Regulacijski mehanizmi ekspresije vaskularnoga endotelnog čimbenika rasta u karcinomu bubrega : doktorska disertacija

Abstract

Uvod: Angiogeneza, ili stvaranje novih krvnih i limfnih žila, je neophodna za rast i progresiju tumora, a karcinom bubrežnih stanica idealan model za evaluaciju novih strategija antiangiogene terapije zbog njegove bogate vaskularizacije i visoke ekspresije angiogenih čimbenika. Jedan od najvažnijih promotora angiogeneze je vaskularni endotelni čimbenik rasta ( VEGF) i njegove srodne molekule. VEGF je prekomjerno eksprimiran i na razini mRNA i na proteinskom nivou u humanim tumorima, a brojni čimbenici kroz intracelularne signalne puteve mogu regulirati njegovu pojavnost uključujući hipoksiju i regulatorni transkripcijski faktor HIF1 α, citokine , hormone te modulatore protein kinaze C. Mehanizmi VEGF ekspresije uz sudjelovanje tumor supresorskih gena kao što je p53 ili čimbenika rasta kao na primjer epidermalnog čimbenika rasta (EGF) samo su djelomično poznati. Cilj ovog istraživanja je određivanje imunohistokemijskog izražaja VEGF-A i VEGF-C u svijetlostaničnim karcinomima bubrežnih stanica ( SKBS) u usporedbi s okolnim normalnim tkivom bubrega, te utvrđivanje ekspresije regulacijskih čimbenika HIF1 α, receptora za epidermalni čimbenik rasta (EGFR), p53 tumor supresorskog proteina u tumorskom tkivu uz utvrđivanje odnosa ovih proteina sa angiogenim faktorima VEGF-A i C te odnosa VEGF-A sa gustoćom novostvorenih patoloških krvnih žila. Cilj je također utvrditi metodom fluorescentne in situ hibridizacije da li postoji amplifikacija gena za EGFR u SSKBS te napokon usporediti angiogenezu i ove čimbenike regulacije angiogeneze s kliničko-patološkim parametrima kao što su nuklearni gradus, Ki67 indeks proliferacije tumorskih stanica, veličina tumora, patološki stadij i petogodišnje preživljenje. Materijal i postupci: Na standardnim rezovima 93 SKBS određena je gustoća krvnih žila imunohistokemijskom metodom uz korištenje antitijela CD31 za endotel i na istim rezovima uspoređena sa imunohistokemijskim izražajem VEGF-A.. Izražaj proteina VEGF-A, VEGF-C, HIF1 α, EGFR 1, p53, istražen je metodom imunohistokemije na tkivnim mikroarejima (TMA) istih SKBS-a te uspoređen standardnim statističkim metodama međusobno kao i s kliničkopatološkim parametrima. Metodom fluorescentne in situ hibridizacije (FISH) vizualizirana je amplifikacija EGFR gena u 43 odabrana tumora koji su pokazivali različitu ekspresiju ovog receptora na razini proteina u tumorskim stanicama. Rezultati: Gradus tumora i stadij bolesti su kao glavni prognostički faktori pokazali inverznu korelaciju u odnosu na preživljenje. Studija gustoće krvnih žila u usporedbi s ekspresijom VEGF-A na standardnim rezovima pokazala je da su tumori s vrlo visokom ekspresijom VEGF-A (>75%) povezani s manjim brojem krvnih žila (p=0,034). Na TMA je potvrđena promjenjiva ekspresija sva tri analizirana proteina u tumorskim stanicama SSKBS-ova. Angiogeni čimbenici pokazali su međusobnu povezanost (p<0,031), a njihova ekspresija se uočavala kao perimembranozno ili difuzno citoplazmatsko obojenje. Nuklearna ispoljenost HIF1α (n HIF1α) pokazivala je obrnutu povezanost s difuznim citoplazmatskim obojenjem VEGF-A (p=0,002) i VEGF-C (p=0,053), dok je citoplazmatska ekspresija HIF1α ( cHIF1α) pokazivala pozitivnu korelaciju s difuznim obojenjem oba angiogena čimbenika (p<0,001 odnosno p<0,001). U usporedbi s kliničkopatološkim parametrima, prekomjerna ispoljenost citoplazmatskog cHIF1α i difuznog citoplazmatskog VEGF-A bila je udružena s višim nuklearnim gradusom (p=0,006 odnosno p<0,001), većim tumorima (p<0,001 odnosno p<0,001), višim stadijem bolesti (p=0,023, odnosno p=0,0027) te kraćim preživljenjem (p=0,018, odnosno, p=0,024). Nasuprot tome