Measurement of cyclin E genomic copy number and strand length in cell-free DNA distinguish malignant versus benign effusions.

PURPOSE: Previous studies have shown that the concentration of cell-free DNA was higher and its strand length longer in body fluids obtained from patients with cancer as compared to patients with benign diseases. We hypothesized that analysis of both DNA copy number and strand length of cell-free DNA from an amplified chromosomal region could improve the diagnosis of malignant diseases in body fluids. EXPERIMENTAL DESIGN: To test this hypothesis, we used ovarian cancer effusion as an example and applied a quantitative real-time PCR to measure the relative copy number and strand length of DNA fragments from one of the most frequently amplified genes, cyclin E, in ovarian serous carcinomas. RESULTS: As compared with nonamplified chromosomal loci, including beta-actin, p53, 2p24.1, and 4p15.31, measurement of cyclin E DNA copy number (100 bp) had the best performance in distinguishing malignant (n = 88) from benign (n = 70) effusions after normalization to effusion volume or Line-1 DNA with areas under the receiver operating characteristics curve (AUC) of 0.832 and 0.847, respectively. Different DNA lengths of the cyclin E locus were further analyzed and we found that the AUC was highest by measuring the 400-bp cyclin E locus (AUC = 0.896). The AUC was improved to 0.936 when it was combined with the length integrity index as defined by the relative abundance of 400 bp cyclin E to 100 bp p53 loci. Cyclin E real-time PCR assay had a higher sensitivity (95.6%) than routine cytology examination (73.9%) and was able to diagnose false-negative cytology cases in this study. CONCLUSIONS: The above findings indicate that measurement of the DNA copy number and strand length of the cyclin E locus is a useful cancer diagnostic tool.     

Clinical evaluation of four tumor markers in malignant and benign pleural effusions.

Total sialic acid (TSA), lipid-bound sialic acid (LSA), ferritin and carcinoembryonic antigen (CEA) were evaluated in 55 patients with malignant pleural effusions and in 32 patients with benign (exudative) pleural effusions. No significant differences were found in the pleural fluid TSA, LSA and ferritin levels between malignant and benign conditions. CEA levels in malignant effusions were significantly higher than those in benign effusions (43.13 +/- 72.8 ng/ml versus 2.6 +/- 5.56 ng/ml, p less than 0.01). At a cut-off level of 5 ng/ml, 60% of the patients with cancer showed elevated pleural fluid CEA levels. The specificity and diagnostic accuracy of CEA in distinguishing malignant from benign pleural exudates were both very high (91% and 71% respectively). Therefore, of the four markers investigated, only CEA could be a valuable tool in the detection of pleural malignancy.     

Evaluation of inflammatory cytokines in malignant and benign pleural effusions

We measured the levels of inflammatory cytokines interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) in pleural effusions and serum in 65 consecutive patients: 32 with malignant pleural effusion (MPE) (group A), and 33 with inflammatory benign pleural effusion (BPE) (group B). Serum levels of 15 healthy individuals served as control. Concentrations of IL-1alpha were higher in serum compared to pleural fluid in both groups (47.1+/-33.9 vs. 25.9+/-1.7 fmol/ml, p<0.001, in group A; and 39.9+/-30.9 vs. 25.4+/-16.3 fmol/ml, p<0.02, in group B). Similarly, concentrations of IL-1beta and IL-2 were significantly higher in serum compared to pleural fluid in both groups. In contrast, IL-6, IL-8 and TNF-alpha were found at high concentration in MPE in comparison to serum IL-6: 171.8+/-60.4 vs. 7. 2+/-7 fmol/ml (p<0.001), IL-8: 1175.15+/-2385.6 vs. 285.2+/-187.2 pg/ml (p<0.05), TNF-alpha: 204.9+/-82.9 vs. 79.4+/-31.9 fmol/ml (p<0. 001). Similarly, pleural concentrations of IL-6, IL-8 and TNF-alpha were higher in BPE patients in comparison to serum IL-6: 124.3+/-56. 2 vs. 8.6+/-6.4 fmol/ml (p<0.001) IL-8: 2109.2+/-4121.5 vs. 291. 6+/-197.9 pg/ml (p<0.02), TNF-alpha: 183.8+/-28.2 vs. 86.2+/-23.9 fmol/ml (p<0.001). These data suggest that IL-6, IL-8 and TNF-alpha might be secreted locally at the site of active disease both in benign and malignant pleural effusions.     

