| Background and objective:Pheochromocytoma is a rare tumor originating from the chromaffin cells and characterized by excessive production of catecholamines, which often leads to increased blood pressure and symptoms of catecholamine excess. Human chromogranin A(CgA), a 48kD protein comprising 439 amino acids, is an acidic protein costored and coreleased with catecholamines from chromaffin granules of normal adrenal medulla and pheochromocytoma. CgA is regarded as a useful tissue marker for a variety of neuroendocrine cells and a possible sensitive circulating marker of neuroendocrine tumors. In this study, we investigate:1) the tissue distribution of immunoreactive-CgA in pheochromocytoma, 2) the behavior of serum CgA levels in patients with pheochromocytomas compared with the levels in healthy subjects and patients with adrenocortical tumors, and 3) the correlation between serum CgA, tumor mass and CA levels to evaluate the utility of CgA in the management of pheochromocytoma, both diagnosis and and follow-up.Design and methods:This study involved 106 patients of whom 52 with pheochromocytoma and 54 with adrenalcortical tumors. In addition, 40 normal subjects were taken as controls. The primary tumor arose from the adrenal gland in 39 patients and from extra-adrenal sites in 13 patients. 41 patients with benign pheochromocytoma and 11 patients with malignant pheochromocytoma .33 benign tumors and 6 malignant tumors were adrenal, 8 benign tumors and 5 malignant tumors were extra-adrenal. Because no reliable gross or microscopic features distinguish benign from malignant pheochromocytoma, the diagnosis of malignant pheochromocytoma was based on the presence of regional or distant metastases. Blood samples were collected for measurement of serum CgA and histological data were obtained following surgical removal of tumor. We evaluated CgA in patients with pheochromocytoma, both benign and malignant before and after surgical excision , and in patients with adrenocortical tumors and healthy subjects. CgA was measured by commercial enzyme-linked immunosorbent assay(ELISA) kit. The cut-off value of 100ng/ml was selected. Tumor tissue were collected for immunohistochemical staining and Western blot analysis for the expression of CgA. Statistical calculation was performed with SPSS, P values less than 0.05 were considered statistically significant.Results:In patients with a histopathologically confirmed diagnosis of pheochromocytoma, immunohistochemistry revealed cytoplasmic immunoreactivity for CgA in all cases, and there was no expression in adrenocortical tumors . The immunoactivity in malignant pheochromocytomas were stronger than that in benign pheochromocytomas. The molecular weight of the protein band revealed by the anti-CgA antibody was determined based on the electrophoretic migration in relation to molecular weight standard bands. Immunoblotting of total proteins from pheochromocytoma with antiserum against human CgA revealed a specific 48-kD band. The mean serum CgA concentration in patients with pheochromocytomas (446. 5±197. 2ng/ml) was significantly higher (P<0.01) than those measured in normal subjects (41.6±10.7ng/ml) and in patients with adrenocortical tumors(77.3±15.6ng/ml). No difference (P>0. 05)in CgA levels was found between patients with adrenocortical tumors and healthy subjects. CgA was significantly different in benign versus malignant pheochromocytoma. Criculating CgA had a sensitivity of 86. 5%, a specificity of 94. 7%, and an accuracy of 91.8 %, respectively in diagnosis of pheochromocytoma. Both CgA and CA showed high sensitivity , specificity and diagnostic accuracy. The CgA assay also detected 4 asymptomatic nonfunctioning pheochromocytoma. A statistically significant relationship was seen between tumor mass and CgA levels(r=0. 71, P<0.001). Compares the results of CgA assay with the results of the urinary norepinephrine and epinephrine assays , a significant relationship was seen between serum levels of CgA and urinary levels of NE with a linear model (r=0. 638, P<0.001). The areas under the ROC curves were 0. 915 for CgA, 0. 930 for NE, and 0. 905 for E, these values were not significant different. In patients with pheochromocytomas the serum CgA level decreased significantly after tumor removal, from 446. 5+197. 2 ng/ml to 65.5±15.9ng/ml (P<0.001). After excision of benign pheochromocytoma, CgA fell to values near normal. In malignant pheochromocytoma, CgA fell but above the normal range. The postoperative CgA level was an early and accurate predictor of curative surgery or relapse.Conclusions:In immunohistochemistry, there was a statistically significant difference of CgA expression between adrenalcortical tumors and pheochromocytomas, also between benign and malignant pheochromocytomas. Serum CgA is a sensitive and specific marker in the diagnosis of pheochromocytoma, moreover, serum CgA levels correlated with urinary CA levels in all patients with pheochromocytomas . Increased levels strongly correlate with tumor mass and a markedly elevated CgA may suggest the diagnosis of malignant pheochromocytoma. CgA may also assist in ascertaining the surgical response in malignant disease. In the follow-up of pheochromocytoma, the serum CgA assay should be used as an alternative to urinary catecholamine measurement.
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