Acute myeloid leukemia (AML) is an onco-haematological disease characterized by acute onset and poor prognosis. Molecular characterization of the leukemic clone is the primary matter to define the prognostic classification of the disease, choose the therapeutic strategy and monitor minimal residual disease (MRD). In this thesis were studied the most frequent mutations in the Isocitrate Dehydrogenase genes (IDH1 and IDH2), which are involved in epigenetic regulation mechanisms. The application of 4 commercial assays was verified with digital droplet PCR (ddPCR) in 90 blood samples from 14 patients with AML who presented mutations in IDH genes at diagnosis. The operative procedure was defined, optimizing amplification conditions and setting the optimal annealing temperature at 56.5°C for all 4 assays. Limits of Detection (LoD) were verified, achieving a LoD of 0.1% for IDH2 R140Q, IDH1 R132H, and IDH2 R132K mutations, and 0.2% for IDH1 R132C mutation. In eight patients, both in subjects with mutations in IDH, and in patients with mutations in NPM1, it was observed that the presence of the molecular marker IDH correlates with clinic state. The implementation of these molecular investigations finds its application in the study of MMR, mainly in those cases where there are no other useful molecular markers. Nowadays, the meaning and reliability of IDH markers are still debated, but the availability of a quantitative molecular assay could be useful to study their prognostic value, expanding the panel of markers for the MRD monitoring in AML.
La Leucemia Acuta Mieloide (LAM) è una patologia oncoematologica caratterizzata da esordio acuto e prognosi infausta. La caratterizzazione molecolare del clone leucemico è di fondamentale importanza per l’inquadramento prognostico della malattia, la strategia terapeutica e il monitoraggio della malattia minima residua (MMR). In questa tesi sono state studiate le più frequenti mutazioni nei geni delle Isocitrato Deidrogenasi (IDH1 e IDH2), coinvolti nei meccanismi di regolazione epigenetica. È stata verificata l’applicazione di 4 saggi commerciali mediante tecnica di digital droplet PCR (ddPCR) su 90 campioni di sangue relativi a 14 pazienti con LAM che presentavano alla diagnosi mutazioni nei geni IDH. Sono state ottimizzate le condizioni di amplificazione, stabilita la procedura operativa e verificati i Limits of Detection. È stata stabilita la temperatura di annealing ottimale a 56.5°C per tutti i saggi, ed è stato raggiunto il Limit of Detection di 0.1% per le mutazioni IDH2 R140Q, IDH1 R132H e IDH2 R172K e di 0.2% per la mutazione IDH1 R132C. In un sottogruppo di otto pazienti si è osservato che la presenza del marcatore molecolare IDH correla con la clinica, sia nei pazienti con sola mutazione nei geni IDH, sia in compresenza delle mutazioni a carico del gene NPM1, marcatore molecolare più conosciuto e studiato. L’implementazione di queste indagini molecolari trova applicazione nello studio della MMR principalmente in quei pazienti che non presentano altri marcatori molecolari utili. Attualmente, il significato e l’attendibilità dei marcatori IDH sono ancora in discussione, ma avere a disposizione un saggio molecolare quantitativo apre la possibilità di studiare il loro valore prognostico, ampliando il pannello di marcatori per il monitoraggio della MMR nella LAM.
DIGITAL DROPLET PCR (ddPCR): APPLICAZIONE DI SAGGI SPECIFICI PER L’ANALISI MOLECOLARE DELLE MUTAZIONI A CARICO DEI GENI IDH1 E IDH2 NELLA LEUCEMIA ACUTA MIELOIDE
CAROLLO, CAMILLA
2022/2023
Abstract
Acute myeloid leukemia (AML) is an onco-haematological disease characterized by acute onset and poor prognosis. Molecular characterization of the leukemic clone is the primary matter to define the prognostic classification of the disease, choose the therapeutic strategy and monitor minimal residual disease (MRD). In this thesis were studied the most frequent mutations in the Isocitrate Dehydrogenase genes (IDH1 and IDH2), which are involved in epigenetic regulation mechanisms. The application of 4 commercial assays was verified with digital droplet PCR (ddPCR) in 90 blood samples from 14 patients with AML who presented mutations in IDH genes at diagnosis. The operative procedure was defined, optimizing amplification conditions and setting the optimal annealing temperature at 56.5°C for all 4 assays. Limits of Detection (LoD) were verified, achieving a LoD of 0.1% for IDH2 R140Q, IDH1 R132H, and IDH2 R132K mutations, and 0.2% for IDH1 R132C mutation. In eight patients, both in subjects with mutations in IDH, and in patients with mutations in NPM1, it was observed that the presence of the molecular marker IDH correlates with clinic state. The implementation of these molecular investigations finds its application in the study of MMR, mainly in those cases where there are no other useful molecular markers. Nowadays, the meaning and reliability of IDH markers are still debated, but the availability of a quantitative molecular assay could be useful to study their prognostic value, expanding the panel of markers for the MRD monitoring in AML.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/54651