胸水脱落细胞DNA含量及VEGF、p53表达对肺癌胸腔积液治疗和预后的预测

目的:探讨胸水细胞的DNA倍体以及VEGF、p53表达与恶性胸腔积液治疗疗效的关系和对湿性肺癌预后的预测价值。方法:随访43例恶性胸腔积液患者的生存期,39例用图像细胞光度技术(ICM)检测DNA含量,免疫组化Envision法检测VEGF、p53,29例经胸腔引流术后胸腔内药物治疗。结果:DNA异倍体与胸水治疗的疗效有相关趋势(P=0.054)。经Cox多因素分析,仅p53是肺癌胸腔积液患者影响预后的独立因素(P=0.05)。p53阴性组与p53阳性组的中位生存期分别为(10.4±3.5)个月,(2.8±0.6)个月(logrank=0.0132)。一年生存率p53阴性组与阳性组分别为17.7%和0。结论:ICM检测胸水细胞的DNA倍体与胸水治疗的疗效有相关趋势。免疫组化检测p53能预测肺癌胸腔积液患者的预后。     

流式细胞术用于良恶性胸腔积液鉴别诊断的研究--DNA倍体分析和突变型p53蛋白检测

目的研究流式细胞术DNA倍体和细胞周期分析、突变型p53检测用于良恶性胸腔积液鉴别诊断的临床可行性.方法 24例标本(12例恶性,12例良性)采用流式细胞术细胞内抗原检测的直接免疫荧光标记法检测胸腔积液细胞中表达突变型p53的阳性细胞百分率和p53表达的平均荧光强度,并且通过PI染色分析细胞倍型和周期分布.结果流式细胞术DNA倍体分析用于恶性胸腔积液诊断的敏感性为66.7%,特异性100%.倍体分析和细胞学检查联合应用能将诊断的敏感性提高到100%,特异性仍为100%.良性胸腔积液和恶性胸腔积液p53表达有显著性差异.以p53阳性细胞百分率＞90%为阳性标准,p53用于良恶性胸腔积液鉴别诊断的敏感性和特异性分别为83.3%和91.7%.联合p53阳性细胞百分率和DNA倍体分析对恶性胸腔积液诊断的敏感性能提高到91.7%,特异性91.7%.细胞周期SPF(S期时相百分比)在良恶性胸腔积液之间没有显著差异.结论流式细胞术DNA倍体分析和突变型p53检测可以作为临床良恶性胸腔积液鉴别诊断的辅助手段,结合细胞学检查更有意义.     

DNA倍体联合肿瘤标志物检测在胸腔积液诊断中的应用

目的探讨DNA倍体分析联合肿瘤标志物在良、恶性胸腔积液诊断中的价值。方法将108例胸腔积液分为恶性组（68例）和良性组（40例）。除常规细胞学检查外,以流式细胞术（flowcytometry,FCM）检测患者胸腔积液中的DNA倍体,采用化学发光法测定胸腔积液中CEA、CA199、NSE、CYFRA211、SCC、CA125等肿瘤标志物含量。比较DNA倍体联合肿瘤标志物诊断与细胞学诊断的优劣。结果DNA倍体诊断恶性胸腔积液的敏感性、特异性分别为70．6％、95．0％,Youden’s指数为0．656,敏感度稍高于细胞学诊断的65．1％,差异无统计学意义（P〉0．05）。除NSE外,其他5种肿瘤标志物在恶性胸腔积液中浓度均高于良性,差异有统计学意义（P〈0．05）。CYFRA211、CEA、CAl99、CAl25、SCC、NSE的AUC分别为：0．893,0．828,0．759,0．691,0．524及0．490;COV分别为：149．2ng／mL,53．6ng／mL,78．2IU／mL,1559．0IU／mL,48．72ng／mL及78．3ng／mL;敏感性分别为：44．1％,44．1％,35．3％,29．4％,13．2％,5．9％,特异性均为100％。4种肿瘤标志物联合检测＋DNA倍体检测的敏感性为88．2％（60／68）,特异性95％,显著高于细胞学诊断。结论DNA倍体联合CEA、CA199、CYFRA211和CA125检测诊断恶性胸腔积液有较高敏感性,具有定量、快速、价廉、易标准化的特点,且操作简单。     

