Analysis of the expression of antiangiogenic FKBPL protein, vascular endothelial growth factor and estrogen receptor in uterine endometrioid carcinoma and benign endometrial hyperplasia

Abstract

ANALIZA EKSPRESIJE ANTIANGIOGENOG FKBPL PROTEINA, VASKULARNOG ENDOTELNOG FAKTORA RASTA I ESTROGENOG RECEPTORA U ENDOMETRIOIDNOM KARCINOMU UTERUSA I BENIGNOJ HIPERPLAZIJI ENDOMETRIJUMA Sažetak Uvod: FKBPL (FK506-binding protein-like), pripada familiji imunofilina, FK506 vezujućih proteina, i predstavlja aktuelno široko ispitivani negativni regulator rasta tumora, angiogeneze i metastaza, sa perspektivom u razvoju ciljane onkološke terapije. Viši nivo ekspresije FKBPL kod pacijenata sa karcinomom dojke je dokazan kao nezavisni prognostički faktor dužeg preživljavanja, kao i dužeg intervala bez relapsa u grupi sa visokom ekspresijom estrogenskog receptora, dok je niži nivo ekspresije FKBPL povezan sa lošijom prognozom preživljavanja. Pokazano je da je povećana ekspresija FKBPL kod pacijenata sa karcinomom dojke praćena nižom ekspresijom estrogenog receptora (ER), većom zavisnošću rasta tumora od estrogene stimulacije i većom senzitivnošću tumora na endokrinu terapiju Tamoksifenom. U okviru predkliničkih studija dokazano je inhibitorno dejstvo FKBPL i njegovih derivata na rast kancerskih stem ćelija karcinoma dojke. Ekspresija FKBPL takođe je ispitivana na kulturama kancerskih stem ćelija karcinoma ovarijuma i ciljana terapija bazirana na FKBPL je pokazala antiangiogeno dejstvo kojim dovodi do usporavanja rasta kolonija stem ćelija karcinoma ovarijuma posredstvom inhibicije migracije endotelnih ćelija. Dokazana je genska ekspresija FKBPL u stromi karcinoma endometrijuma, dok su trenutno jedini izvor o imunohistohemijskoj ekspresiji FKBPL u endometrijumu i karcinomu endometrijuma enciklopedijske baze podataka. Prema dostupnim i relevantnim izvorima, do sada nije izvedena analiza značaja nivoa ekspresije antiangiogenog FKBPL proteina u karcinomima endometrijuma, sa detaljnim sagledavanjem ključnih prognostičkih parametara. Ciljevi: Ciljevi ovog istraživanja su bili da se ispita nivo ekspresije FKBPL, VEGF-A i ER , kao i pojedinačna povezanost nivoa ekspresije VEGF-A i ER sa nivoom eks presije FKBPL, u endometrioidnom karcinomu i benignoj hiperplaziji endometrijuma; da se ispita povezanost nivoa ekspresije FKBPL sa prosečnom gustinom krvnih sudova u endometrioidnom karcinomu i benignoj hiperplaziji endometrijuma; i da se ispita povezanost nivoa ekspresije FKBPL u endometrioidnom karcinomu endometrijuma sa dubinom invazije miometrijuma, prisustvom limfovaskularne invazije, histološkim gradusom tumora i kliničkim stadijumom bolesti. Metode: Studijsku populaciju je činilo 90 slučajeva endometrioidnog karcinoma i 40 slučajeva benigne hiperplazije endometrijuma. Kalupi su izdvojeni iz arhive službe za patologiju a klinički i histološki podaci su dobijeni uvidom u dokumentaciju pacijenata u Kliničko - bolničkom centru Zemun. Imunohistohemijska bojenja na FKBPL, VEGF-A, ERα i CD34 su urađena, prema uputstvima proizvođača, u laboratoriji Instituta za patologiju, Medicinskog fakulteta u Beogradu. Intenziteti ekspresije FKBPL i VEGF-A su izvedena dvostruko slepom analizom od strane dva patologa, kao i morfometrijskom metodom određivanja procenta površine žlezdanog tkiva sa pozitivnom reakcijom antitela. Nivo ERα je određen u okviru dvostruko slepe analize određivanjem intenziteta skalom 0 - 3, procentualne zastupljenosti ERα pozitivnih epitelnih ćelija skalom 0 - 5 i Allered-ovim skorom koji je suma vrednosti prethodne dve skale i u rasponu je 0 - 8. Krvni sudovi obeleženi antitelom CD34 su analizirani određivanjem vaskularne gustine morfometrijskom metodom, na delu uzorka. Morfometrijska merenja su rađena softverom za analizu slike Fiji: an open-source platform for biological-image analysis. Rezultati: Intenzitet ekspresije FKBPL je pokazao statisticki značajno (p<0.001) niže vrednosti u endometrioidnom karcinomu nego u benignoj hiperplaziji endometrijuma, umerenu pozitivnu korelaciju (p<0.05) sa sva tri parametra ekspresije ERα, i umerenu negativnu korelaciju (p<0.05) sa nivoom ekspresije VEGF-A na nivou celog uzorka...ANALYSIS OF THE EXPRESSION OF ANTIANGIOGENIC FKBPL PROTEIN, VASCULAR ENDOTHELIAL GROWTH FACTOR