摘要
目的:观察嗜铬粒蛋白A(CgA)在嗜铬细胞瘤组织中的表达,检测嗜铬细胞瘤患者的血清CgA水平,评价CgA的组织免疫活性在嗜铬细胞瘤组织病理学中的应用,血清CgA浓度在嗜铬细胞瘤诊断、功能评估及术后随访中的作用。
方法:肿瘤组织标本取自北京协和医院泌尿外科手术切除的嗜铬细胞瘤52例及肾上腺皮质肿瘤54例,采用免疫组织化学方法研究CgA在肿瘤组织中的表达,并通过Western Blot方法对嗜铬细胞瘤组织内的CgA蛋白进行分离、纯化及测定。采集同组病例及40名健康对照的血清,采用酶联免疫吸附分析法(ELISA)对血清CaA水平进行检测,应用SPSS11.5软件对血清CgA水平的检测结果与肿瘤临床特性进行统计学分析。
结果:免疫组化染色在所有嗜铬细胞瘤病例中CgA均呈阳性表达,而在肾上腺皮质肿瘤组未见CgA表达。良性嗜铬细胞瘤组CgA阳性表达率为68.3%(28/41),强阳性表达率为31.7%(13/41);恶性嗜铬细胞瘤组的阳性表达率为27.3%(3/11),强阳性表达率为72.7%(8/11)。恶性组CgA的强阳性表达率明显高于良性嗜铬细胞瘤组(P＜0.05)。通过蛋白印记分析发现在嗜铬细胞瘤瘤体组织内存在CgA蛋白的表达,与蛋白标准品对比,确定其分子量为48KD。ELISA检测健康对照组血清CgA浓度为41.6±10.7ng/ml,肾上腺皮质肿瘤组为77.3±15.6 ng/ml,嗜铬细胞瘤组为446.5±197.2 ng/ml。嗜铬细胞瘤组明显高于肾上腺皮质肿瘤组及健康对照组,统计学检验有显著性差异(P＜0.01);恶性嗜铬细胞瘤组血清CgA浓度为687.3+777.1ng/ml,明显高于良性嗜铬细胞瘤组354.6±244.3 ng/ml,(P＜0.01)。血清CgA浓度用于诊断嗜铬细胞瘤的灵敏度为86.5%(45/52)、特异度为94.7%(89/94)、准确度为91.8%(134/146)、阳性预测值为90.0%(45/50)、阴性预测值为92.7%(89/96)。血清CgA浓度与肿瘤重量呈正相关(r=0.71,P＜0.001),与NE呈正相关(r=0.638,P＜0.001),与免疫组化结果呈正相关(r=0.69,P＜0.001),与肿瘤功能分级无相关(r=0.0043,P＞0.05),与患者血压、心率及E与无明显相关性(P＞0.05)。嗜铬细胞瘤患者术后血清CgA浓度由术前446.5±197.2 ng/ml下降至65.5±15.9ng/ml(P＜0.001)。其中良性组由术前254.6±244.3ng/ml下降至术后57.3±11.6ng/ml(P＜0.001)。而恶性组仅由术前687.3±777.1ng/ml下降至术后454.6±274.2 ng/ml(P＞0.05)。
结论:免疫组化检测CgA的表达,可用于诊断嗜铬细胞瘤,且强阳性表达者肿瘤为恶性的可能性大。血清CgA浓度与CgA在嗜铬细胞瘤组织中的表达强度相关,与尿NE水平、肿瘤重量呈正相关,高水平的血清CgA浓度提示肿瘤可能为恶性。良性嗜铬细胞瘤患者术后血清CgA浓度下降明显,而恶性嗜铬细胞瘤术后下降不明显。CgA在嗜铬细胞瘤诊断及术后随访中可与尿CA联合应用。
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.
引文
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