20.500.12556/RUL-152735 Določanje različic v genih, ki povzročajo odpornost na tarčno zdravljenje kronične limfatične levkemije Determination of variants in genes responsible for resistance to targeted chronic lymphatic leukemia therapy Kronična limfatična levkemija (KLL) je najpogosteje diagnosticirana levkemija zahodnega sveta, povprečna starost bolnikov ob diagnozi pa presega 70 let. V splošnem se kaže kot razrast limfocitov B v krvi, kostnem mozgu in limfatičnih organih. KLL velja za neozdravljivo bolezen, njen potek pa je med bolniki zelo raznolik in močno povezan z njihovimi genetskimi in citogenetskimi značilnostmi. Bolniki sprva ne prejemajo zdravljenja in jih spremljamo do nastopa bolezenskih znakov. Klasično zdravljenje zajema kemoimunoterapijo oz. kombinacijo kemoterapevtikov (fludarabin, ciklofosfamid) in bioloških zdravil (rituksimab), vendar se bolniki z del(17p) ali različicami v genu TP53 slabo odzivajo na tovrstno zdravljenje. Tarčna zdravila ciljajo posamezne ključne molekule v celičnem signaliziranju in tako omejijo razrast levkemičnih celic. Za tarčno zdravljenje KLL uporabljamo zaviralce Brutonove tirozinske kinaze (BTK) (npr. ibrutinib, akalbrutinib), BCL- 2 (venetoklaks) in fosfatidilinositol 3-kinaze (PI3K) (idelalizib). Kljub veliki uspešnosti tarčnih učinkovin se pri določenem deležu bolnikov po nekaj letih pojavi odpornost na zdravljenje, kar je posledica različic, ki nastanejo v vezavnem mestu na tarčni beljakovini. Odpornost na ibrutinib najpogosteje povzročajo različice v genu BTK, sledijo pa ji različice v genu PLCG2, ki povzroča aktivacijo signalizacije tudi ob vezavi ibrutiniba. V magistrski nalogi smo v Specializiranem hematološkem laboratoriju Kliničnega oddelka za hematologijo Univerzitetnega kliničnega centra v Ljubljani izvedli določanje različic v genih, ki povzročajo odpornost na tarčno zdravljenje KLL pri bolnikih, ki vsaj dve leti prejemajo ibrutinib. Vzorce smo zbrali po predhodni privolitvi in poučitvi bolnika, iz celic periferne krvi izolirali DNA in jo analizirali z metodo NGS. Pridobljene odčitke smo obdelali z ustrezno programsko opremo. Pri večini bolnikov smo odkrili različice, ki bodisi vplivajo na razvoj odpornosti na zdravljenje z ibrutinibom, bodisi predstavljajo neugoden klinični pomen. Pri 28, 6 % bolnikov smo odkrili različice v BTK, klinično pomembnih različic v genih PLCG2 in BCL2 pa nismo zaznali. Analizirali smo tudi gene, ki vplivajo na potek bolezni (TP53, NOTCH1, SF3B1), in pri dobri tretjini obravnavanih bolnikov odkrili različice v TP53, pri večini pa smo odkrili različice v genih NOTCH1 oz. SF3B1. Dokazali smo, da je določanje genskih različic, ki vplivajo na uspešnost zdravljenja in potek bolezni, smiselno in potrebno določati tudi med zdravljenjem oz. ob napredovanju bolezni. Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in the Western world. CLL most commonly occurs in older adults, as the median age of diagnosis is 70 years. The diagnosis is based on persistent lymphocytosis in blood, bone marrow and lymphatic organs. With duration of the disease patients develop “B symptoms”, that are characteristic for CLL progression. Progress of the disease vastly differs between patients, which is usually based on their individual genetic and cytogenetic characteristics. In asymptomatic stages of CLL patients do not receive therapy and are closely monitored until the onset of further complications. While CLL is generally considered incurable, it is commonly treated with chemoimmunotherapy which combines chemotherapeutics (fludarabine, cyclophosphamide) and monoclonal antibodies (rituximab). Patients with certain genetic characteristics, such as del(17p) or TP53 variants do not respond well to chemoimmunotherapy. Targeted therapy targets individual molecules that play a key role in cell signaling resulting in decreased proliferation of leukemic cells. Targeted therapy of CLL includes Bruton's tytosine kinase (BTK) inhibitors (ibrutinib, acalabrutinib), BCL-2 inhibitors (venetoclax) and phosphatidylinositol 3-kinase (PI3K) inhibitors (idelalisib). Despite improvement in clinical outcomes a significant percentage of patients develops resistance to targeted therapy. Resistance most likely occurs as a consequence of genetic variants in a drug's binding site on the targeted protein. The leading cause of ibrutinib resistance is a BTK variant in the binding- site for ibrutinib, followed by the PLCG2 variant that causes constant activation of cell signaling even in the presence of ibrutinib. In the Specialised Haematology Laboratory of the Clinical Department of Haematology, University Clinical Centre Ljubljana we performed determination of genetic variants that cause resistance to targeted therapy of CLL in patients who have been receiving ibrutinib for more than two years. We collected blood samples from patients following informed consent, performed DNA isolation and analysis by NGS. After software analysis of the results we determined the presence of multiple genetic variants that impact prognosis and therapy response in most patients. We found BTK variants in 28,6 % of observed patients while clinically significant PLCG2 and BCL2 variants weren't found. Genes that may predict clinical outcome were also analysed (TP53, NOTCH1, SF3B1), where we found TP53 variants in more than a third of patients, while either NOTCH1 or SF3B1 variants were found in majority of patients. We have shown that determining specific genetic variants is crucial for prediction of disease progression and therapeutic response in CLL patients. kronična limfatična levkemija ibrutinib odpornost različice NGS chronic lymphocytic leukemia ibrutinib variants resistance NGS true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2023-12-05 08:45:04 2023-12-05 08:45:19 2023-12-06 03:40:30 0000-00-00 00:00:00 2023 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 104648 20627.pdf 20627.pdf 1 E9BED6BC1FA0CF3C8440AE20F2563175 def9866f86e363cf0743c33a9327d27c49fb0d76b3a7ec6c34acec6701114997 3c6b7e01-9342-11ee-867f-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=178350 Fakulteta za farmacijo 0 0 0