Expression And Clinical Significance Of COX-2and Heparanase-1in Adrenal Tumors

Background:In recent years, With the continuous development of medical science technology, especially in imaging (CT, MRI, etc.) and biochemical detection methods of improved and people’s healthy check-up awareness of continuous improvement, the rate of diagnosis of adrenal tumors is increasing year by year. Adrenal tumors including adrenocortical tumors and adrenal medullary tumors.The adrenocortical tumors contain adrenal cortical adenoma and adrenal cortical carcinoma.The adrenal medullary tumors are divided into benign pheochromocytoma, malignant pheochromocytoma and paraganglioma.Among the adrenocortical tumors, adrenal cortical adenoma has a good prognosis and can be cured by surgery,but the adrenal cortical carcinoma has a higher degree of malignancy, invasive strong, early diagnosis and its treatment is still radical surgery-based,while lacks of effective adjuvant therapy, especially in the early stage it can be transfer to the lung, lymph nodes, liver, bone, etc. its annual incidence of approximately4to12per million population,5-year Survival<35%.According to the organizational structure and morphology of the adrenal cortical carcinoma and adrenocortical adenoma is similar, so the identification of the benign or malignant adrenocortical tumors is difficult, and sometimes need to combine their clinical manifestations, gross specimens, imaging and microscopic histopathological morphology and immunohistochemistry to make a comprehensive judgment. At present the pathogenesis of adrenocortical tumors is not clear,sometimes only according to the histopathological examination is difficult to determine the degree of malignancy, thus a lot of hardship can bring to the treatment.At present the pathogenesis of Pheochromocytoma is still unclear. So far the diagnosis of malignant adrenal pheochromocytoma is still according to the rumor invasion of adjacent organs or metastasis to the without chromaffin tissue of chromaffin cells.The degree of Tumor cell differentiation, such as filamentous mitotic activity, nucleic acid polymorphism and so on,which are not apply to used to distinguish between benign and malignant pheochromocytoma. Malignant pheochromocytoma tumor diagnostic pathology and imaging are lack of specific index, the recognized gold standard for the Malignant pheochromocytoma is appear the metastases in the area without pheochromocytoma.From the clinical manifestations, imaging findings and histopathologic features to distinguish between benign and malignant pheochromocytoma are very difficult.At present,there are no reliable histological or biological markers can be used to distinguish between benign and malignant pheochromocytoma.COX-2(cyclooxygenese-2) is also known as prostaglandin peroxide synthase,which is an important rate-limiting enzyme in prostaglandin (PG) synthesis process and can be metabolized arachidonic acid to the product of a variety of prostaglandins, thus play a role in physiological and pathological processes in the body.In addition,through by the promotion of cell proliferation, inhibit apoptosis, promote tumor angiogenesis, promoting tumor cell infiltration pathways,which is involved in tumorigenesis and development process.Heparanase-1is an endo-glucuronide cut enzymes, which can role in the extracellular matrix and basement membrane,and in a particular site of cracking the sulfate heparan proteoglycans. HSPGs are widely exist in various tissues surface, cell surface, extracellular matrix(ECM) and basement membrane (BM), also are the main component of glycosaminoglycans for the ECM and BM composition. Tumor metastasis must be the two most important steps of angiogenesis, as well as through the basement membrane and extracellular matrix. At the tumor status, the Heparanase-1(HPA-1) has a important role in the promotion of tumor cell invasion, metastasis and angiogenesis.COX-2and Heparanase-1is the the hot spot of the research in recent years,and many researches have indicated that they are high expression in most human malignant tumors,such as head and neck malignancy, breast cancer, colon cancer, and stomach cancer, melanoma and prostate cancer,, bladder cancer, kidney cancer, etc. They are associated with the tumor invasion, tumor angiogenesis and prognosis, However, about the possible mechanism of the COX-2and Heparanase-1in the development of the adrenocortical tumors, and if they can be reliable biological indicators for early distinguishing between the benign and malignant of the adrenal cortical tumors and estimating the prognosis, at present the domestic and foreign have not been reported. In addition,about the expression and correlation of COX-2and Heparanase-1in the adrenal pheochromocytoma is rare reported in the domestic and foreign, pending further research and discussion.Objective:1.To investigate the expression and clinical significance of COX-2and Heparanase-1in the normal adrenal gland, adrenal cortical adenoma and adrenal cortical carcinoma;2.To investigate the expression and clinical significance of COX-2and Heparanase-1in the normal adrenal gland, adrenal benign pheochromocytoma, adrenal malignant pheochromocytoma;3.To investigate the expression of COX-2and Heparanase-1in the development of adrenal tumors, and to understand whether they can become a reliable biological indicators of early identifying adrenal benign and malignant tumors, and estimating the prognosis,thus to provide a new means of the clinical diagnosis and treatment for adrenal tumors.Methods:1.47cases of adrenal cortex tumors specimens resected from patients diagnosed as adrenal cortex tumors from Guangzhou General Hospital of Guangzhou Command from Apr.1990to Dec.2010,who have surgical treatments complete