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ID naloge: 62 Letnik: 1999 Predmet: patologija in histologija
Ugotavljanje proliferacijske in apoptotske aktivnosti hepatocitov
Avtor: Jože Pižem
Mentor: doc.dr. Andrej Cör
Izhodišca. V normalnih tkivih je število celic konstantno zaradi uravnoteženosti med celicno delitvijo in celicno izgubo (apoptozo). Bcl-2 in p53 sta kljucna proteina pri regulaciji tako celicne delitve, kot tudi apoptoze. Za tumorska tkiva je znacilna nenadzorovana celicna delitev. Hepatocelularni karcinom (HCC) je najpogostejši primarni jetrni tumor. Pri nas se na leto pojavi 50 novih primerov. Namen naloge je bil ugotoviti stopnjo proliferacijske in apoptotske aktivnosti v HCC in netumorskem jetrnem tkivu in preveriti, ali obstajajo korelacije med histopatološkimi znacilnostmi HCC in proliferacijsko ter apoptotsko aktivnostjo. S tem bi bilo ugotavljanje proliferacijske in apoptotske aktivnosti uporabno v rutinski patohistološki diagnostiki. Želeli smo ugotoviti tudi vlogo proteinov p53 in Bcl-2 pri uravnavanju apoptoze in celicne delitve.
Metode. Iz arhiva Inštituta za patologijo v Ljubljani smo izbrali resekcijske vzorce jetrnih tumorjev 16 pacientov s HCC. Od vsakega pacienta smo analizirali vzorec HCC in vzorec netumorskega tkiva, v 11 primerih pa tudi vzorec netumorskega tkiva v 400µm pasu ob tumorju. V preparatih, barvanih s hematoksilinom in eozinom ter s trikromsko metodo po Massonu, smo dolocili gradus tumorja, stopnjo vnetja v tumorju, prisotnost svetlih celic, prisotnost kapsule in površinski delež veziva v netumorskem tkivu. Proliferacijsko aktivnost smo dolocili z imunohistokemicnim barvanjem na PCNA (jedrni antigen proliferirajocih celic) in Ki67. Apoptotsko aktivnost smo dolocili z apoptotskim indeksom (AI), in sicer po morfoloških znacilnostih apoptotskih celic in z metodo TUNEL. Izražanje p53 in Bcl-2 v jetrnem tkivu smo ugotavljali z imunohistokemicnim barvanjem na p53 in Bcl-2.
Rezultati. V HCC smo ugotovili statisticno pomembno korelacijo med indeksom PCNA in indeksom Ki67 (p<0,01). Indeks PCNA je bil v netumorskih jetrih 0,21% ± 0,80% (aritmeticna sredina ± SD) in v HCC 7,41% ± 8,22% , indeks Ki67 pa je bil v netumorskih jetrih 0,19% ± 0,26% ter v HCC 9,67% ± 7,70%. Razlike med netumorskim tkivom jeter in HCC so bile tako pri indeksu PCNA kot tudi pri indeksu Ki67 statisticno pomembne (p<0,05 in p<0,01). Povprecne vrednosti indeksa PCNA so se med posameznimi gradusi tumorjev statisticno pomembno razlikovale (p<0,05). V HCC in netumorskih jetrih je bil AI, dolocen po morfoloških znacilnostih, višji od AI, dolocenega z metodo TUNEL (p<0,01). Med AI (ne glede na nacin dolocanja) in gradusom HCC ni bilo korelacije. Barvanje na p53 je bilo pozitivno v 7 primerih HCC (43,8%), vendar je bil delež pozitivnih celic nizek (od 0,1% do 4,6%). Barvanje na Bcl-2 je bilo pozitivno v 1 primeru netumorskih jeter (6,3%) in v 4 HCC (25%). V HCC sta bila izražanje Bcl-2 in AI v negativni korelaciji (p<0,05).
