Cyclooxygenase-2 Expression Determines Neoangiogenesis in Gallbladder Carcinomas

Neoangiogenesis may have an important role in the poor prognosis of gallbladder carcinoma. An enhanced expression of COX- was found in precancerous lesions and in gallbladder carcinoma, likely to be involved in carcinogenesis as well as in angiogenesis. To study the relationships between the COX- expression and degree of vascularization, as well as to evaluate their role in the prognosis of patients with gallbladder carcinoma.  cases of gallbladder adenocarcinoma were included, classifi ed grading I-III according the WHO classifi cation. Th e COX- and endothelial antigen CD expressions were assessed immunohistochemically. COX- expression was evaluated according to the percentage and staining intensity of positive cells into “COX- positive” and “COX- negative” groups. In order to assess tumor microvessel density (MVD), CD positively stained microvessels were counted for each specimen in predominantly vascular areas (hot spots) at x magnifi cation. Th e MVD ranged from  to  microvessels/fi eld.  tumors belonged to the hypervascular group (MVD ≥ ) and  to the hypovascular group. Th ere were  (,) COX- positive cases. Th ere was diff erence in the degree of angiogenesis between COX- positive vs. COX- negative group:  (,) out of  “COX- positive” tumors were hypervascular, in comparison with just  (,) of “COX negative” tumors. Our data show that the MVD corresponds to the COX- overexpression in gallbladder carcinomas. Augmented tumor neovascularization induced by COX- might be responsible for the poor prognosis in gallbladder carcinoma patients.


Introduction
Carcinoma of the gallbladder is an agressive tumor with an overall -year survival rate of less than .Activation of the angiogenic pathways in malignant tumors is common and, by inducing new blood vessel formation, may have a decisive role in determining local invasion, metastasis and clinical outcome (, ).CD (endoglin) is a new endothelial cell marker, to visualize neovasculature and to count the intratumor microvessel density (MVD).CD is only weakly expressed in normal tissues, but strongly in tumor endothelia (, ).It is superior to other panendothelial markers because it distinguishes between newborn tumor vessels and preexsisting parenteral vessels, detecting only tumor neoangiogenesis (, ).Expression of the COX- enzyme is responsible for enhanced tumor growth and angiogenesis in various tumours (-), having the role both in early carcinogenesis as well as in angiogenesis.Th e role of COX- in the process of carcinogenesis has been dramatically emphasized by confirmation that non-steroidal antiinflammatory drugs decrease the number of colonic polypes in humans with adenomatous polyposis and lower the incidency of colorectal cancer ().Data on gallbladder carcinoma biology are scarce (, , ).Enhanced expression of cyclooxygenase- (COX-) was found in precancerous lesions and in gallbladder carcinoma ().Th e aim of our recent study was to investigate the relationships between the expression of COX- and angiogenesis in gallbladder carcinoma as well as to evaluate their role in the prognosis of patients with gallbladder carcinoma.

Materials and Methods
We performed a retrospective study of  gallbladder adenocarcinoma tissue specimens obtained from patients operatively treated between  and  (Table ).IMMUNOHISTOCHEMISTRY COX- and CD expressions were investigated by immunohistochemistry. Formalin-fixed, paraffin-embedded specimens were sectioned in series at a thickness of  μm.After deparafi nisation, the slides were immersed for  min in , hydrogen peroxide/methanol to deplete endogenous peroxidase.Antigen retrieval was achieved using the pressure-cooking method for  min in a citrate buff er (pH= ,).For COX- expression, polyclonal anti-human COX- antibody (Cayman Chemicals, Ann Arbor, MI) was used in a dilution :.For CD expression, the monoclonal mouse anti-human CD antibody (Novocastra Neomarkers, United Kingdom) in a dilution : was used.After the primary antibody incubation, the specimens were treated with a biotinylated secondary antibody for  min.Antigen visualisation was achieved by applying a standard streptavidin-biotin complex (ABC, DAKO, Denmark) for  minutes, followed by diaminobenzidin chromogen (DAB, Sigma Chemical CO, Germany) in , HO PBS solution.The specimens were counterstained with haematoxylin.Th e specifi city of the applied antibodies was checked with positive or negative controls.
VESSEL ENUMERATION All sections were stained immunohistochemically with CD and were examined using a Nikon Eclipse E microscope.Th e slide was searched for the hot spots rich in vessels, located in the area of tumor tissue under a low power microscope.Images were captured using a Nikon digital camera and analysed with LUCIA measurement software package.MVD was counted under a x magnification, according to the standards that any stained endothelial cell or cells were identified as an independent vessel.These vessels have to be clearly separated from each other.Ten different vision fields were chosen on each tu-mour sample, and the stained vessels were counted by two observers separately.The results were averaged, the value was the amount of vessels per unit area.

COX- ANALYSIS
Immunostaining was independently evaluated by two investigators (AC and ML) and a consensus agreement was achieved.COX- was evaluated according to the percentage ( to  degree; : , : <, : -, : -, : >) and staining intensity (-; : negative, : weak, : moderate, : strong) of positive cells.For the immunoreactive score (IRS), the percentage of positive cells and staining intensity were multiplied, resulting in a value between  and .We united samples with an IRS of  to  into the group with negative to weak COX- expression (the COX- negative group) and those with an IRS - (the COX- positive group).

