20.500.12556/RUL-179214 Pomen prisotnosti mutacij plazemske prostocelične DNA in DNA zunajceličnih veziklov za napovedovanje kliničnega izida pri bolnikih z rakom trebušne slinavke Prognostic relevance of mutations in plasma cell-free DNA and extracellular vesicle DNA in patients with pancreatic cancer​ Uvod. Rak trebušne slinavke kljub napredku na področju diagnostike in zdravljenja ostaja bolezen s slabo prognozo. Z analizo krožečih oblik DNA v tekočinski biopsiji bi lahko bolje predvideli potek bolezni in posamezniku ustrezno prilagodili zdravljenje. V naši raziskavi smo želeli ovrednotiti vlogo tekočinske biopsije pri zaznavanju za tumor značilnih genetskih sprememb (mutacij), spremljanju odgovora na zdravljenje in napovedovanju kliničnega izida pri bolnikih z rakom trebušne slinavke. Hipoteze. 1. Z analizo prostocelične DNA in DNA zunajceličnih veziklov dosežemo enako uspešnost določanja tumorskih mutacij kot z analizo DNA, izolirane iz ob operaciji odvzetega tumorskega tkiva. 2. Večji delež za tumor značilnih mutacij v prostocelični DNA oziroma DNA zunajceličnih veziklov pred resekcijo tumorja odraža večjo razširjenost oz. neresektabilnost tumorja in je povezan s slabšim celokupnim preživetjem. 3. Po radikalni resekciji tumorja v prostocelični DNA oziroma DNA zunajceličnih veziklov ne zaznamo za tumor značilnih mutacij, prisotnost za tumor značilnih mutacij v prostocelični DNA oziroma DNA zunajceličnih veziklov pa omogoča zgodnje zaznavanje ponovitve ali napredovanja bolezni. Metode. V raziskavo smo vključili 83 bolnikov s histološko potrjenim rakom trebušne slinavke, ki so bili operirani z namenom kirurške resekcije tumorja. Bolnikom smo pred operacijo ter na kontrolnih pregledih en, šest in dvanajst mesecev po operaciji odvzeli vzorce krvi, iz katerih smo izolirali prostocelično DNA in DNA zunajceličnih veziklov. Kot referenčne vzorce za iskanje za tumor značilnih mutacij smo uporabili arhivirane vzorce tumorskega tkiva, odvzetega ob operaciji, fiksirane v formalinu in vklopljene v parafin. V izolatih DNA smo določili prisotnost in delež najpogostejših za tumor značilnih mutacij v genih KRAS, TP53, SMAD4 in CDKN2A. Molekularno-genetske analize smo izvedli s pomnoževanjem tarčnih odsekov DNA s kompetitivno alelno-specifično verižno reakcijo s polimerazo (castPCR) in z digitalno verižno reakcijo s polimerazo (dPCR). Rezultati. S castPCR smo v DNA, izolirani iz tumorskega tkiva, najpogosteje zaznali mutaciji KRAS p.G12D in TP53 p.R273H. Ti mutaciji smo izbrali za nadaljnjo analizo DNA iz tumorskega tkiva in longitudinalnih vzorcev prostocelične DNA z dPCR. Izolacijo in analizo DNA zunajceličnih veziklov smo izvedli v podskupini 10 izbranih bolnikov, vendar zaradi majhnega števila izoliranih kopij analiza DNA ni bila uspešna. V prostocelični DNA smo ob operaciji izbrani mutaciji zaznali pri manjšem deležu bolnikov kot v tumorskem tkivu. Zaradi nizkega deleža mutacij v prostocelični DNA nismo zaznali statistično značilne povezave med deležem izbranih mutacij v DNA iz tumorskega tkiva in prostocelični DNA ob operaciji. Delež mutacij v prostocelični DNA je bil ob operaciji deloma povezan s predoperativno razširjenostjo bolezni. Delež mutacij v longitudinalnih vzorcih prostocelične DNA ni kazal jasne povezanosti s kliničnim potekom bolezni. Zaključek. S tekočinsko biopsijo nismo mogli v celoti nadomestiti analize tkivne DNA, še zlasti pri omejeni bolezni. Večji delež mutacij v prostocelični DNA ob operaciji je sovpadal z večjo razširjenostjo bolezni, ni pa bil povezan s preživetjem. Delež mutacij v prostocelični DNA tudi ni bil uporaben za spremljanje poteka bolezni. Izsledki naše raziskave v okviru uporabljene metodologije ne podpirajo uporabe tekočinske biopsije z analizo krožečih oblik DNA pri obravnavi bolnikov z rakom trebušne slinavke. Introduction. Despite advances in diagnostics and treatment, the pancreatic cancer prognosis remains poor. Circulating DNA analysis in liquid biopsy could enable better prediction of disease course and allow for personalized treatment. In our study, we aimed to evaluate the role of liquid biopsy in detecting tumour-specific mutations, monitoring treatment response, and predicting clinical outcome in patients with pancreatic cancer. Hypotheses. 