Zakljucki. Proliferacijska aktivnost je v HCC višja kot v netumorskem jetrnem tkivu. Tako PCNA kot tudi Ki67 sta uporabna za ugotavljanje proliferacijske aktivnosti tumorskih hepatocitov. Oba oznacevalca celicnega razmnoževanja sta lahko v pomoc v rutinski patohistološki diagnostiki, predvsem pri klasifikaciji HCC v razlicne graduse. Pri nastanku in rasti HCC ima kljucno vlogo nekontrolirana delitev tumorskih celic, saj se apoptotska aktivnost med tumorskim in netumorskim jetrnim tkivom ne razlikuje. Pri uravnavanju apoptoze v HCC je pomemben predvsem Bcl-2, medtem ko je mutacija ali prekomerno izražanje p53 prisotna le v posamezni tumorskih hepatocitih
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[Abstract / English version]
The assessment of proliferative and apoptotic activity of hepatocytes
Author: Jože Pižem
Mentor: doc.dr. Andrej Cör
Background. The number of cells in normal tissue remains constant because of the balance between cell division and cell loss (apoptosis). Bcl-2 and p53 play an important role in cell cycle and apoptotic process regulation. Uncontrolled cell division is characteristic of neoplastic tissue. Hepatocellular carcinoma (HCC) is one of the most common liver cancers. In Slovenia 50 new cases of HCC emerge each year. The aim of our study was to determine the proliferative and apoptotic activity of neoplastic and non-neoplastic hepatocytes and to ascertain, whether there was a correlation between the histopathological characteristics of HCC and its proliferative or apoptotic activity. We wanted to discover whether proliferative and apoptotic activity assessment could be useful in routine pathological practice. We also wanted to establish the role of p53 and Bcl-2 in hepatocyte cell division and apoptosis mechanisms.
Methods. Liver tissue resection samples from 16 patients were selected from the archive of the Institute of Pathology. From each patient one sample of HCC and one sample of non-neoplastic liver were analysed. From 11 patients the liver tissue close to the tumour (not more than 400 mm from the tumour) was additionally analysed. The grade of the tumours, the degree of inflammation and the number of clear cells in the tumour, the fibrous capsule around the tumour and the density of fibrous tissue in non-neoplstic livers were determined on haematoxylin-eosin and trichrome stained slides. Proliferative activity was established by immunohistochemical staining against PCNA (proliferating cell nuclear antigen) and Ki67. Apoptotic activity was determined with an apoptotic index (AI), with morphological and by TUNEL methods. Bcl-2 and p53 expressions were assessed with immunohistochemical staining.
Results. A statistically significant correlation between PCNA and Ki67 proliferative indexes was found in HCC (p<0.01). PCNA index was 0.21% ± 0.80% (Mean ± SD) in non-neoplastic liver and 7.41% ± 8.22% in HCC meanwhile Ki67 index was 0.19% ± 0.26% in non-neoplastic liver and 9.67% ± 7.70% in HCC. The differences between HCC and non-neoplastic liver were statistically significant (p<0.05 and p<0.01). Differences in PCNA index between different HCC grades were statistically significant (p<0.05). In HCC and non-neoplastic liver samples the AI assessed by the morphological method were higher than AI determined by the TUNEL method. Differences in the AI (irrespective of the method used) between different HCC grades were not significant. In 7 HCC cases (43.8%) p53 staining was positive, but the number of positive cells was 0.1% - 4.6%. Bcl-2 staining was positive in 1 non-neoplastic liver sample (6.3%) and in 4 HCC samples (25%). In HCC cases bcl-2 expression and AI were in negative correlation.
Conclusions. Proliferative activity was higher in HCC than in non-neoplastic liver samples. PCNA and Ki67 were useful for prolifearative activity assessment of hepatocytes. Both markers can help in routine pathological practice, especially in HCC grading. There were no differences in apoptotic activity between HCC and non neoplastic tissue, so it seems that uncontrolled tumour cell division plays an important role in HCC growth. In the regulation of the apoptotic process in HCC, Bcl-2 is important. Mutation or overexpression of p53 was present in only a few neoplastic hepatocytes.
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