Results
The positive expression of CD was presented as brownish granules in a cytoplasm of vascular endothelial cells.The microvessels were heterogeneouslly distributed in malignant tissues.In our gallbladder carcinoma tumors, MVD ranged from  to  microvessels per x optical fi eld.Th e mean MVD was  ±  (mean ± SD).We classifi ed tumors into hypovascular (MVD < ) and hypervascular group (MVD ≥ ):  tumors belonged to the hypovascular (Figure ) and  to the hypervascular group (Figure ).COX- was located as brownish-yellow stained granules in a cytoplasm of tumor cells (Figure ).Th ere were  (,) COX- positive and  (,) negative cases.Th e expression rate of high MVD in  cases of "COX- positive" gallbladder carcinoma was  (,) in com-parison with only  (,) out of  "COX- negative" cases (Graph ).Tumor grading was observed in hypovascular vs. hypervascular group of tumors (Graph ): tumor grade was insignifi cantly higher (Table ) in hypervascular group ( out of  poorly diff erentiated tumors had high MVD whereas the majority of grade I tumors were hypovascular).A correlation was observed between the COX- expression and tumor grade:  out  poorly diff erentiated (grade III) gallbladder carcinomas were COX- positive (Graph ); however, the statistical diff erence in COX- expression regarding tumor grading was non-signifi cant (p= ,).Th e COX- expression as well as MVD did not correlate with age or gender.The mean survival time in our patients was  ±  months (it diff ered in patients with hypovascular vs. hypervascular tumor, Table ).Only  patient is still alive,  patients dropped out of medical control (alive?).Th ese  patients tumors belonged to the hypovascular group.

Conclusion
Our study presented on a small group of patients with gallbladder carcinomas shows a clear association between COX- overexpression and neovascularization.Augmented tumor blood vessels induced by COX- might be responsible for the poor prognosis of human gallbladder carcinomas.

Discussion
Angiogenesis, the formation of new blood vessels within and adjacent to a tumor, is essential for tumor growth by providing nutrients and eliminating metabolic waste products.The quantity of microvessels identified by anti-CD monoclonal antibodies is nowadays an ideal instrument to quantify new microvessels in tumors; being specifi c to angiogenic tissues and as such superior to panendothelial markers such as CD, CD, F etc. (, ).CD is a proliferation-associated endothelial marker, a new kind of adhesion molecule, a receptor that is strongly up-regulated in proliferating endothelial cells (also in tumor neovascualture) (, ).Immunohistochemical staining for CD is very selective for the blood vessel endothelium and reacts specifi cally with endothelial cells without signifi cant cross-reactivity of infl ammatory or stromal cells within the neoplasm (, ).The extent of angiogenesis varied considerably in our gallbladder carcinomas.Dividing tumors into hypovascular and hypervascular groups showed that the grade III carcinoma were commonly hypervascular, whereas grade I carcinoma usually belonged to the hypovascular group (p= ,, Table ).Our results confi rm the hypothesis that COX- plays an important role in tumor angiogenesis.COX- is an isoform of enzyme cyclooxygenase (COX), a prostaglandin (PG) synthase that catalyzes rate limiting steps in the production of PG and tromboxanes.Since another isoform COX- is constitutively expressed in most mammalian tissues and responsible for normal kidney and platelet function and for the maintenance of gastrointestinal mucosa, COX- is not detected in most normal tissues ().It is induced by mitogenic and infl ammatory stimuli, which results in the enhanced synthesis of PGs in neoplastic and infl ammed tissues ().COX- is overexpressed in several premalignant and malignant conditions (), in many gastrointestinal tumors, including gallbladder carcinoma (, ).Th ere are many COX- signalling pathways that promote tumorigenesis.Our opinion is that probably the most important role of COX- in tumorigenicity is enhancing angiogenesis of the tumor ().COX- (induced by hypoxia) appears to play a key role in the release of proangiogenic proteins, such as prostaglandin E, basic fi broblast growth factor, nitric oxide and VEGF, thus stimulating endothelial cell migration and angiogenesis in vivo ().Our results show a tendency that high COX- expression in gallbladder carcinomas is related to hypervascularity of the tumor.On the other hand, hypovascular tumors were more likely to be COX- negative.Our fi nding is in accordance with the study of Zhi et al. (), who found MVD and VEGF positive rate in the COX- positive gallbladder carcinoma tissue higher than that in the COX- negative tissues.We believe that the fi nding of possible positive association between COX- and angiogenesis, also in gallbladder carcinomas, is very important since therapeutic targeting of COX- with COX- selective inhibitors is a possible future direction for fi ghting not only in early carcinogenesis (), but also tumor angiogenesis ().Gallbladder carcinoma is by now a tumor with a weak prognosis, dependent mostly on surgical treatment, with no relevant guides for the use of additional pharmaceutical therapy.Angiogenesis has been proposed as a prognostic marker in a variety of human neoplasms, including gastrointestinal (, ).Only a few studies investigated the clinical implications of tumor neoangiogenesis in gallbladder carcinomas (, , ).Some studies reported MVD as an independent prognostic factor for gallbladder carcinomas (), others failed to fi nd such a correlation ().Our results suggest that neovascularization may refl ect the malignant potential of gallbladder tumors since hypervascular tumours predicted a (insignifi cantly) shorter survival time.However, we are aware that our study lacks other parameters for prognosis such as the general condition of the patient, lymph node involvement, presence of the metastasis at the operation.