in which progenitor cells this is initiated. To address these questions, we used targeted single-cell sequencing of total bone marrow cells and CD34+CD38− multipotent progenitor cells for four T-ALL cases. Hierarchical clustering detected a dominant leukemia cluster at diagnosis, accompanied by a few smaller clusters harboring only a fraction of the mutations. We developed a graph-based algorithm to determine the order of mutation acquisition. Two of the four patients had an early event in a known oncogene (MED12, STAT5B) among various pre-leukemic events. Intermediate events included loss of 9p21 (CDKN2A/B) and acquisition of fusion genes, while NOTCH1 mutations were typically late events. Analysis of CD34+CD38− cells and myeloid progenitors revealed that in half of the cases somatic mutations were detectable in multipotent progenitor cells. We demonstrate that targeted single-cell sequencing can elucidate the order of mutation acquisition in T-ALL and that T-ALL development can start in a multipotent progenitor cell.

Highlights

These authors contributed : Jolien De Bie, Sofie Demeyer.Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Leuven, Belgium 4 Department of Pediatric Hemato-Oncology, UZ Leuven, Leuven, Belgium 5 Department of Hematology, UZ Leuven, Leuven, Belgium 6 Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK 7 Department of Oncology, KU Leuven, Leuven, Belgium 8 Department of Laboratory Medicine, UZ Leuven, Leuven, BelgiumT-cell acute lymphoblastic leukemia (T-ALL) is a common childhood malignancy caused by clonal proliferation of immature T cells
Several JAK1, JAK2, JAK3, NRAS and KRAS mutations have been described, as well as mutations in IL7R and DNM2, which result in activation of the JAK/STAT pathway [1, 8, 9]
Identification of somatic variants using bulk whole genome and transcriptome sequencing Whole-genome sequencing (WGS) and RNA sequencing was performed on bone marrow (BM) samples obtained at diagnosis and remission from four childhood T-ALL cases (Supplemental table S1)

Summary

Introduction

These authors contributed : Jolien De Bie, Sofie Demeyer. CDKN2A/2B and NOTCH1 are the most frequently affected genes in T-ALL, with 60% of T-ALL patients showing activation of the NOTCH1 signaling pathway and up to 80% harboring deletions and/or mutations inactivating the CDKN2A/B genes at chromosome 9p [4, 5]. Other pathways that are frequently mutated in T-ALL include the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and RAS (Rat Sarcoma oncogene) signaling pathways [1, 3, 6, 7]. Several JAK1, JAK2, JAK3, NRAS and KRAS mutations have been described, as well as mutations in IL7R and DNM2, which result in activation of the JAK/STAT pathway [1, 8, 9].

Methods

Results

Conclusion