Klonale Heterogeniteit en Evolutie Tijdens Behandeling in Pediatrische Acute Lymfatische Leukemie - Een Single-Cell Karakterisatie

Acute lymphoblastic leukemia (ALL) represents the most common pediatric cancer, arising from the stepwise accumulation of mutations in developing B cells (B-ALL) or T cells (T-ALL). Despite significant improvements in the outcome of patients with ALL over the past few decades, relapse still occurs in approximately 10%-15% of patients and is associated with a poor prognosis. Previous single-cell DNA sequencing (scDNA-seq) in B-ALL cases demonstrated a particularly high degree of subclonal heterogeneity in high hyperdiploid (HeH) B-ALL. HeH B-ALL represents the most common subtype of pediatric B-ALL, and although it is generally associated with a good prognosis, it also accounts for the largest absolute number of relapses. We aimed to assess whether a higher degree of clonal heterogeneity at diagnosis could be associated to subsequent development of therapy resistance or relapse. In the first part of this thesis, we aimed to extensively characterize the subclonal complexity of HeH B-ALL at diagnosis and during treatment. We performed targeted scDNA-seq using our custom amplicon panel in 13 HeH B-ALL patients, which identified RAS (KRAS/NRAS) mutations that were acquired in parallel and occurring mutually exclusive in all cases. Moreover, single-cell analysis demonstrated that chromosomal copy number changes were mostly stable and early events, whereas point mutations, including RAS mutations, were usually subclonal and late events. ScDNA-seq could also aid in the detection of measurable residual disease (MRD) in bone marrow samples at early treatment phases. In the second part of this thesis, we examined the clonal evolution patterns in 9 diagnosis-relapse pairs of patients with HeH B-ALL at single-cell level. Although RAS mutations were omnipresent at diagnosis, they were present in the dominant relapse clone in only half of the cases. This observation suggested that RAS mutations are not the main drivers of relapse in HeH B-ALL. We did, however, observe a trend of a higher degree of clonal heterogeneity at diagnosis in patients that relapsed as compared to those that did not. Additionally, in cases of patients with other B-ALL subtypes that relapsed with a CD19-negative B-ALL following anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, profiling of the surface proteins at single-cell level could track CD19-negative populations before and after relapse following CAR T-cell therapy. In the last part of this thesis, we present a case series of 4 patients with B-ALL that experienced CD22-positive relapse following anti-CD19 CAR T-cell therapy. All patients received inotuzumab ozogamicin, a CD22-targeting antibody conjugated to the cytotoxic agent calicheamicin, and were successfully brought into remission to subsequently receive allogeneic hematopoietic stem cell transplantation. This illustrates the potential of a personalized treatment approach in these heavily pretreated patients. In conclusion, single-cell analysis characterized the clonal heterogeneity in patients with HeH B-ALL at diagnosis, during treatment and at relapse. Despite the limited cohort size, our findings offer novel insights into the clonal architecture and evolution of HeH B-ALL and lays the groundwork for future research aimed at enhancing risk stratification and clinical outcomes in affected children.