| Abstract | CILJ: Tumorska angiogeneza je prognostički čimbenik preživljenja bolesnika koji boluju od nesitnostaničnog raka pluća. Međutim, u literaturi nedostaje radova koji se odnose na procjenu angiogeneze u primarnim tumorima i metastatski promijenjenim limfnim čvorovima sredoprsja te njezinog utjecaja na preživljenje bolesnika s nesitnostaničnim rakom pluća. Cilj je ove studije izvršiti procjenu tumorske angiogeneze određivanjem srednje gustoće krvnih žila (MVD), imunohistokemijski utvrditi ekspresiju vaskularnog endotelnog čimbenika rasta (VEGF-A) i receptora iz obitelji receptora epidermalnog čimbenika rasta (EGFR i HER-2) na tumorskim stanicama, proteinsku ekspresiju VEGF-A, EGFR i HER-2 usporediti s gustoćom krvnih žila; proteinsku ekspresiju EGFR i HER-2 usporediti s genskom amplifikacijom utvrđenom FISH analizom, a potom utvrditi značaj svih navedenih parametera u kontekstu patohistoloških karakteristika raka i kliničkih osobina bolesnika odnosno utjecaj na preživljenje bolesnika s nesitnostaničnim rakom pluća. MATERIJALI I METODE: Ukupno je ispitano 90 uzoraka nesitnostaničnog raka pluća; 30 uzoraka primarnih tumora bolesnika koji su imali metastaze u limfnim čvorovima sredoprsja, 30 uzoraka metastatski promijenjenih limfnih čvorova i 30 uzoraka tumora bolesnika bez metastaza. Nakon izrade tkivnih mikroareja (TMA, tissue microarray) pristupilo se imunohistokemijskom bojanju tkiva na EGFR, HER-2 i VEGF-A. Imunohistokemijska markacija CD31 (PECAM-1; platelet/endothelial cell adhesion molecule) izvršena je korištenjem monoklonalnog anti-CD31 antitijela te se na taj način izvršila procjena gustoće tumorskih krvnih žila ( engl. microvessel density, MVD). Evaluacija statusa gena HER-2 i EGFR rađena je FISH metodom koristeći Vysis probe (Downers Grove, IL, SAD). Podatci o preživljenju prikupljeni su iz Registra za rak Hrvatskog zavoda za javno zdravstvo. V REZULTATI: Ukupno dvogodišnje preživljenje ispitivanih bolesnika bilo je 59%. Uočena je statistički značajna razlika u preživljenju bolesnika sa metastazama u limfnim čvorovima sredoprsja i bolesnika bez metastaza ( 47% u odnosu na 73%). Prosječno dvogodišnje preživljenje svih ispitivanih bolesnika koji su imali visoki MVD je bilo 45% dok je preživljenje bolesnika sa niskim MVD bilo 69%. Ova razlika u preživljenju je statistički značajna (p=0,02). U skupini bolesnika bez metastaza njih 15 (50%) imalo je visoko vaskularizirane tumore (visoki MVD), a 15 bolesnika je imalo tumore sa niskim MVD. Razlika u preživljenju u odnosu na MVD u ovoj grupi bolesnika nije statistički značajna. U grupi bolesnika s metastazama njih 13 (43%) imalo je niski MVD, a 17 bolesnika imalo je visoki MVD. Razlika u preživljenju u ovoj grupi bolesnika je statistički značajna: dvogodišnje preživljenje bolesnika s visokim MVD u primarnim tumorima bilo je 23%, a bolesnika s niskim MVD u primarnim tumorima 74% što čini statistički značajnu razliku preživljenja ( p=0,01). Uočena je visoka korelacija MVD u primarnim tumorima i metastatski promijenjenim limfnim čvorovima (r=0,57) što može imati značajna kliničke implikacije. Uočena je umjereno snažna i snažna ekspresija VEGF-A ( više od 50% tumorskih stanica) u tumorima svih ispitivanih bolesnika-83%. Ovakva VEGF-A ekspresija korelira s MVD u metastatski promijenjenim limfnim čvorovima. Korelacija VEGF-A s MVD u primarnom tumoru uočena je tek kod snažne ekspresije VEGF-A (više od 75% pozitivnih tumorskih stanica) na granici statističke značajnosti (p=0,064). Nije uočena korelacija izražaja VEGF-A s MVD tumora bolesnika bez metastaza. Nije uočena povezanost ekspresije VEGF-A s preživljenjem bolesnika. Ukupna ekspresija EGFR kao i ekspresija EGFR po grupama bolesnika bila je 20%. Nije uočena statistički značajna korelacija ekspresije EGFR sa kliničkopatološkim osobinama bolesnika, preživljenjem niti MVD. Nije uočena niti korelacija ekspresije EGFR u primarnom VI tumoru i metastazama. Ukupna ekspresija HER-2 bila je 13%, u grupi bolesnika s metastazama 20% dok je u grupi bolesnike bez metastaza bila 10% što nije statistički značajna razlika. Nije bilo korelacije ekspresije HER-2 s kliničkopatološkim osobinama bolesnika, preživljenjem niti MVD. FISH analizom otkrivena je amplifikacija HER-2 gena u 3 tumora (5%), dok se amplifikacija EGFR gena nije uočena (0%). ZAKLJUČAK: Univarijantna analiza pokazala je da bolesnici s visokim MVD imaju statistički značajno lošije preživljenje u odnosu na bolesnike s niskim MVD, dok je multivarijantna analiza pokazala da MVD ima neovisni prognostički značaj u ukupnom broju ispitivanih bolesnika i u grupi bolesnika s metastazama. Dobiveni rezultati ukazuju da dobro vaskularizirani tumori imaju agresivniji klinički tijek i posljedično lošije preživljenje te da su bolesnici s takvim tumorima kandidati za agresivno liječenje neovisno o kliničkom stadiju bolesti. |