AND ESTROGEN RECEPTOR IN UTERINE ENDOMETRIOID CARCINOMA AND BENIGN ENDOMETRIAL HYPERPLASIA Abstract Background: FKBPL (FK506 binding protein-like), is a divergent member of the immunophilin family, FK506 binding proteins, and is currently a widely studied negative regulator of tumor growth, angiogenesis, and metastasis, with a perspective in the development of targeted therapy in gynecological oncology. A higher level of FKBPL expression in breast cancer patients has been proven to be an independent prognostic factor of longer survival, as well as a longer relapse-free interval in the group with high estrogen receptor expression, while a lower level of FKBPL expression is associated with a worse survival prognosis. It has been shown that increased expression of FKBPL in patients with breast cancer is accompanied by lower expression of ER, greater dependence of tumor growth on estrogen stimulation, and greater sensitivity of tumors to endocrine therapy. In the framework of preclinical studies, the inhibitory effect of FKBPL and its derivatives on the growth of breast cancer stem cells has been proven. The expression of FKBPL was also examined on the cultures of ovarian cancer stem cells and the targeted therapy based on FKBPL showed an antiangiogenic effect, which leads to the slower growth of ovarian cancer stem cell colonies, by inhibiting the migration of endothelial cells. The gene expression of FKBPL in the stroma of endometrial carcinoma has been proven, while currently, the only available source on the immunohistochemical expression of FKBPL in the endometrium and endometrial carcinoma is the encyclopedic human protein database. According to available and relevant sources, there are no publications offering a comprehensive analysis on the subject of expression of the anti-angiogenic FKBPL protein in endometrial cancers has been performed, with a detailed review of the key prognostic parameters. Objectives: The objectives of this study were to examine the expression level of FKBPL, VEGF -A, and ER, as well as the individual association of the expression level of VEGF-A and ER with the expression level of FKBPL, in endometrioid carcinoma and benign endometrial hyperplasia; to examine the association of FKBPL expression levels with vascular density in endometrioid carcinoma and benign endometrial hyperplasia; and to explore the association of FKBPL expression levels in endometrioid endometrial carcinoma with the depth of myometrial invasion, presence of lymphovascular invasion, histological tumor grade and clinical stage of the disease. Methods: The study population consisted of 90 cases of endometrioid carcinoma and 40 cases of benign endometrial hyperplasia. Paraffin molds were obtained from the archives of the pathology service, and clinical and histological data were obtained from patient documentation at the Zemun Clinical Hospital Center. Immunohistochemical stainings for FKBPL, VEGF-A, ERα, and CD34 were performed, according to the manufacturer's instructions, in the laboratory of the Institute of Pathology, Faculty of Medicine in Belgrade. Expression intensities of FKBPL and VEGF-A were performed by double-blind analysis by two pathologists, as well as by the morphometric method of determining the percentage of the glandular tissue surface with a positive antibody reaction. The level of ERα was determined in a double-blind analysis by determining the intensity on a scale of 0- 3, the percentage of ERα-positive epithelial cells on a scale of 0-5, and Allered's score, which is the sum of the values of the previous two scales and is in the range of O-8. Blood vessels labeled with the CD34 antibody were analyzed by determining the vascular density using the morphometric method, on part of the sample. All morphometric measurements were made with Fiji image analysis software: an open-source platform for biological-image analysis. Results: FKBPL expression intensity showed statistically significantly (p<0.001) lower values in endometrioid carcinoma than in benign endometrial hyperplasia, moderate positive correlation (p<0.05) with all three parameters of ERα expression, and moderate negative correlation (p<0.05) with the level of VEGF-A expression observed analyzing the whole sample..