prekomjerna ekspresija nHIF1α kao i perimembranozna ispoljenost VEGF-C su bili povezani s boljim dijagnostičkim parametrima kao što su niži nuklearni gradus (p=0,006, odnosno, p<0,001 ), manji tumori (p=0,057, odnosno, p= 0,007) i dulje preživljenje ( p=0,005, odnosno,p=0,008). Nadalje, ispoljenost nHIF1α je pokazivala pozitivnu korelaciju s indeksom proliferacije (p=0,022). Veća ekspresija tumor supresorskog proteina p53 bila je povezana sa većim tumorima (p=0,007),višim nuklearnim gradusom ( p=0,048) i stadijem bolesti (p=0,036), kao i veći izražaj EGFR koji je kroz histoskor korelirao s višim nuklearnim gradusom ( p=0,0002) s većim tumorima (p=0,018) višim pT(p=0,036) te kraćim preživljenjem (p=0,046) kod tumora koji su pokazivali samo kontinuirano membransko bojenje. p53 je pokazivao pozitivnu povezanost sa postotkom ekspresije VEGF-C ( (p=0,037), a EGFR je bio proporcionalan sa postotkom difuznog izražaja VEGF-A/C te obrnuto proporcionalno povezan sa postotkom perimembranoznog izražaja. FISH metoda je uočila povezanost polizomije 7 i prekomjerne membranske ekspresije EGFR u SSKBS. Zaključak: Rezultati studije na TMA ukazali su na agresivniji subtip SSKBS-a višeg nuklearnog gradusa koji pokazuje prekomjernu ekspresiju VEGF –A i cHIF1α i na razini multrivarijantne analize što se može smatrati neovisnim prognostičkim značenjem sa mogućim kliničkim implikacijama. Značenje nHIF1α ekspresije povezane sa bolje diferenciranim tumorima i višim proliferativnim indeksom trebalo bi bolje istražiti. Nadalje, subcelularna lokalizacija angiogenih proteina kao i njihovog transkripcijskog faktora pokazala se važnom u odnosu na prognozu bolesti. Naš tumorski model nije potvrdio jednostavnu povezanost VEGF-A i angiogeneze kroz gustoću krvnih žila, ali se i na standardnim rezovima pokazalo da njegova prekomjerna ekspresija predstavlja lošiji prognostički parametar. Difuzna subcelularna lokalizacija VEGF-A i VEGF-C povezana s EGFR najvjerojatnije je povezana sa mehanizmima tumorigeneze.AIM: The role of angiogenesis in the pathogenesis of renal cell carcinoma is well recognized, however the influence of tumor cells in this activity is still not well-known. First, the purpose of this investigation was to analyze and correlate the immunohistochemical pattern of vascular endothelial growth factor (VEGF) expression with the average of microvessel density (MVD) and other clinicopathologic parameters in clear cell renal cell carcinoma (CCRCC) in order to determine its prognostic significance. Secondly we wanted to expand our knowledge on expression of VEGF-A and to compare its value with the VEGF-C expression recognized to be involved in lymph vessel neoangioegenis. Furthermore, the aim was to analyze expression of both angiogenic factors in comparison to Hypoxia inducible factor-1α ( HIF-1α), a regulatory factor of angiogenic switch, p53 tumor supresor protein and epidermal growth factor receptor (EGFR) which are involved in tumor pathogenesis, and finally all analyzed parameters were compared with clinicopathological characteristics of CCRCC including the patients’ survival. We also wanted to investigate the role of EGFR gene copy number changes in relation to EGFR overexpression by means of fluorescence in situ hybridization (FISH). MATERIAL AND METHODS: This study includes tumor specimens of CCRCC obtained from patients undergoing nefrectomy at the Department of Urology, Clinical Hospital Center Rijeka from 1989-1994. All cases were reviewed using WHO tumor classification criteria. First part of our investigation was performed on surgical specimens of 93 CCRCC wich were immunohistochemically analyzed on parafine embedded , whole slide, standard sections for VEGF expression, MVD with anti-CD31, and Ki 67 proliferative