联合检测EGFR、CEA对恶性胸腔积液的诊断价值

目的:评价联合检测胸腔积液患者的胸水细胞块表皮生长因子受体（epidermal growth factor receptor,EGFR）基因拷贝数,以及胸水、血清CEA水平对良恶性胸腔积液鉴别诊断的价值。方法:应用荧光原位杂交技术（Fish法）检测恶性胸腔积液（n=35）、良性胸腔积液（n=30)组患者胸水细胞块EGFR基因拷贝数水平。采用电化学发光全自动生化分析仪检测胸水及血清中CEA水平,根据受试者工作特性曲线（ROC）选取最佳灵敏性和特异性的点作为临界值,评价CEA及联合检测EGFR基因拷贝数对良恶性胸腔积液的诊断价值。结果:35例恶性胸腔积液完成Fish检测。恶性胸腔积液中15例阴性,20例阳性,阳性率为57.1％。其中13例为EGFR基因高度多体性,7例为EGFR基因扩增。肺腺癌16例中,EGFR基因高度多体性、扩增14例,肺腺癌扩增率为87.5％;肺鳞癌14例中,EGFR基因簇状扩增3例(21.4％),点状扩增3例(21.4％),无扩增8例(57.1％),肺鳞癌扩增率为42.9％。腺癌Fish阳性率(87.5％)高于鳞癌(42.9％）,P<0．01。30例良性胸腔积液中有1例脓胸患者EGFR Fish检测阳性,阳性率为3.3%,余检测结果均阴性。恶性胸腔积液患者胸水及血清CEA分别为（220.9±71.65）ng/ml、(18.11±11.38）ng/ml,显著高于良性胸腔积液组（2.31±1.29)ng/ml、(1.67±1.06）ng/ml,差异有统计学意义（P<0.01）。其中,恶性胸腔积液胸水CEA明显高于血清CEA,而良性胸腔积液组中,胸水CEA与血清CEA无明显差异。肺腺癌所致胸水及血清CEA分别为（441.02±102.65)ng/ml、(32.87±28.66）ng/ml,鳞癌所致胸水及血清CEA分别为（28.75±21.39）ng/ml、（5.99±5.32）ng/ml,腺癌显著高于鳞癌,差异有统计学意义（P<0.01）。比较胸水EGFR、CEA对良恶性胸腔积液诊断的效能,两者之间无明显差异（P=0.453＞0.05）。Spearman相关性分析胸水EGFR同CEA之间存在显著正相关。结论:EGFR在恶性胸腔积液的形成中起重要作用,通过Fish技术检测胸水细胞块EGFR基因拷贝数可行,其敏感性为57.1%。对肺腺癌导致恶性胸腔积液的诊断敏感性为87.5%。CEA（临界值5.0ng/ml）在恶性胸腔积液及血清中显著高于良性,其中胸水CEA检测诊断敏感性为65.7%,而在腺癌中为87.5%,其在胸水及血清中的比值＞1.5有助于恶性胸腔积液的诊断。EGFR基因突变阳性与肿瘤标记物CEA阳性表达呈正相关,尤见于肺腺癌患者,两者联合检测可提高诊断性试验的准确性。     

细胞角蛋白19片断与癌胚抗原联合检测对良恶性胸腔积液的诊断价值

目的：探讨角蛋白19片断(CYFRRA21．1)与癌胚抗原(CEA)的联合检测对良恶性胸腔积液的鉴别诊断价值。方法：采用放射免疫分析法对40例恶性胸腔积液组、32例良性胸腔积液组，分别测定其血清及胸液中CYFRA21．1和CEA浓度。结果：恶性胸腔积液组血清及胸液CYFRA21．1和CEA浓度均显著高于良性胸腔积液组。结论：联合检测CYFRA21-与CEA有利于良恶性胸液的鉴别诊断。     

CK19mRNA和CK19蛋白联合检测在恶性胸腔积液诊断中的价值

[目的]探讨联合检测细胞角蛋白19（CK19）mRNA和CK19蛋白在恶性胸腔积液患者诊断中的价值。[方法]32例恶性胸腔积液患者和30例良性胸腔积液患者的胸水标本,以定量逆转录-聚合酶链反应法（RT—PCR）测定胸液CK19mRNA和酶联免疫吸附法（ELISA）测定胸液CK19蛋白。[结果]恶性良性胸腔积液组CK19mRNA的阳性率分别为62．5％和16．7％：恶性良性胸液组CK19蛋白的阳性率为43．8％和10．0％（P〈0．01）。联合检测胸液CK19mRNA和CK19蛋白诊断恶性胸腔积液敏感性和准确性分别为81.3％和79．0％。[结论]CK19mRNA和CK19蛋白联合检测可提高恶性胸腔积液诊断的准确性．对良恶性胸腔积液的鉴别诊断有重要参考价值。     

