Importance of testing proliferative markers of cyclin A and Ki-67 and telomerase in renal cell carcinoma

[J. A. Trifunović]

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Molekularna medicina - Patologija / Molecular medicine - Pathology

Karcinom bubrežnih ćelija (RCC) je najrasprostranjeniji oblik karcinoma bubrega i čini oko 2-3% svih karcinoma kod odraslih, češće kod muškaraca. U svetu se beleži porast obolelih usled primene savremene tehnologije u dijagnostici koja omogućava raniju i precizniju dijagnostiku RCC. Najčešći histološki podtipovi RCC su: svetloćelijski, papilarni, hromofobni, karcinom sabirnih kanala i neklasifikovani. Proliferativni markeri ciklin A, Ki-67 i telomeraza su bitni u dijagnostici RCC. Cilj: Imunohistohemijski ispitati intenzitet ekspresije ciklina A, Ki67 i telomeraze kod RCC i analizirati korelaciju ovih ekspresija sa kliničko-morfološkim karakteristikama RCC. Analizirati prisustvo mutacija i polimorfizma, amplifikaciju i metilacioni status u promotorskom regionu hTERT gena kod ispitivanih pacijenata sa RCC. Materijal i metode: Za istraživanje je korišćen operativni materijal 74 pacijenta obolela od RCC dobijen parcijalnom nefrektomijom koji je posle obrade i kalupljenja, najpre obojen hematoksilin-eozinom i postavljena je histopatološka dijagnoza RCC, a kasnije su rađene imunohistohemijske metode i molekularno-genetičko istraživanja. Rezultati: Imunohistohemijska ispitivanja ekspresije ciklina A u tkivu RCC pokazala su da postoji statistički značajna razlika ekspresije ciklina A kod različitih histopatoloških tipova. Najintenzivnija ekspresija uočena je u slučajevima papilarnog RCC. Imunohistohemijska ispitivanja ekspresije Ki67 pokazala je da je kod papilarnog RCC dobijena je izrazita pozitivnost u odnosu na preostala dva analizirana histopatološka tipa. Takođe, imunohistohemijska ekspresija telomeraze drastično je veća kod papilarnog RCC u odnosu na ostala dva histopatološka tipa. Sekvenciranje 343 bp dugog promotorskog regiona nije ukazalo na aktivaciju mutacija ni u jednom od ispitivanih uzorka RCC, ali je otkriveno prisustvo tranzicije T/C na poziciji -245 bp. Genska amplifikacija je uočena kod 19.4% pacijenata. HTERT promotor je bio metilovan u 54,8% uzoraka. Zaključak: Proliferativni markeri ciklin A, Ki67 i telomeraze imaju veliki prognostički značaj kod RCC, naročito kod papilarnog RCC. Sekvenciranjem je otkriveno prisustvo tranzicije T/C na poziciji -245 bp. Mutirani alel bio je prisutan u homozigotnom obliku u 7,4%, u heterozigotnom obliku u 40,7%, a 51.9% bilo je homozigotno za nemutirani tip gena (wt alel). Nije uočena povezanost između hTERT amplifikacije i epidemioloških, kliničkih i patoloških nalaza. Značajna razlika zapažena je između histopatoloških tipova tumora, pošto je kod hromofobnog histopatološkog tipa, kod svih 5 pacijenata hTERT promotor bio metilovan.

Renal Cell Carcinoma (RCC) is the most common form of renal cancer and makes approximately 2-3% of all carcinoma in adults, being expresed more often in males. An increase in the number of deseased is detected due to the implementation of most contemporary technology in diagnostics that enables an early and accurate diagnostics of RCC. The most common histological subtypes of RCC are the following: clear cell, papillary, chromophobe, collecting duct carcinoma and unclassified. The proliferative markers cycline A, Ki-67 and telomerase are significant in RCC diagnostics. Aims: The goal was to immunohistochemically investigate into the intensity of the cycline A, Ki-67 and telomerase expression in RCC and analyse the correlation of these expressions with clinical-morphological characteristics of RCC. The goal was also to analyse the presence of mutations and polymorphism, gene amplification and metilation status in the hTERT promotor gene region in the examined RCC patients. Materials and Methods: The operational material used in the research was that taken from 74 patients diagnosed with RCC, obtained through partial nephrectomy, which was, following the processing and moulding, first stained by hematoxylin and eosin and then RCC was diagnosed; immunohistochemical methods and molecular-genetic research ensued. Results: Immunohistochemical investigation into the cycline A expression in the RCC tissue have shown a statistically significant discrepancies in the cycline A expression among different histopathological types. The most intensive expression is observed in the cases of papillary RCC. The immunohistochemical investigations into the Ki-67 expression have demonstrated a distinct positivity in comparison with the two other analyzed histopathological types. Similarly, the immunohistopathological expression of telomerase is dramatically higher in papillary RCC in comparisson with the other two histopathological types. The sequencing of 343 bp long promotor region has failed to demonstrate any activation of mutations in any of the examined RCC samples, however, it has shown the presence of T/C transition at the -245 bp position. The gene amplification has been observed in 19.4% patients. HTERT promotor was methylated in 54.8% samples. Conclusion: Proliferative markers, cyclin A, Ki67, and telomerases have great prognostic significance in RCC, especially in papillary RCC. Sequencing detected the presence of the T/C transition at -245 bp position. The variant allele was present in homozygous form in 7.4%, in heterozygous in 40.7%, and 51.9% were homozygous for the wild type allele. There was no association between hTERT amplification and epidemiological, clinical and pathological findings. A significant difference was observed between histopathological types of tumors, since in chromophobe histopathological type, in all 5 patients, the hTERT promoter was methylated.

RCC, proliferativni markeri, ciklin A, Ki67, telomeraza, hTERT, mutacije, polimorfizam, genska amplifikacija, metilacija

RCC, proliferative markers, cyclin A, Ki67, telomerase, hTERT, mutations, polymorphism, gene amplification, methylation

616.61-006.6:577.2(043.3)

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