Detekcija i karakterizacija genomske nestabilnosti tumora mozga glijalnog porekla

Abstract

Glioblastomi (GBM) predstavljaju najčešći i najmaligniji tip tumora mozga. Iako imaju istu histopatološku sliku, primarni i sekundarni glioblastomi se razlikuju po mehanizmu nastanka i setu karakterističnih genetičkih promena. Iako je tokom poslednjih godina došlo do određenog poboljšanja u sagledavanju njihove kompleksne prirode, glioblastomi i dalje predstavljaju neizlečivu bolest. Osnovni cilj ove doktorske disertacije bio je detekcija i kvantifikacija genomske nestabilnosti, kao i identifikacija specifičnih genetičkih promena odgovornih za promociju i progresiju malignih glioma, a sa dugoročnim ciljem da se definišu potencijalni molekularni markeri za dijagnozu i prognozu bolesti. Sa tim u vezi, izvršena je i analiza najčešćih poznatih genetičkih promena u gliomima: detekcija inaktivacije ključnih tumor supresor gena (p53, PTEN i p16) i amplifikacije EGFR onkogena. Promene dva ili više navedenih gena su prisutne u većini analiziranih uzoraka, što potvrđuje značaj višestrukih genskih alteracija u patogenezi glioma. Upoređivanjem AP-PCR DNK profila tumorskog i zdravog tkiva pacijenata uočena su dva tipa razlika: kvalitativne razlike koje nastaju usled promena u sekvenci DNK molekula i predstavljaju manifestaciju mikrosatelitske nestabilnosti i nestabilnosti pojedinačnih nukleotida (MIN-PIN), kao i kvantitativne razlike nastale usled amplifikacija ili delecija većih hromozomskih regiona, pokazatelj hromozomske nestabilnosti (CIN). Oba tipa promena su prisutna u svim analiziranim uzorcima, doprinoseći podjednako visokom stepenu ukupne genomske nestabilnosti u oba histološka podtipa GBM. Detaljnija analiza DNK profila je omogućila identifikaciju specifičnih promena 11 novih gena, koji do sada nisu povezani sa progresijom glioma: LHFPL3, SGCG, HTR4, ITGB1, CPS1, PROS1, GP2, KCNG2, PDE4D, KIR3DL3 i INPP5A. Većina identifikovanih gena ima značajnu ulogu u procesima signalne transdukcije i ćelijske adhezije, koji su veoma značajni za nastanak i progresiju kancera. Prisustvo promena u identifikovanim genima je, dalje, korelisano sa kliničko-patološkim parametrima, stepenom genomske nestabilnosti i preživljavanjem pacijenata, kao i sa statusom p53, PTEN, p16 i EGFR gena. Pokazane su statistički značajne ko-alteracije nekih od identifikovanih gena sa p53, p16 i EGFR genima, ali nije pokazana statistički značajna povezanost između inaktiviranog PTEN-a i bilo kojeg novog identifikovanog gena...

Glioblastoma is the most frequent and the most malignant human brain tumor. Despite a similar histological appearance, primary and secondary glioblastomas are distinct tumor entities with different genetic alterations but none being specific enough to distinguish them. Despite better insight in its complex genetic nature, glioblastoma is still incurable disease, with extremely short median survival. The purpose of this study was to detect specific genetic changes, as well as to quantify overall level of genomic instability in samples of malignant glioma patients. Besides, we analyzed genetic alterations of key tumor suppressors (p53, PTEN and p16) and EGFR oncogene, commonly aberrant in glioma samples. Alterations of two or more genes were present in majority of analyzed samples indicating importance of multiple changes for gliomagenesis. AP-PCR DNA profiling revealed two types of genetic differences between tumor and normal tissue: qualitative changes which represent accumulation of changes in DNA sequence and are the manifestation of microsatellite and point mutation instability (MIN-PIN) and quantitative changes which represent amplifications or deletions of existing chromosomal material and are the manifestation of chromosomal instability (CIN). Both types of alterations were present in all analyzed samples contributing almost equally to the total level of genomic instability, and showing no differences between histological subtypes. Further investigation of alterations in DNA profiles revealed specific changes in the following 11 genes that were not previously associated with glioma pathogenesis: LHFPL3, SGCG, HTR4, ITGB1, CPS1, PROS1, GP2, KCNG2, PDE4D, KIR3DL3, and INPP5A. Most of identified genes have significant role in signal transduction or cell adhesion, which are important processes for cancer development and progression. The frequencies of observed alterations were correlated with clinicopathologic parameters, the level of genomic instability and patient survival, as well as with presence of alterations in p53, PTEN, p16 and EGFR genes. Some of the identified genes showed significant association with p53 and p16 tumor suppressors, as well as with EGFR, but there was no significant correlation between loss of PTEN and any of identified genes. In conclusion, our results confirmed complexity of glioma genetic nature, emphasizing high level of genomic instability as hallmark of this tumor type. Identified novel genes could be used as potential biomarkers for diagnosis of primary and secondary glioblastoma, as well as predictors of patients’ outcome.