血管内皮生长因子和癌胚抗原测定对良、恶性胸腔积液诊断价值的对比研究

目的 研究胸腔积液血管内皮生长因子（VEGF）对良，恶性胸腔积液鉴别诊断的价值。并与癌胚抗原（CEA）进行对比。方法 采用双抗体夹心法检测胸腔积液VEGF和CEA的水平。根据最终诊断结果。30名患者分为两组：（1）良性胸腔积液组16例；（2）恶性胸腔积液组14例，并将VEGF检测结果与CEA水平进行对比。结果 恶性胸腔积液组有13例患者的VEGF呈高活性（D值118-482pg/L)。结论 胸腔积液VEGF的检测对鉴别良，恶性胸腔积液明显优于CEA的测定，可作为一种诊断恶性胸腔积液的有力手段。     

应用ROC曲线评价肿瘤标志物对恶性胸腔积液的诊断价值

目的：探讨癌胚抗原（CEA）、细胞角蛋白（CYRFA21—1）、神经元烯醇化酶（NSE）单独和联合检测在良、恶性胸腔积液中的鉴别诊断价值。方法：应用受试者工作曲线（ROC）对恶性31例及良性30例胸水的CEA、CYRFA21—1、NSE进行分析。结果：恶性胸水CEA、CYRFA21—1和NSE浓度较良性明显增高（P〈0．05）;CEA、NSE、CYRFA21—1单独检测AUC分别为0．831、0．747、0．900,灵敏度分别为67．7％、80．6％、74．2％,特异度分别为96．7％、73．3％、86．7％;NSE联合CEA、NSE联合CYRFA21—1、CEA联合CYRFA21—1检测的AUC分别为0．887、0．916、0．978,灵敏度分别为64．5％、74．2％、93．5％,特异度分别为100．0％、96．7％、90．0％;三项联合检测的AUC为0．985,灵敏度为96．8％,特异度为66．7％。结论：检测胸水中CEA、CYRFA21—1和NSE对良、恶性胸水鉴别均有诊断意义。三项联合检测对恶性胸水诊断准确性高于二项联合检测及单独检测。     

胸水CA125、CA199、CEA、NSE、CYFRA21-1、CA72-4对原发性肺癌的诊断价值

目的探讨胸水中糖链抗原125（CA125）、糖链抗原199（CA199）、癌胚抗原（CEA）、神经元特异性烯醇化酶（NSE）、细胞角蛋白19片段（CYFRA21—1）和糖链抗原72-4（CA72-4）在原发性肺癌并胸腔积液的诊断和鉴别诊断、病理分型中的价值。方法采用电化学免疫荧光发光法同时检测90例原发性肺癌并胸腔积液患者（恶性胸腔积液组）和64例良性胸腔积液患者（良性胸腔积液组）胸水中CA125、CA199、CEA、NSE、CYFRA21-1和CA72-4水平。结果恶性胸腔积液组各胸水肿瘤标志物水平均高于良性胸腔积液组（P〈0．05）,其中CEA、CYFRA21-1、NSE分别对腺癌、鳞癌、小细胞肺癌最敏感。联合检测以CEA＋NSE＋CYFRA21-1最优,可使敏感性达98．9％,阴性预测值至96．6％,准确性提高至76．0％。结论胸水肿瘤标志物在原发性肺癌的诊断中价值较高,其中CEA的诊断价值最大,联合检测诊断准确性优于单项检测。     

P53-immunoreactive cells in benign and malignant effusions: diagnostic value using a panel of monoclonal antibodies and comparison with CEA-staining

