The clinical impact of clonal diversity in infant leukemia

The clinical impactof clonal diversity in infant leukemia

Acute leukemia arising in infants,i.e. children aged 0-12 months, is characterized by KMT2A-generearrangements (KMT2A-r) and a very poor prognosis with 70% of patients succumbing to disease. The cause of this poor prognosis is not known. Although over 90% of infants reach clinical remission, they are prone to rapid relapse,and our understanding of the mechanisms driving relapse remains limited. The overall goal of this proposal is to improve our mechanistic understanding of infant leukemia development and relapse and thereby impact how it is diagnosed and treated in the future.

Molecular genetic studies, in recent years accelerated by high-through put sequencing technologies, have significantly improved our understanding of the molecular pathogenesis of acute leukemia. However, these methods sequence cells mixed together and typically do not have the resolution to identify and characterize distinct subpopulations. Some of these cell subpopulations may play a fundamental role in disease evolution and progression and in acquired resistance to treatment. Recently,single-cell RNA-sequencing (scRNA-seq) and single-cell DNA sequencing have provided unique possibilities to study these processes.

We have completed a study of the genetic mechanisms of relapse in infant and childhood KMT2A-r acute leukemia (Ahlgren L et al, In Press, Nature Communications). We performed wholegenome and exome sequencing of relapsed KMT2A-r cases (n=36), and longitudinal deep-seq of 258 samples from 30 patients. Somatic alterations indrug-response genes, most commonly in TP53 and IKZF1 (64%), were highly enriched in early relapse ALL (79%, 9-36 months after diagnosis), but virtually absent in very early relapse ALL (<9 months). By single-cell DNA sequencing we could determine the mutational order and which mutations that occurred together in the same clone at relapse. We also tracked residual leukemia cells, clonal drug responses, and the upcoming relapse, thereby providing new insights into mechanisms of relapse in this highly lethal form of pediatric acute leukemia.

Further, scDNA- and scRNA-sequencing have been used to study lineage plasticity. The data show that genetic evolution is independent of the cellular immunophenotype, suggesting an inherent lineage plasticity of the transformed cells. Further, the molecular and cellular characteristics of the cells that expanded on day 15 were shared by a small subset of diagnostic cells(manuscript in preparation).

Finally, scDNA whole exome sequencing on 26 cells was used to providei nsights into clonal diversity and identified a total of five RAS-pathway mutations in a single patient and each of these mutations were identified in distinct cells. We also inferred mutational history, showing that the leukemia had evolved through branching evolution with at least 8 diagnostic clones (manuscript in preparation).

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