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.     

Regulatory T cells and cytokines in malignant pleural effusions secondary to mesothelioma and carcinoma

Immunotherapy against a variety of malignancies, including pleural-based malignancies, has shown promise in animal models and early human clinical trials, but successful efforts will need to address immunosuppressive factors of the tumor and host, particularly certain cytokines and CD4(+) CD25(+) regulatory T cells (Treg). Here, we evaluated the cellular and cytokine components of malignant pleural effusions from 44 patients with previously diagnosed mesothelioma, non-small cell lung cancer (NSCLC), or breast cancer and found significant differences in the immune profile of pleural effusions secondary to mesothelioma vs. carcinoma. Although a high prevalence of functionally suppressive CD4(+) CD25(+) T cells was found in carcinomatous pleural effusions, mesothelioma pleural effusions contained significantly fewer CD4(+) CD25(+) T cells. Activated CD8(+) T cells in pleural fluid were significantly more prevalent in mesothelioma than carcinoma. However, there is clear patient-to-patient variability and occasional mesothelioma patients with high percentages of CD4(+) CD25(+) pleural effusion T cells and low percentages of CD8(+) CD25(+) pleural effusion T cells can be identified. Mesothelioma pleural effusions contained the highest concentrations of the immunosuppressive cytokine transforming growth factor (TGF)-beta. Thus, the contribution of cellular and cytokine components of immunosuppression associated with malignant pleural effusions varies by tumor histology and by the individual patient. These results have implications for the development of immunotherapy directed to the malignant pleural space, and suggest the need to tailor immunotherapy to overcome immunosuppressive mechanisms in tumor environments.     

肿瘤标志物联合检测在良恶性胸腔积液鉴别诊断中的价值

 目的　探讨胸腔积液4种肿瘤标志物联合检测在良恶性胸腔积液鉴别诊断中的价值。方法　采用电化学发光免疫法检测126例胸腔积液患者（其中恶性组52例,良性组74例）癌胚抗原（CEA）、糖类抗原125（CA125）、糖类抗原15-3（CA15-3）和细胞角蛋白片段19（CYFRA21-1）水平, 并计算上述指标单独和与CEA联合检测在诊断中的敏感度、特异度、准确度和约登指数（YI）。结果　恶性组4种肿瘤标志物水平均明显高于良性组（P＜0.01）。单项检测各种肿瘤标志物的敏感度以CA125最高（90.4 %）,特异度以CYFRA21-1最高（79.7 %）,诊断准确度以CEA和CYFRA21-1最高（71.4 %）,YI以CEA最高（0.41）。联合检测较单项检测敏感度、准确度和YI明显提高,其中CEA、CYFRA21-1和CA15-3三项联合效果最好,敏感度为92.3 %,特异度为78.4 %,准确度为84.1 %,YI值最高为0.71。四项联合敏感度为94.2 %,特异度为75.7 %,准确度为83.3 %,YI值为0.70,与三项联合结果相比差异无统计学意义（P＞0.05）。结论　单项检测的诊断价值有限,CEA、CYFRA21-1和CA15-3三项联合效果最好、最经济,可指导患者恰当选择进一步的侵入性检查手段。     

Tissue culture studies of malignant effusions.

This study reports attempts to culture tumour cells from 51 malignant effusions using standard tissue culture techniques. Cultures proliferating for more than one month were derived from 42 effusions including 24/32 from breast cancer patients and 5/6 from colon carcinomata. The morphology of these cells and their culture characteristics were compared with that of cells derived from a benign effusion. A common cell type--believed to be of mesothelial origin--was found in all cultures. In addition, fibroblastic cells were common and smaller pleomorphic cells, possibly tumour cells, were found in many effusions. The mesothelial cells were often multinucleated and grew for long periods. Although the tumour cells grew in conjunction with the mesothelial cells, all attempts at separation have failed. These studies indicate that cells derived from malignant effusions may be largely of mesothelial origin although tumour cells may also be present. The use of short-term cultures of malignant effusions as the source of cells for use as target cells in cytotoxicity tests and in chemotherapy assays is disscussed.     

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.     

Effect of host-cell interactions on clonogenic carcinoma cells in human malignant effusions.

