Expression of ZEB1 and LOXL2 in rectal carcinoma and their correlation with extramural venous invasion (EMVI): preliminary study

Abstract

Introduction: Venous invasion has consistently been shown to be associated with poor prognosis in rectal carcinoma (RC), both when detected by pathology and radiology. Extramural venous invasion (EMVI) is characterized by the presence of tumor cells within veins outside the bowel wall and is strongly associated with poor survival and increased risk of local recurrence and distant metastases. Molecular basis of EMVI is still unexplored and genes that regulate tumor microenvironment interactions may have significant role in this process. ZEB1 is transcriptional factor that promote cancerogenesis by indirect regulation of epithelial-mesenchymal transition (EMT) process, while LOXL2 contributes to tumor invasion and metastasis due to its role in the stabilization of the extracellular matrix. Aim: This study aimed to compare the expression level of ZEB1 and LOXL2 genes in relation to EMVI status and other clinic-pathological parameters of RC patients. Methods: We conducted preliminary study on 21 untreated RC patients (9 EMVI+ and 11 EMVI- ) who underwent curative resection in 2016-2018 at Oncology Institute of Vojvodina. The presence of EMVI was assessed on standard hematoxylin and eosin-stained histolological sections of postoperative tumor specimen samples, from which RNA was isolated. Expression of ZEB1 and LOXL2 mRNA was measured using quantitative real-time PCR. Results: Comparative analysis revealed higher expression level of ZEB1 in EMVI positive samples and in patients in TNMIII stage, however the observed differences had no statistic significance (p=0.323 and p=0.197, respectively). Significant difference in LOXL2 expression according to the EMVI status was not detected (p=0.915), while we noted higher LOXL2 expression in late stages of disease, but without statistic significance (p=0.342). Relative expression of these two genes was not associated with metastases frequency and death outcome. Conclusion: Further analyses on larger number of samples with more potential molecular targets included are required and planed.

Uvod: Venska invazija je kontinuirano asocirana sa lošom prognozom kod obolelih od karcinoma rektuma (KR), bilo da je detektovana patološkim ili radiološkim metodama. Ekstramuralna venska invazija (EMVI) se karakteriše kao prisustvo tumorskih ćelija u venskim sudovima izvan zida debelog creva koje je značajno asocirano sa lošim preživljavanjem i povećanim rizikom za nastanak lokalnih recidiva i udaljenih metastaza. Molekularna osnova EMVI procesa nije dovoljno ispitana, a geni koji regulišu interakcije u tumorskoj mikrosredini mogu imati potencijalnu ulogu. ZEB1 je transkripcioni factor koji stimuliše kancerogenezu indirektnom regulacijom epitelnomezenhimske tranzicije (EMT), dok LOXL2 doprinosi procesu tumorske invazije ulogom u stabilizaciji ektracelularnog matriksa. Cilj: Cilj ove studije je uporediti relativnu ekspresije gena ZEB1 i LOXL2 u odnosu na EMVI status i druge kliničko-patološke parametre KR pacijenta. Metode: Ova preliminarna studija obuhvatila je 21 netretiranih KR pacijenata (9 EMVI+ i 11 EMVI-) koji su lečeni operativnim putem u periodu 2016-2018. god. u Institutu za onkologiju Vojvodina. Prisustvo EMVI je utvrđeno na standardno hematoksilinom i eozinom bojenim isečcima postoperativnog tumorskog tkiva iz kojih je izolovana RNK. Ekspresija ZEB1 i LOXL2 iRNA izmerena je kvantitativnom PCR metodom u realnom vremenu. Rezultati: Uporednom analizom uočena je povišena ekspresija ZEB1 kod EMVI+ uzoraka i kod obolelih u TNMIII stadijumu, ali uočene razlike nisu bile statistički značajne (p=0,323 i p=0,197, respektivno). Znčajna razlika u ekspresiji LOXL2 u odnosu na EMVI status nije detektovana (p=0,915), a zabeležena je i povećana ekspresija LOXL2 u kasnim stadijumima bolesti, ali bez statističke značajnosti (p=0,342). Relativna ekspresija ova dva gena nije značajno povezana sa pojavom metstaza i krajnjim ishodom bolesti. Zaključak: Dalje analize na većem broju uzoraka sa više uključenih molekularnih targeta u studiju su neophodne i planirane u budućnosti.