The diagnosis of malignancy in peritoneal and pleural effusions can be difficult, because activated mesothelial cells may resemble malignant cells. P53 mutations are the most frequent genetic changes in human cancer and translate into an overexpression of the p53 protein detectable by immunocytochemistry. In this study, we investigated the sensitivity and specificity of 4 different monoclonal antibodies (moAB) against p53 for the diagnosis of malignancy in effusions. We also compared p53 staining with CEA immunocytochemistry which previous work had established as a specific marker of malignancy in body cavity effusions. Normal and malignant cells from 28 benign and 52 malignant effusions (pleura and ascites) were examined by indirect immuno-alkaline phosphatase method. Four different moAB against p53 (PAB 1801, PAB 240, DO-1 and DO-7) and one moAB against CEA (CEA-84) were used. Antibodies p1801, p240 and DO-1 react with 52-75% of cases of effusions with malignant cells, but also with 38-80% of benign effusions. Only the antibody DO-7 and the CEA moAB show specificity for malignant cells reacting respectively with 20 (55%) of cases. A combination of these 2 markers does not enhance the sensitivity for the detection of tumor cells. No direct correlation between CEA and p53 immunostaining could be established. The sensitivity and specificity of staining p53 in malignant cells by immunocytochemistry depend strongly on the antibody used. Some p53 moAB are positive with reactive cells in ascites and pleural effusions. Currently, p53 staining of expressing cells does not improve the identification of malignant cells in comparison with CEA immunocytochemistry, but may help to screen for patients with p53 mutations.     

Potential diagnostic value of methylation profile in pleural fluid and serum from cancer patients with pleural effusion

BACKGROUND: The objective of this study was to investigate the diagnostic value of methylation profiles for discrimination between malignant and benign pleural effusions. A secondary objective was to examine the concordance of methylation in samples of serum and pleural fluid. METHODS: The authors used methylation-specific polymerase chain reaction (MSP) analysis to examine the promoter methylation status of 4 genes in patients with pleural effusion: death-associated protein kinase (DAPK), Ras association domain family 1A (RASSF1A), retinoic acid receptor beta (RARbeta), and p16/INK4a. Pleural effusions were collected from 87 patients who had their diagnoses confirmed on cytologic and/or histologic examinations and clinical evolution. Pleural effusions were classified as malignant (n = 53 patients) or benign (n = 34 patients). RESULTS: Methylation was detected in serum from 45.3% of patients with malignant pleural effusions and from 0% of patients with benign pleural effusions, and it was detected in pleural fluid samples from 58.5% of patients with malignant pleural effusions and from 0% of patients with benign pleural effusions (P = .001). The sensitivity of MSP was greater than that of cytologic examination alone (39.1%; P = .001). When MSP was used together with cytologic examination, sensitivity increased to 69.8% (P = .001). CONCLUSIONS: Cell-free methylated DNA in pleural fluid can be detected in patients with neoplastic malignancy in a single extraction by thoracocentesis. Adequate management of the extracted pleural fluid can provide a rapid and reliable diagnosis in patients with pleural effusions who have suspected malignancy. MSP, used together with cytologic examination, may obviate the need for other invasive diagnostic tests.     

胸腔积液中肿瘤坏死因子、癌胚抗原和神经元特异性烯醇化酶的检测及诊断价值研究

目的探讨检测胸水中肿瘤坏死因子（TNF-α）、癌胚抗原（CEA）和神经元特异性烯醇化酶（NSE）对胸腔积液的诊断价值。方法采用电化学发光酶免疫分析法检测59例结核性胸水和48例肺癌性胸水患者胸水中TNF-α、CEA和NSE水平。结果结核性胸水中TNF-α水平显著高于肺癌性胸水（P〈0.05）。肺癌性胸水中CEA和NSE明显高于结核性胸水（P〈0.01）。肺腺癌胸水中CEA升高最明显,非小细胞肺癌胸水中NSE升高最显著。联合检测CEA及NSE,诊断敏感度92.0%,准确度86.3%。结论检测TNF-α、CEA和NSE对结核性胸水和肺癌性胸水的诊断及鉴别诊断有较高的临床价值,联合检测胸水CEA和NSE可提高肺癌诊断敏感度。     

联合检测p53,ras,c—myc癌基因蛋白对恶性胸腔积液诊断价值的研究

目的：为了早期、快速、准确地诊断恶性胸腔积液。方法：用免疫组织化学ＡＢＣ法联合检测了２７例恶性胸腔积液和良性胸腔积液细胞中的ｐ５３、ｒａｓ、ｃ－ｍｙｃ癌基因蛋白的表达水平，恶性胸腔积液同时还与常用的四种诊断方法包括乳酸脱氢酶、癌胚抗原、细胞学、胸膜活检相对照。结果：恶性胸腔积液上述三种癌基因蛋白联合表达的敏感性为８５．３％，特异性为１００％，明显高于常用方法，而良性胸腔积液上述三种癌基因蛋白均为阴性表达。结论：联合检测上述三种癌基因蛋白为诊断恶性胸腔积液提供一条新的可靠途径。     

