| 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. |
| Abstract (english) | 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. |
