Periodic Reporting for period 1 - scTALLmap (Single-cell map of the composition and evolution of T-cell acute lymphoblastic leukemia)
Reporting period: 2019-04-01 to 2021-03-31
Spaniards have their daily siesta, Germans like sausages and Belgians love beer. Stereotypes can certainly be misleading, just like judging a cell by its membership to a particular cell type. For instance, we now know that tumors are tremendously heterogeneous and, in the era of single-cell sequencing, we have the exquisite opportunity to judge each individual cell with unprecedented resolution. This ability to profile single cells can specially advance our understanding of blood malignancies, a perfect model for single-cell studies since blood cells are already in suspension. In particular, we have investigated acute lymphoblastic leukemia (ALL), a frequent type of cancer in children in which the bone marrow produces too many lymphocytes (a type of white blood cell). ALL is a success story in pediatric oncology, since the prognosis (chance of recovery) for children suffering from ALL has remarkably improved over the last decades and is now > 90%. However, these children undergo a very intense 2-year treatment, based on a combination of high-dose chemotherapy drugs, and they suffer from long-term side-effects. Still, approximately 10% of the cases do not respond to the treatment and have high chances of relapse (disease recurrence) with a fatal outcome.
This blood cancer shows extensive intratumoral heterogeneity, meaning that is composed by a mixture of clones (or leukemia subpopulations) with different mutation combinations. This heterogeneity might be the underlying reason for an incomplete response to treatment and for the development of relapse. To facilitate the clinical implementation of better risk classification methods accounting for patient’s heterogeneity, it is essential to: 1) generate a reference single-cell map for leukemia and 2) accumulate evidence on how the leukemia composition at the time of diagnosis affects the response to treatment. With this aim, in this European MSCA funded project, we have built a comprehensive single-cell overview of the composition, development and response to therapy for the childhood T-cell (T-ALL) aggressive subtype.
This blood cancer shows extensive intratumoral heterogeneity, meaning that is composed by a mixture of clones (or leukemia subpopulations) with different mutation combinations. This heterogeneity might be the underlying reason for an incomplete response to treatment and for the development of relapse. To facilitate the clinical implementation of better risk classification methods accounting for patient’s heterogeneity, it is essential to: 1) generate a reference single-cell map for leukemia and 2) accumulate evidence on how the leukemia composition at the time of diagnosis affects the response to treatment. With this aim, in this European MSCA funded project, we have built a comprehensive single-cell overview of the composition, development and response to therapy for the childhood T-cell (T-ALL) aggressive subtype.
In order to identify the clonal composition of T-ALL at diagnosis (and during chemotherapy treatment), we performed single-cell sequencing using state-of-the-art technologies and investigated two molecular dimensions: DNA and RNA in thousands of leukemic cells per patient. In total, we sequenced more than 150,000 cells from 25 blood and bone marrow samples from 8 leukemia patients (between 2 and 12 years old diagnosed at University Hospital Leuven). Our results show that T-ALL is indeed heterogeneous at diagnosis and that the clonal and cellular composition of the leukemia is, to a large extent, patient specific. Moreover, the data illustrate a surprising degree of heterogeneity for mutations of a particular gene (NOTCH1), with multiple cells from the same sample acquiring independent NOTCH1 mutations, highlighting the importance of this mutated gene in T-ALL progression. We also provide evidence that the clonal and cellular composition in bone marrow and blood at diagnosis is largely similar, indicating that there is little or no differential clonal selection of the leukemia cells in blood versus bone marrow and that molecular analysis of a blood sample is representative of the blasts in the bone marrow. Importantly, we sequenced serial samples from the same patient at diagnosis, during chemotherapy and in relapse, a feature which is lacking in most single-cell studies up to date. This approach provided real temporal information about how leukemia develops, the exact types of cells that comprise it, and the sensitivity of the different cells to the therapy. We could identify sensitive clones as well as resistant clones that were already present at diagnosis at low frequency. Remarkably, we could also detect the existence of residual leukemic cells that were missed in routine clinical assessment and monitor the appearance of new resistant clones.
Note: article manuscript accepted for publication in Blood, URL link will follow.
Note: article manuscript accepted for publication in Blood, URL link will follow.
Overall, our study provides a comprehensive view on the clonal composition and evolution of T-ALL at the single-cell level, giving insights on the pathogenesis of blood cell transformation and its clonal complexity. Moreover, we provide evidence that blood sampling would be sufficient for diagnostic purposes and could prevent invasive bone marrow aspirate procedures. Finally, the results of this project together with the sequencing of many more patients, provide the basis for identifying clones that correlate with drug resistance and can predict the development of relapse. This has the potential to translate into improved risk-stratification methods based on individualized patient molecular profiles. Ultimately, it will enable the development of novel treatment strategies targeting the full diversity of leukemia clones.