| Abstract (english) | AIM: Tumor angiogenesis is a significant prognostic factor for survival in patients with non-small cell lung cancer (NSCLC). Data on evaluation of angiogenesis in primary tumor and lymph node metastases in mediastinum and its impact on survival in patients with NSCLC are lacking. The aims of this study are to evaluate angiogenesis by detemination of microvessel density (MVD); to immunohistochemicay determine the expression of vascular endothelial growth factor (VEGF-A) and the expression of two members of epidermal growth factor receptor family (EGFR and HER2) in tumor cells; to correlate protein expression of VEGF-A, EGFR and HER2 with microvessel density and to correlate protein expression of EGFR and HER2 with gene amplification determined with FISH assay. Significance of all above mentioned parameters was evaluated in the context of histopatological characteristics of tumor and clinical features of patients and their impact on survival of patients with NSCLC was determinated. MATERIALS AND METHODS: Total of 90 samples of NSCLC were examined; 30 samples of primary tumors of patients with lymph node metastases, 30 samples of metastatic lymph nodes and 30 samples of primary tumors of patients without lymph node metastases. Tissue microarrays (TMA) were created and immunohistochemical analysis of EGFR, HER-2 and VEGF-A was performed. To evaluate microvessel density (MVD) immunohistochemical marking of CD31 molecule (PECAM-1, platelet/endothelial cell adhesion molecule) using monoclonal anti-CD 31 antibody was performed. For evaluation of HER-2 and EGFR gene amplification FISH assay using Vysis probe (Downers Grove, IL, USA) was used. Data on patients' survival were obtained from Croatian National Cancer Registry. VIII RESULTS: Total two-year survival of patients was 59%. There was statisticaly significant difference in survival of patients with mediastinal lymph node metastases and patients without lymph node metastases (47% compared to 73%). Two-year survival of all patients with high MVD was 45% and of patients with low MVD was 69%. This difference in survival is statisticaly significant (p=0.02). The group of patients without lymph node mestastases comprised 15 (50%) patients with highly vasularised tumors (high MVD) and 15 patients with low MVD. Difference in survival in this group in relation to MVD was not statisticaly significant. The group of patients with lymph node metastases comprised 13 (43%) patients with low MVD and 17 patients with high MVD. Difference in survival in this group is statisticaly significant; two-year survival in patients with high MVD in primary tumor was 23% and in patients with low MVD in primary tumor was 74% (p=0,01).There was high correlation between MVD in primary tumor and in lymph node metastases (r=0,57) which may have significant clinical implications. There was moderately strong and strong expression of VEGF-A (>50% of tumor cells) in tumor specimens -83%. This VEGF-A expression correlates with MVD in metastatic lymph nodes. Correlation of VEGF-A and MVD was noticed in primary tumors with strong VEGF-A espression (>75% of tumor cells) but did not reach statistical significance (p=0,064). There was no correlation between VEGF-A expression and MVD in tumors of patients without metastases. Expression of VEGF-A was not associated with patients' survival. Overall EGFR expression and the expression in groups was 20%. There was no statistically significant correlation of EGFR expression with clinicopathological features of patients, survival nor with MVD. EGFR expression in primary tumor was not correlated with EGFR expression in lymph node metastases. Total expression of HER-2 was 13%; in the group of patients with lymph node metastases it was 20% IX and in patients without metastases it was 10%. The difference between groups was not statisticaly significant. There was no correlation of HER-2 expression with clinicopathological features of patients, survival nor with MVD. Using FISH analysis HER-2 gene amplification was discovered in 3 tumors (5%), while EGFR gene amplification wasn't noticed. CONCLUSION:Univariate analysis revealed that patients with high MVD have statisticaly lower survival rate than patients with low MVD. Multivariate analysis demonstrated that MVD is an independent prognostic factor in overall patient population included and in the group of patients with lymph node metastases. Obtained results indicate that highly vascularised tumors have more aggressive clinical course and consequently worse survival . Patients with such tumors are candidates for aggressive treatment irrespective of clinical stage of disease. |
