Optimiranje izolacije slobodne cirkulirajuće DNA u bolesnica oboljelih od raka dojke

master's thesis

• Title (english): Optimization of isolation of circulating cell-free DNA in breast cancer
• Author: Teo Morožin
• Mentor: Sandra Šupraha Goreta (mentor)
• Committee member: Sandra Šupraha Goreta (predsjednik povjerenstva)
• Committee member: Lidija Bach-Rojecky (član povjerenstva)
• Committee member: Olga Gornik (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2017-07-11, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Rak dojke najčešći je maligni tumor te prvi po mortalitetu od raka u žena u Hrvatskoj. Po svojoj molekularnoj osnovi, postoji 5 podtipova raka dojke: luminalni tip A, luminalni tip B, bazalni tip, HER2 pozitivan i normalni tip. Ovi tipovi se međusobno razlikuju prema prisutnosti i zastupljenosti pojedinih vrsta receptora, a njihovo određivanje je bitno za definiranje terapije te procjenu ishoda liječenja. Trenutne metode koje se koriste pri dijagnostici raka dojke su: citološka dijagnostika, patohistološka dijagnostika, radiološka dijagnostika, magnetska rezonancija dojki, biopsija sentinelnog limfnog čvora. Slobodna cirkulirajuća DNA obuhvaća fragmente nukleinskih kiselina koji cirkuliraju plazmom, serumom ili nekom drugom tjelesnom tekućinom izvan stanice te se uglavnom radi o fragmentima dvolančane DNA veličine do 200 baznih parova. Različita istraživanja su pokazala kako je razina slobodne cirkulirajuće DNA povišena u različitim malignim stanjima pa tako i u raku dojke. Međutim i dalje postoje prepreke za njenu upotrebu kao novog biomarkera u svakodnevnoj kliničkoj praksi. Cilj ovog istraživanja bio je pridonijeti optimiranju metode izolacije slobodne cirkulirajuće DNA u raku dojke. U tu svrhu su korištene različite metode tj. kolone za izolaciju slobodne cirkulirajuće DNA. Istraživanje je pokazalo kako je za izolaciju slobodne cirkulirajuće DNA potrebna
upotreba specifičnih kolona kako bi izolacija bila provedena bez tragova genomske DNA koji ometaju detekciju i kvantifikaciju. Također potrebno je oprezno rukovanje s uzorcima, bez opetovanih odmarzavanja i zamrzavanja, s obzirom na to da je slobodna cirkulirajuća DNA manje stabilna od genomske DNA. Nadalje pri usporedbi s kontrolnim uzorcima zdravih dobrovoljaca potreban je njihov pažljiv odabir te dobra diferencijalna dijagnostika jer razina slobodna cirkulirajuća DNA može biti povišena i pri određenim fiziološkim stanjima.

Abstract (english)

Breast cancer is the most common malignancy in women and the first most common cause of cancer death among the female population in Croatia. On molecular basis, there are 5 subtypes of breast cancer: luminal A, luminal B, basal, HER positive and normal type. Those types have different expression level of certain receptors what is important for the choice of therapy and its outcome. Current methods used in breast cancer diagnosis are: cytological diagnostic, histopathological diagnostic, radiological diagnostic, magnet resonance of breasts, the biopsy of sentinel lymph node. Circulating cell free DNA are nucleic acids fragments that circulate in plasma, serum and other bodily fluids outside of cells. Those fragments are composed of double stranded DNA and their size is up to 200 base pairs. Various researches have demonstrated that the level of circulating cell free DNA is elevated in malignancy therefore in breast cancer as well. However, there are still obstacles for its everyday use in clinical practice as a new biomarker. The aim of this research was to contribute to the optimization of isolation of circulating cell free DNA in breast cancer. Different methods and kits for isolations were used. The results showed that for isolation of circulating cell free DNA the specific kits for isolation should be used to eliminate
the traces of genomic DNA which may interrupt detection and quantification of cell free DNA. Moreover, the handling with the samples should be very careful, without several freezing and defrosting steps because cell free DNA has the lower level of stability than genomic DNA. Last but not least, considering that level of circulating cell free DNA can be elevated in several physiological conditions, the choice of healthy volunteers should be done very carefully and the differential diagnostic should be done as well.

• Keywords: rak dojke; slobodna cirkulirajuća DNA; biomarker; optimiranje
• Keywords (english): breast cancer; circulating cell-free DNA; biomarker; optimization
• Language: croatian
• URN:NBN: urn:nbn:hr:163:163244
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-11-16 16:08:12