Approximately one-third of patients with acute myeloid leukemia (AML) have a mutation in the FLT3 gene. The most common mutation in FLT3 is an internal tandem duplication (ITD), which occurs in exons 14 and 15, encoding the juxtamembrane domain of the FLT3. The ITD varies between patients in length and location of insertion. Determining the presence of the FLT3-ITD variant at the time of diagnosis of AML by molecular testing is crucial for appropriate treatment selection. Early incorporation of targeted FLT3 inhibitors is highly recommended. They can be used in therapy as monotherapy or in combination with chemotherapy. Treatment is divided into induction, consolidation and maintenance therapy.
Measurable residual disease (MRD) serves as a biological marker of treatment efficacy. It is defined as the amount of leukemic cells remaining after treatment and it is also a key piece of information for disease monitoring. It allows for early assessment of remission status as well as the likelihood of disease recurrence, and also assists in therapy decisions. FLT3-ITD has long been considered an unreliable marker for MRD monitoring, mainly due to the high diversity of ITDs among patients and the instability of the mutation throughout the progression of the disease. Advances in sequencing have made molecular detection of FLT3-ITD MRD possible. The aim of the master's thesis was to introduce and verify a next-generation sequencing (NGS)-based test for MRD monitoring. Currently, the test is among the new and investigational methods for monitoring MRD in patients with FLT3-ITD. For the verification of the method, we analyzed samples from international controls UK NEQAS and pacient samples. With the introduced method, we obtained comparable results compared to the results of international laboratories. This confirmed the accuracy of the introduced method. We also assessed the reproducibility of the method within run and between runs. We found that the method is sufficiently accurate, sensitive and specific, which represents an important advancement for monitoring patients with AML. Increasingly sensitive methods are key to detecting MRD at lower cutoff values and also for improving treatment decisions. The advantage of the test, which is a combination of PCR and NGS, is also the relatively rapid data acquisition and efficient analysis using software. However, the method is associated with relatively high analysis costs. The introduction of the method into clinical practice for MRD monitoring will significantly improve the clinical management of patients with AML and the FLT3-ITD variant. Especially those who do not have variants in NPM1, meaning that FLT3-ITD is their only available marker for MRD detection.
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