20.500.12556/RUL-178452 Overitev metode sekvenciranja naslednje generacije za spremljanje merljivega preostanka bolezni pri akutni mieloblastni levkemiji z različico FLT3-ITD Verification of the method for detection of minimal measurable disease by next generation sequencing in acute myeloid leukemia with FLT3-ITD mutation Pri približno eni tretjini bolnikov z akutno mieloblastno levkemijo (AML) se pojavi mutacija v genu FLT3. Najpogostejša mutacija v FLT3 je notranja tandemska podvojitev (ITD), ki se pojavlja v eksonih 14 in 15 v področju gena, ki kodira jukstamembransko domeno FLT3. Med bolniki se ITD razlikujejo po dolžini in mestu vstavitve. Določitev prisotnosti mutacije FLT3-ITD z molekularnimi testi je ob diagnozi AML ključna za ustrezno izbiro zdravljenja. Priporočljiva je zgodnejša vključitev zaviralcev FLT3, ki delujejo tarčno. V terapiji se lahko uporabijo samostojno ali v kombinaciji s kemoterapijo. Zdravljenje je razdeljeno na indukcijsko, konsolidacijsko in vzdrževalno terapijo. Merljivi preostanek bolezni (MRD) služi kot biološki označevalec učinkovitosti zdravljenja. Opredeljen je kot količina preostalih levkemičnih celic po zdravljenju in je ključen podatek za spremljanje poteka bolezni. Omogoča zgodnjo oceno statusa remisije kot tudi verjetnost ponovitve bolezni, pripomore pa tudi pri odločitvah glede terapije. FLT3-ITD dolgo ni veljal za zanesljiv označevalec za spremljanje MRD, predvsem zaradi velike raznolikosti ITD med bolniki in nestabilnosti mutacije glede na potek bolezen. Zaradi napredka v sekvenciranju je postala molekularna zaznava FLT3-ITD MRD mogoča. Cilj magistrske naloge je bil vpeljava in overitev testa za spremljanje MRD, ki temelji na sekvenciranju naslednje generacije (NGS). Trenutno test spada med nove in raziskovalne metode za spremljanje MRD pri bolnikih s FLT3-ITD. Za overitev metode smo analizirali vzorce mednarodnih kontrol UK NEQAS in vzorce bolnikov. Z vpeljano metodo smo dobili primerljive rezultate za vzorce mednarodnih kontrol z rezultati vključenih mednarodnih laboratorijev. S tem smo potrdili točnost vpeljane metode. Izvedli smo tudi ponovljivost metode znotraj serije in med serijami. Ugotovili smo, da je metoda dovolj natančna, občutljiva in specifična, kar predstavlja pomemben napredek za spremljanje bolnikov z AML. Vse bolj občutljive metode so ključne za odkrivanje MRD pri nižjih mejnih vrednostih in za izboljšavo odločitev o zdravljenju. Prednost testa, ki je kombinacija PCR in NGS-a, je tudi relativno hitro pridobivanje podatkov ter njihovo učinkovito analiziranje s pomočjo programske opreme. Vendar pa je metoda povezana z relativno visokimi stroški analize. Z vpeljavo metode za spremljanje MRD v klinično prakso se bo bistveno izboljšala klinična obravnava pacientov z AML in FLT3-ITD, predvsem tistih, ki nimajo različic v NPM1, kjer je FLT3-ITD edini razpoložljivi označevalec za določanje MRD. 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. akutna mieloblastna levkemija različica FLT3-ITD merljivi preostanek bolezni sekvenciranje naslednje generacije acute myeloid leukemia FLT3-ITD mutation measurable residual disease next generation sequencing true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-01-28 07:45:25 2026-01-28 07:45:34 2026-01-29 04:45:44 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 124712 31779.pdf 31779.pdf 1 8767515A669077680D0284273526B048 2eba9a23ba5f670906e55c736c8b74acb3d3f85c0cb36d7152148aff1c0c2028 ec8915e9-fc14-11f0-a1ba-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=226337 Fakulteta za farmacijo 0 0 0