University of Rijeka. Faculty of Medicine. Department of General Pathology and Pathological Anatomy.
Abstract
Cilj istraživanja: Imunohistokemijski analizirati ekspresiju apoptotičkih biljega, bcl-2-a, bax-a i p53 te transkripcijskog faktora c-myc-a na tumorskim stanicama meduloblastoma. Utvrditi međuovisnost analiziranih parametara kao i njihov odnos prema spolu i dobi bolesnika, histološkom tipu meduloblastoma i broju krvnih žila unutar tumora. Materijali i metode: Parafinski rezovi 39 uzoraka meduloblastoma obojeni su streptavidin imunoperoksidaza tehnikom na automatskom imunobojaču primjenom poliklonskih (za bax) i monoklonskih (za bcl-2, p53 i c-myc) protutijela. Evaluacija imunohistokemijskog bojenja vršena je na temelju utvrđivanja intenziteta bojenja odnosno imunohistokemijske reakcije (IR), postotka reaktivnih stanica (PRS) te konačno indeksa imunohistokemijskog bojenja koji je dobiven zbrojem IR i PRS. Broj krvnih žila mjeren je na 10 vidnih polja najaktivnije vaskularizacije, a vrijednost je izražena kao gustoća krvnih žila na mm2. Rezultati: Pozitivno nuklearno obojenje s p53 je utvrđeno u 14, citoplazmatsko s bcl-2 u 9, citoplazmatsko i membransko s bax-om u 39 te nuklearno i citoplazmatsko s c-myc-om u 26 uzoraka, nije koreliralo sa spolom bolesnika. Starija dobna skupina bila je obilježena tumorima jače p53 ekspresije (p=0,036), a neklasični oblici meduloblastoma jačom ekspresijom bcl-2 (p=0,046). Međusobnim uspoređivanjem biljega nađena je pozitivna korelacija između IIB za p53 i IR za bcl-2 (p<0,001). Ekspresija c-myc-a utvrđena je, osim na tumorskim stanicama i na endotelu krvnih žila u 22 uzoraka meduloblastoma. Jači izražaj ovog transkripcijskog faktora bio je udružen s većom gustoćom krvnih žila (p=0,021). ii Uspoređujući ekspresiju p53, broj i gustoću krvnih žila utvrđeno je da tumori s više pozitivnih stanica imaju u prosjeku veći broj (p=0,0435) i veću gustoću krvnih žila (p=0,0528). Isto tako, utvrđena je povezanost, iako bez statističke značajnosti, između IIB za p53 na stanicama meduloblastoma i broja (p=0,0728) te gustoće krvnih žila (p=0,0948) unutar tumora. Zaključak: Istraživanja pokazuju heterogenu ekspresiju apoptotičkih proteina i transkripcijskog faktora c-myc-a na tumorskim stanicama. Veća ekspresija p53 proteina u starijoj dobi bolesnika, veći broj i gustoća krvnih žila kao i veća ekspresija antiapoptotičkog bcl-2 u neklasičnom obliku meduloblastoma mogli bi imati značaj za biološko ponašanje meduloblastoma, odnosno odgovor na terapiju tumora, dok bi pojačana ekspresija c-myc-a na endotelu, koja je udružena s jačom vaskularizacijom, mogla ukazivati na značaj ovog transkripcijskog faktora na progresiju tumora.The aim of the research was to conduct an immunohistochemical analysis of the expression of apoptotic markers, bcl-2, bax and p53 and of the transcription factor c-myc in the medulloblastoma tumour cells, and to determine the interdependence of the analysed parameters and their relationship according to the patients' sex and age, histological type of medulloblastoma and the number of blood vessels in the tumours. Materials and methods: Paraffin slices of 39 medulloblastoma samples were stained by streptavidin immunoperoxidasemethod in the automatic immunostainer using the polyclonal (for bax) and monoclonal (for bcl-2, p53 and c-myc) antibodies. The evaluation of the immunohistochemical staining done by establishing the intensity of the staining, i.e. of the immunohistochemical reaction (IR), percentage of reactive cells (PRS) and the final index of the immunohistochemical staining being the sum of the IR and PRS. The number of blood vessels was measured in the 10 visible fields of the most active vascularisation, and the value was expressed as the blood vessel density per mm2. Results. The positive nuclear staining with p53 which was established in 14 samples, cytoplasmatic with bcl-2 in 9 samples, cytoplasmatic and membrane with bax in 39 samples and nuclear and cytoplasmatic with c-myc in 26 samples did not correlate with the patients' sex. The older age group was characterised with the tumours of stronger p53 expression (p=0.036), and the nonclassical forms of the medulloblastoma with stronger expression of the bcl-2 (p=0.046). The markers' comparison proved a positive correlation between the IIB for p53 and IR for bcl-2 (p < 0.001). iv The c-myc expression was found not only in the tumour cells but also in the endothelium of the blood vessels in the 22 medulloblastoma samples. The stronger expression of this transcription factor was associated with the greater density of the blood vessels (p=0.021). The comparison of the p53 expression and the number and density of blood vessels showed that the tumours with more p53 positive cells have, on average, the greater number (p=0.0435) and density of blood vessels (p=0.0528). A connection, of no statistical relevance though, was established between IIB for p53 in the medulloblastoma cells and the number (p=0.0782), and the density of blood vessel in the tumour (p=0.0948). Conclusion: The study has shown a heterogeneous expression of the apoptotic proteins and the transcription factor c-myc in medulloblastoma tumour cells. A greater expression of the proapoptotic p53 protein commonly found in the medulloblastoma of old age group patients, the greater number and density of blood vessels and the greater expression of the antiapoptotic bcl-2 protein in a nonclassical form of the medulloblastoma may be relevant for the biological behaviour i.e. for the tumour response to therapy, while the connection between stronger tumour vascularisation and the greater number of p53 positive tumour cells, i.e. stronger expression of the c-myc in the endothelium may indicate the significance of these proteins in the tumour progression