We have studied the clonogenic capacity of tumour cells in agar from 38 malignant effusions from 31 patients with epithelial tumours. Colony formation of unfractionated cells, varies considerably from patient to patient, and is positively correlated with the percentage of tumour cells in the sample. Clonogenicity was shown to be reduced in 8/9 cases by removal of plastic-adherent and iron-phagocytic cells. In the ninth case, increased clonogenicity occurred after this procedure. When the autologous adherent cells were removed from the effusion and used in reconstitution experiments as an underlayer in a two-layer agar system, they were able to reverse the effect of the initial fractionation in a dose-dependent fashion. This indicates cellular communication based on release of a diffusible product of plastic-adherent cells. Morphological, phagocytic and prostaglandin-synthetic analysis of the fractions involved in the reconstitution experiments implicate the macrophage as the operative cell in this interaction. However, an accessory role for lymphoid cells or tumour cells themselves cannot be excluded.     

胸部CT胸膜区病变征象在鉴别良恶性胸腔积液中的价值

目的:探讨胸膜区CT病变征象在鉴别良恶性胸腔积液中的价值。方法:收集比较良恶性胸腔积液的胸部 CT平扫图像,观察胸膜区病变征象,包括胸膜结节、纵隔胸膜增厚、病变突入胸膜外脂肪间隙、胸膜与胸膜外脂肪交界面不光整、肋间淋巴结肿大、胸内筋膜及最内肋间肌增厚、肋间血管增粗、单侧胸腔积液,胸膜外脂肪间隙密度增高。单因素分析应用χ2检验,并将单因素分析差异有统计学意义的因素纳入Logistic回归进行多因素分析,并绘制受试者工作特征曲线（ROC）,通过计算曲线下面积（AUC）判断诊断效能。结果:单因素分析中胸膜结节、纵隔胸膜增厚、病变突入胸膜外脂肪间隙、胸膜与胸膜外脂肪交界面不光整、肋间淋巴结肿大、单侧胸腔积液这些因素在良恶性胸腔积液间的差异有统计学意义（P＜0.05）。纳入多因素分析的变量包括胸膜结节、纵隔胸膜增厚、病变突入胸膜外脂肪间隙、胸膜与胸膜外脂肪交界面不光整、单侧胸腔积液,其联合诊断恶性胸腔积液的灵敏度为85.4%,特异度为97.7%,ROC曲线下面积（AUC）为0.961。结论:胸膜区的病变征象对于胸腔积液良恶性诊断有较高价值,综合应用这些征象可为良恶性胸腔积液的鉴别诊断提供一种简便高效的方法。     

Percoll density gradient separation of cells from human malignant effusions

A simple method is described for the separation of cells derived from effusions of patients with adenocarcinomas in discontinuous density gradients of Percoll. After separation, cells from different fractions were analyzed by morphologic, histochemical and immunologic criteria. Total cell recovery from 27 experiments was 67 +/- 4%. Macrophages (82%) were recovered in the intermediate density fraction (1.056-1.067 g ml-1) with a purity of 90%. Recovered lymphocytes (98%) were found in the high density fraction (1.067-1.077 g ml-1) with a purity of 92%. The majority of the lymphocytes recovered were T cells. Malignant adenocarcinoma cells (90%) were recovered in the lowest density fractions (up to 1.056 g ml-1) with a purity of 79%. Use of effective cell separation procedures should facilitate the analysis of the functional capacities of both normal and neoplastic cells derived from human malignant effusions.     

免疫细胞化学鉴别良恶性体腔积液的抗体选择和组合

目的 优化免疫细胞化学鉴别良、恶性体腔积液的抗体选择和组合.方法 对病理学诊断明确的67例恶性和22例良性体腔积液标本采用石蜡细胞块和免疫细胞化学技术检测13种抗体.单因素方法分析每个抗体的敏感性和特异性.Logistic回归分析不同抗体组合对良、恶性体腔积液诊断的准确性.结果 检测体腔积液内肿瘤细胞敏感性最高的抗体是E-cadherin,为85.1%.其次是BerEP4和CK7,敏感性分别为82.1%和80.6%.检测肿瘤细胞特异性最强的抗体是E-cadherin、BerEP4和CK20,特异性均达到100%.Calretinin检测间皮细胞的敏感性和特异性分别为100%和95.5%,Desmin检测间皮细胞的敏感性和特异性分别为90.9%和100%.Logistic回归分析显示Calretinin是统计学上判断良、恶性体腔积液最佳的单个抗体,Calretinin和E-cadherin是区分良、恶性体腔积液最有效率的抗体组合.结论 结合单因素和多因素分析结果,我们推荐E-cadherin、BerEP4、Calretinin和Desmin作为日常免疫细胞化学鉴别良、恶性体腔积液的抗体组合.     