1. Analysis of cell-free DNA and extracellular vesicle-associated DNA achieves comparable success rate in identifying tumour-specific mutations as the analysis of DNA isolated from surgically resected tumour tissue. 2. A higher proportion of tumour-specific mutations in cell-free DNA or extracellular vesicle-associated DNA prior to tumour resection reflects greater disease burden or tumour unresectability and is associated with poorer overall survival. 3. After radical tumour resection, tumour-specific mutations cannot be detected in cell-free DNA or extracellular vesicle-associated DNA, while their presence enables early detection of disease recurrence or progression. Methods. We included 83 patients with histologically confirmed pancreatic ductal adenocarcinoma who underwent surgery with curative intent. Blood samples were collected before surgery and at follow-up visits one, six, and twelve months postoperatively for cell-free DNA and extracellular vesicle-associated DNA isolation. Archived formalin-fixed paraffin-embedded tissue samples were used as reference material for identifying tumour-specific mutations. Molecular analyses were performed using competitive allele-specific PCR (castPCR) and digital PCR (dPCR) to determine the presence and frequency of selected common somatic mutations in four pancreatic cancer driver genes: KRAS, TP53, SMAD4, and CDKN2A. Results. Using castPCR, the most frequently detected mutations in tumour tissue DNA were KRAS p.G12D and TP53 p.R273H. These mutations were selected for further analysis of DNA from tumour tissue and longitudinal samples of cell-free DNA using dPCR. Isolation and analysis of extracellular vesicle-associated DNA were performed in a subgroup of 10 patients; however, mutation detection was limited by low DNA yield. In contrast to primary tumour analysis, the detection of selected mutations in cell-free DNA was markedly lower. Due to the low frequency of mutant alleles in cell-free DNA, we did not observe a statistically significant correlation between the mutation frequency in tumour tissue and cell-free DNA at surgery. The frequency of mutations in cell-free DNA samples at surgery was partly associated with preoperative disease burden. No consistent association was observed between the mutation frequency in longitudinal cell-free DNA samples and the clinical course of the disease. Conclusion. Liquid biopsy could not completely replace tissue DNA analysis, especially in cases of limited disease. A higher proportion of mutations in cell-free DNA at the time of surgery correlated with greater disease burden but was not associated with survival. The proportion of mutations in cell-free DNA was also not useful for monitoring disease progression. The results of our study, within the framework of the methodology used, do not support the use of liquid biopsy with analysis of circulating DNA forms in the treatment of patients with pancreatic cancer. rak trebušne slinavke tekočinska biopsija prostocelična DNA zunajcelični vezikli pancreatic cancer liquid biopsy cell-free DNA extracellular vesicles true false false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2026-02-08 07:15:18 2026-02-08 07:15:45 2026-02-12 04:36:00 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 38564 8527.pdf 8527.pdf 1 11DB32087C73662B9DE3AF15868365A4 231c1a2190142c1f1d04860137c4b5c84837920511e7c91472959895613f9b7b 82b6e820-04b5-11f1-a1ba-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=227505 Medicinska fakulteta 0 0 0