MEASURABLE RESIDUAL DISEASE AND LEUKEMIC STEM CELLS IN ACUTE MYELOID LEUKEMIA

Nearly all fit patients with acute myeloid leukemia (AML) receive intense chemotherapy, followed by consolidation therapy which can be either additional cycle(s) of chemotherapy, autologous stem cell transplantation or allogeneic stem cell transplantation. In this order, anti-leukemic efficacy increases together with toxicity. While, fortunately, most patients achieve complete remission, unfortunately, 40-50% of patients experience a relapse. Patients who relapse have a dismal prognosis since the relapse is mostly difficult to eradicate. A correct understanding of the risk to relapse is vital for selecting the correct therapy intensity. Risk stratification at diagnosis is based on factors such as age, white blood cell (WBC) count and genetic (mutations and cytogenetic aberrations) characteristics.1 This risk assessment at diagnosis does not suffice for an accurate estimation of patients that relapse, therefore, more specific and sensitive methods (both by flow cytometry and molecular techniques) are widely used to assess possible residual disease during and after therapy. When this residual disease (termed measurable residual disease or minimal residual disease, MRD) is present above a critical level, patients have a higher chance of experiencing a relapse. The overall aim of the studies described in this thesis is to investigate the role of measurable residual disease (MRD) and leukemic stem cells (LSC), and several initiatives to improve the MRD assessment to be used for relapse prediction for the individual patient. Chapter 2 covers a review on several aspects of LSCs in AML and its considered role in relapse progression. Moreover, it discusses how these relatively rare cells can be detected by flow cytometry, and furthermore discusses how this detection is currently used in clinical application. In chapter 3-4 we investigated if the LSC frequency harbors prognostic information for improved relapse prediction for AML. In chapter 3 we present the clinical significance of the presence and frequency of CD34+CD38- LSCs at time of diagnosis and in remission bone marrow in adult AML. In addition, the prognostic relevance of the combination of LSC-MRD and MFC-MRD is investigated. In chapter 4 we investigated whether detection of CD34+CD38- LSCs in BM of newly diagnosed pediatric AML bears similar prognostic relevance as shown in adult AML. In chapter 5-6 we elaborate on the importance of standardization of the flow cytometric MRD and LSC detection approaches. In chapter 5 we evaluated the technical and analytical feasibility of the previously designed eight‐color LSC single tube assay, as well as standardization of the process. In chapter 6 we present a new flow cytometric model for standardized and objective MRD calculation, retrospectively applied in a large clinical study. For this, we evaluate if the balance between neoplastic and normal progenitors in CR bone marrow has prognostic relevance. In chapter 7 we evaluate whether next-generation sequencing has clinical value for the prediction of relapse. Since measurements were simultaneously evaluated for MFC-MRD, we investigated whether NGS and MFC-MRD have independent and additive prognostic value. In addition, we studied whether MRD and LSC-MRD is a valid surrogate endpoint in AML. As shown in a recent clinical trial, the new therapeutic clofarabine has clinical beneficial effect in a subgroup of patients. In chapter 8 we investigated whether the prospectively defined MRD and LSC-MRD frequencies were different between patients with clofarabine and patients without clofarabine, and whether MRD levels mirrored the clinical outcome within this subgroup. Finally, in chapter 9 we summarize the results of this thesis and which implications these results may have for future AML relapse prediction. Furthermore, we evaluate the different techniques used in this thesis, discuss how each technique can be further optimized and elaborate on the optimal use for future clinical trials.