Diagnostic value of tumor markers for differentiating malignant and benign pleural effusions of Iranian patients

In order to evaluate the diagnostic yield of tumor markers in differentiating malignant and benign pleural effusions, we carried out a prospective study in a group of Iranian people. Pleural and serum levels of carcinoembryonic antigen (CEA), carbohydrate antigen 15-3 (CA15-3), neuron-specific enolase (NSE) and cancer antigen 125 (CA 125) were assayed prospectively in patients with pleural effusion (40 malignant and 37 benign). The highest sensitivity was obtained with a combination of CA 15-3 in serum, and CA 15-3 and CEA in pleural fluid (80%), also with combination of CA 15-3 in serum, and CA 15-3, NSE and CEA in pleural fluid (80%). The highest specificity was obtained with combination of CA 15-3 in serum, and CA 15-3 and NSE in pleural fluid (100%), and also with combination of CA 15-3 in serum, and CA 15-3, NSE and CEA in pleural fluid (100%).     

Utility of anti-L523S antibody in the diagnosis of benign and malignant serous effusions

BACKGROUND: Immunohistochemistry is helpful in distinguishing metastatic carcinoma from atypical mesothelial cells; however, it is not useful in differentiating atypical mesothelial cells from malignant mesothelial cells. K homolog domain containing protein overexpressed in cancer (KOC), a member of the insulin-like growth factor mRNA-binding protein (IMP) family, also known as L523S and IMP3, is expressed during embryogenesis and in various malignancies. Using a mouse monoclonal antibody (L523S) against KOC, KOC expression was investigated in malignant tumors and reactive mesothelial cells in serous effusions. METHODS: Seventy-six cases with paraffin-embedded pleural, pericardial, and peritoneal serous effusion cell blocks including 60 malignant serous effusions (11 malignant pleural mesotheliomas and 49 metastatic carcinomas) and benign pleural effusions (14 cases with reactive mesothelial cells and 2 cases with atypical cells with uncertain significance) were selected for immunohistochemical analysis with L523S, calretinin, and CK5/6. RESULTS: Immunohistochemical studies showed that positive staining for KOC of variable degrees of intensity was observed in 47 of 60 cases in malignant serous effusions including 10 of 11 mesotheliomas and 36 of 49 metastatic carcinomas. The associated reactive mesothelial cells were negative for KOC but positive for calretinin and CK5/6. All 11 malignant mesotheliomas exhibited positivity for calretinin, and 9 of 11 cases had CK5/6 staining. In addition, 16 cases that were originally diagnosed either as pleural effusions with reactive mesothelial cells (14) or atypical cells with uncertain significance (2) were also tested for KOC expression. Interestingly, 3 of 16 cases exhibited various degrees of positivity for KOC, 2 of which were diagnosed as lung adenocarcinoma with a recurrence after tumor resection and 1 as malignant pleural mesothelioma. CONCLUSIONS: Anti-L523S antibody is a useful marker for the detection of malignant cells in serous effusions and it can have significant utility in differentiating reactive mesothelial cells from malignant mesothelioma and metastatic carcinoma in combination with calretinin and CK5/6 staining.     

The cytologic application of carcinoembryonic antigen for the discrimination of malignant from benign serous effusions

In an attempt to diminish false cytologic diagnosis in discriminating between benign and malignant effusions, the value of intracellular carcinoembryonic antigen (CEA) was estimated in cytologic smears of effusions using a modified immunoperoxidase technique. CEA serum level was estimated by radioimmunoassay technique. In this study we included a group of 42 patients, 23 with a malignant and 19 with a benign effusion. CEA in cells of effusions was positive and useful for confirming malignancy in 65% of malignant effusions. The percentage increased to 83% with the combination of the results of positive CEA in cells and serum. Cytology was positive for malignancy in 16 of the 23 (70%) malignant cases. CEA in cells of benign effusions was negative in 84% and cytology in 100%. It is concluded that intracellular CEA estimation, when combined with CEA estimation in serum, improves the discriminating range of cytology for differentiating malignant and benign effusions.