index. Then, the tissue microarrays (TMA) were built from the same reviewed cohort of 94 archive formalin fixed and paraffin embedded CCRCC. Immunohistochemistry was performed on tissue microarrays for VEGF-A, VEGF-C, HIF- 1α and Ki67,p53 and EGFR expression. The staining was evaluated as a percentage of cytoplasmic, membranous or nuclear positive tumor cells, and as a histoscore (HS). Clinicopathologic data obtained from patient medical records and from files of the Department of Pathology, Rijeka University School of Medicine, Rijeka, Croatia included sex, age, tumor size, TNM stage, histologic subtype and nuclear grade as assessed using Fuhrman nuclear grading system. RESULTS: On the whole tumor slides, VEGF expression was recorded as the percentage of positive tumor cells(≥75% and ≤75%) and as diffuse or perimembranous VEGF expression according to cytoplasmic distribution. Statistical analysis showed that tumors with ≥75% of VEGF expression were characterized by lower MVD value (p = 0.034), higher nuclear grade(p = 0.018), and higher Ki 67 proliferation index (p = 0.023). Moreover, a higher nuclear grade of tumor cells was characterized by diffuse cytoplasmic VEGF distribution (p = 0.005). The TMA analysis showed the variable expression of analyzed protein in tumor cells among different CCRCC. Both angiogenic factors demonstrated perimembranous or diffuse cytoplasmic staining, with diffuse pattern positively associated (p<0.001). Nuclear HIF-1α expression (nHIF-1α) showed inverse correlation with diffuse cytoplasmic VEGF-A (p=0.002) and VEGF-C (p=0.053), while cytoplasmic HIF-1α expression (cHIF-1α) showed positive correlation with diffuse staining of both angiogenic factors (p<0.001; p<0.001, respectively). In comparison to clinicopathological characteristics, a higher nuclear grade (p=0.006; p<0.001, respectively), larger tumor size (p=0.009; p=0.015, respectively), higher stage (p=0.023; p=0.027, respectively) and shorter survival (p=0.018; p=0.024, respectively) were associated with overexpression of diffuse cytoplasmic VEGF-A and cytoplasmic HIF-1α expression. In contrary, overexpression of nHIF-1α was associated with better diagnostic parameters i.e. lower nuclear grade (p=0.006), smaller tumor size (p=0.057), and longer survival (p=0.005). Expression of p53 protein was recorded as percentage of nuclear positivity /500 tumor celss. Overexpression (≥ of median score 40.5%) was connected with biger tumors, higher NG and pT stage (p=0.007, p=0.048, p=0.036 respectively). Intesive membranous EGFR expression in tumors was also related to worse prognostic parameters and lower overal survival (Hscore p=0.046) EGFR showed higher expression in relation to diffuse cytoplasmic distribution of VEGF-A/C but was negatively associated with perimembranous expression of VEGF-A/C. FISH method revealed polisomy 7 associated with protein EGFR overexpression in CCRCC tumor cells. Multivariate analysis underline NG and diffuse cytoplasmic HIF-1α as an independent prognostic factors. CONCLUSION: Study on standard CCRCC tumor slides did not confirm the postulated simple relationship between VEGF overexpression and angiogenesis through high microvessel count. However, the study results indicated that overexpression of VEGF was a worse histologic prognostic parameter in CCRCC. Survey of CCRCC with TMA technique highlights more aggressive subtype of CCRCC with overexpression of VEGF-s and cHIF- 1α, p53 and EGFR in tumors cells that might have some clinical implication. A significance of nHIF-1α expression, associated with better differentiated tumors should be further better understood. FISH analysis showed that EGFR gene dosage can influence on protein ekspresion through polisomy rather than amplification