Microsatellite DNA analysis does not distinguish malignant from benign pleural effusions

Distinguishing malignant from benign pleural effusions using routine cytology is a common diagnostic problem. Recently, genetic alterations, including microsatellite instability (MSI) and loss of heterozygosity (LOH), have been described in malignant pleural effusions and proposed as methods improving diagnostics. The purpose of this study was to evaluate a panel of molecular markers for the detection of genetic alterations of cells in pleural effusions and to determine their diagnostic value as an additional test to cytologic examination. Pleural fluid and peripheral blood from 48 patients (36 male and 12 female, median age 71 years) were analyzed. Twenty-six patients had malignant pleural effusion, including 23 lung cancer and three metastatic non-pulmonary carcinoma. The control group consisted of 22 patients with benign pleural effusions. Only 14 malignancy-associated pleural effusions were cytology-positive for malignant cells (54%), whereas all benign pleural effusions were negative. DNA was extracted from all the samples and analysed for MSI and/or LOH using the following microsatellite markers: D3S1234, D9S171, D12S363, D17S250, D5S346 and TP53Alu, located at five chromosomal regions: 3p, 9p, 12q, 17q, 5q. Microsatellite analysis of the pleural fluid pellet exhibited genetic alterations in two neoplastic pleural fluid cases and in one inflammatory case. Two out of 26 (7.6%) patients with malignant pleural effusion showed genetic alterations. One exhibited MSI in three different microsatellite markers (D17S250, D9S171, D3S134) and the other showed LOH in marker D3S134. One out of 22 (4.5%) patients with benign pleural effusion showed LOH in marker D3S134. In conclusion, genetic alterations at the level of microsatellite DNA, were detected only in very few cases of malignant pleural effusions, and in one case of benign pleural effusion. Thus, our data suggest that microsatellite DNA analysis does not facilitate the diagnosis of malignant pleural effusion.     

流式细胞术在恶性胸腔积液诊断中的价值

目的 探讨胸腔积液脱落细胞ＤＮＡ定量分析对恶性胸腔积液的诊断价值。方法 采用流式细胞仪（ＦＣＭ）对７２例恶性胸腔积液和５８例良性胸腔积液做ＤＮＡ定量分析,并同进与胸膜活检病理诊断,人积液细胞学诊断及胸腔积液ＣＥＡ测定等方法相比较。结果 以异倍体一为标准,ＦＣＭ诊断恶性胸腔积液的敏感性为８０．６％,特异性为１００．０．％,与胸膜活检病理诊断（８６．１％）。无显著性差异（Ｐ〉０．０５）,但显著高于胸腔     

New approaches in the diagnostic procedure of malignant pleural effusions

The content of carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (Ca 19-9), carbohydrate antigen 15-3 (Ca 15-3) and the expression of LewisY related carbohydrate antigens in benign and malignant pleural effusion were determined. These included 35 malignant pleural effusions: 13 breast cancers, 12 lung cancers (6 squamous cell carcinomas, 5 adenocarcinomas and 1 microcytoma), 2 mesotheliomas, 1 epithelioma, 1 kidney cancer, 1 hepatocarcinoma, 1 colon carcinoma, 3 lymphomas, 1 osteosarcoma and 9 benign pleural effusions. We showed that pleural fluid content of CEA, Ca 19-9 and Ca 15-3 were higher in malignant than in benign effusions. However CEA levels in squamous lung cancers were very high in both serum and pleural fluids whereas its levels were only slightly above the cut-off in breast cancers and in lung adenocarcinomas. Serum and pleural fluid Ca 15-3 values were higher in breast and in lung cancers with the highest values in the patients with breast cancer. Furthermore, the LewisY related carbohydrate antigens, evaluated by the reactivity of the cell extracts to MAb B3, were expressed only in breast cancers. These data suggest that pleural fluid content of CEA, and Ca 15-3 associated with the immunoblotting of cell extracts with MAb B3 appear to be very useful to improve the diagnosis of malignant pleural effusions.