clinical,pathological and follow-up data. Adrenal cortex tumors were divided into two groups(33cases of adrenal cortical adenoma and14cases of adrenal cortical carcinoma). All tissue samples were uniform10%formaldehyde solution-fixed, paraffin-embedded tissue section thickness is2.5μm. In addition,10cases of normal adrenal tissue collected from healthy kidney transplant donor as control group. Adrenocortical adenoma group male13cases,20cases of women; adrenocortical carcinoma group,10males and4females. The follow-up time of126days to2319days.2.32cases of adrenal pheochromocytoma specimens resected from patients diagnosed as adrenal pheochromocytoma from Guangzhou General Hospital of Guangzhou Command from Apr.1990to Dec.2010,who have surgical treatment,a complete clinical,pathological and follow-up data, adrenal pheochromocytoma were divided into two groups(26cases of adrenal benign pheochromocytoma and6cases of adrenal malignant pheochromocytoma). All tissue samples were uniform10%formaldehyde solution-fixed, paraffin-embedded tissue section thickness is2.5μm. In addition,10cases of normal adrenal tissue collected from healthy kidney transplant donor as control group. In the adrenal benign pheochromocytoma group(16cases of males and10cases of females),and the adrenal malignant pheochromocytoma group(3cases of males and3cases of females).The follow-up time of403days to3721days.3. To detected the expression of COX-2and Heparanase-1in the adrenal tumors and normal adrenal glands by the immunohistochemistry NovolinkTMPolymer method. In this study, instead of primary antibody with phosphate buffered saline (PBS) as negative control, staining positive expression of gastric biopsy specimens known as COX-2, Heparanase-1positive control, best of COX-2, Heparanase-1diluted concentration of1:200(thinner as the primary antibody diluent).4. The result judgement:The expression of COX-2and Heparanase-1are located in the cell plasma,the positive expression is light yellow,tan and brown particles. In the exclusion of nonspecific dyeing premise,to independently observe slice and assess the results of the immunohistochemistry by two experienced pathologists who don’t know the clinical and pathological material Immunohistochemical results according to the coloring number of positive cells and staining intensity to respectively score.Evaluation method using semi-quantitative integral method to judge the staining results:randomly selected5high power field to count the number of positive cells0-5%and0;6-25%points;26-50%within2 points;51-75%per3points;76-100%and4; positive expression intensity:no staining,0points, yellow,1point; brown yellow scoring2; tan3points. Two points multiplied by integral points0, negative (-); points1-4points, weakly positive (+); points5-8, moderately positive (++); points9-12strongly positive (+++). Determine the results of a difference of3points, then re-determine.5. The use of SPSS13.0software for analysis.To compare the positive expression rate of the data with the χ2test and carry out the rate for multiple comparisons with Fisher’s exact probability method combined with Bonfferoni method,the the new test standards for α=0.05/3=0.0167; Spearmen rank correlation test the correlation between the various factors; during Cox model analysis using the log-rank test on univariate analysis, and then select the univariate analysis, statistically significant variables in the Cox proportional hazards regression analysis, the difference was statistically significant (P<0.05)Results:1. The expression of COX-2and Heparanase-1in adrenal cortical carcinoma were significantly higher than normal adrenal tissue and adrenal cortical adenoma, expression differences were statistically significant (P<0.0167).The expression of COX-2and Heparanase-1in adrenal cortical adenoma and normal adrenal tissue were no significant difference(P>0.0167).The expression of COX-2and Heparanase-1in adrenal cortical adenoma and adrenal cortical carcinoma were positive correlation(P<0.05).The expression of COX-2and Heparanase-1were related to the prognosis of patients with adrenal cortical carcinoma (P<0.05). COX-2and Heparanase-1have existed the statistical collinearity problem, and both react the same information, to use one of them can predict the prognosis of patients.The higher expression of COX-2or Heparanase-1, the worse the prognosis of patients.2. The expression of COX-2in adrenal malignant pheochromocytoma were significantly higher than adrenal benign pheochromocytoma and normal adrenal tissue, expression differences were statistically significant (P<0.0167). The expression of Heparanase-1in adrenal malignant pheochromocytoma expression was significantly higher benign adrenal pheochromocytoma, expression differences were statistically significant (P<0.067), compared with the expression of Heparanase-1 in normal adrenal, the expression of differences were not statistically significant (P>0.0167) The expression of COX-2and Heparanase-1in adrenal benignpheochromocytoma and normal adrenal tissue were no significant difference(p>0.0167).The expression of COX-2and Heparanase-1in adrenal benign pheochromocytoma and adrenal malignant pheochromocytoma were positive correlation(P<0.05). Their correlation coefficients were respectively for rs=0.677, rs=0.898(P<0.05)Conclusion:1. The high expression of COX-2and Heparanase-1are the valuable indicators for the clinical judgment of adrenocortical carcinoma prognosis.2. COX-2and Heparanase-1was highly expressed in the pheochromocytoma patients, their postoperative malignant tendency was more likely, who should be closely followed up after the surgery.3. To detected the expression of COX-2and Heparanase-1,which will contribute to early differential diagnosis of benign and malignant adrenal tumors.