bromochloroacetic-acid and Pleural-Effusion

Pleural effusion (PE), excess fluid in the pleural space, is often observed in lung cancer patients and also forms due to many benign ailments. Classifying it quickly is critical, but this remains an analytical challenge often lengthening the diagnosis process or exposing patients to unnecessary risky invasive procedures. We tested the analysis of PE using a multiplexed cytokeratin (CK) panel with targeted mass spectrometry-based quantitation for its rapid classification. CK markers are often assessed in pathological examinations for cancer diagnosis and guiding treatment course. We developed methods to simultaneously quantify 33 CKs in PE using peptide standards for increased analytical specificity and a simple CK enrichment method to detect their low amounts. Analyzing 121 PEs associated with a variety of lung cancers and noncancerous causes, we show that abundance levels of 10 CKs can be related to PE etiology. CK-6, CK-7, CK-8, CK-18, and CK-19 were found at significantly higher levels in cancer-related PEs. Additionally, elevated levels of vimentin and actin differentiated PEs associated with bacterial infections. A classifier algorithm effectively grouped PEs into cancer-related or benign PEs with 81% sensitivity and 79% specificity. A set of undiagnosed PEs showed that our method has potential to shorten PE diagnosis time. For the first time, we show that a cancer-relevant panel of simple-epithelial CK markers currently used in clinical assessment can also be quantitated in PEs. Additionally, while requiring less invasive sampling, our methodology demonstrated a significant ability to identify cancer-related PEs in clinical samples and thus could improve patient care in the future.

Topics: Actins; Aged; Biomarkers, Tumor; Enzyme-Linked Immunosorbent Assay; Female; Humans; Keratins; Lung Neoplasms; Male; Mass Spectrometry; Middle Aged; Pleural Effusion; Vimentin

A limitation of positive selection strategies to enrich for circulating tumor cells (CTCs) is that there might be CTCs with insufficient expression of the surface target marker which may be missed by the procedure. We optimized a method for enrichment, subsequent detection and characterization of CTCs based on depletion of the leukocyte fraction.. The 2-step protocol was developed for processing 20 mL blood and based on red blood cell lysis followed by leukocyte depletion. The remaining material was stained with the epithelial markers EpCAM and cytokeratin (CK) 7/8 or for the melanoma marker HMW-MAA/MCSP. CTCs were detected by flow cytometry. CTCs enriched from blood of patients with carcinoma were defined as EpCAM+CK+CD45-. CTCs enriched from blood of patients with melanoma were defined as MCSP+CD45-. One-hundred-sixteen consecutive blood samples from 70 patients with metastatic carcinomas (n = 48) or metastatic melanoma (n = 22) were analyzed.. CTCs were detected in 47 of 84 blood samples (56%) drawn from carcinoma patients, and in 17 of 32 samples (53%) from melanoma patients. CD45-EpCAM-CK+ was detected in pleural effusion specimens, as well as in peripheral blood samples of patients with NSCLC. EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin. This was confirmed by CGH. By defining CTCs in carcinoma patients as CD45-CK+ and/or EpCAM+, the detection rate increased to 73% (61/84).. Enriching CTCs using CD45 depletion allowed for detection of epithelial cancer cells not displaying the classical phenotype. This potentially leads to a more accurate estimation of the number of CTCs. If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined.

Topics: Antigens, Neoplasm; Ascites; Biomarkers, Tumor; Calibration; Cell Adhesion Molecules; Cell Line, Tumor; Epithelial Cell Adhesion Molecule; Humans; Immunomagnetic Separation; Keratins; Leukocyte Common Antigens; Lymphocyte Depletion; Melanoma; Microspheres; Nanoparticles; Neoplasms; Neoplastic Cells, Circulating; Pleural Effusion

Mycobacterium kansasii disease was diagnosed in an 85-year-old woman admitted to the hospital for cough and gradually worsening breathlessness. Transbronchial biopsy indicated either non-necrotizing granulomata or bronchiolitis obliterans organizing pneumonia (BOOP). She was cured with combined therapy of specific anti-mycobacterial medications and systemic steroids. To our knowledge, this is the first report of M. kansasii non-tuberculous mycobacterium disease with a BOOP-like pattern on lung biopsy.

Topics: Aged, 80 and over; Bronchi; Cryptogenic Organizing Pneumonia; Female; Granuloma; Humans; Immunohistochemistry; Keratins; Mycobacterium Infections, Nontuberculous; Mycobacterium kansasii; Pleural Effusion; Tomography, X-Ray Computed

Many human breast cancer cell lines have been in culture for several years, serving as model systems for studying aspects of breast cancer biology. Molecular alterations might occur in these cells during cultivation, and it remains unknown to which extent findings in these cell lines can be related to human disease. Hereby, we describe the establishment of a breast cancer cell line, MW1, from malignant pleural effusion. We compare expression patterns of several molecular markers in breast biopsy tissue, in cultivated tumor cells derived from pleural effusion reflecting the metastatic state, and in late passages of a lineage derived from the pleural culture. Our data show that expression of estrogen and progesterone receptors was lost in the cultivated tumor cells derived from pleural effusion as shown by immunohistochemical staining. Cytokeratin expression patterns remained luminal. During cultivation, the growth rate of MW1 cells increased dramatically and the morphology underwent alterations. As shown by Western blotting, E-cadherin expression remained unchanged whereas P-cadherin expression had increased after 4 years of cultivation of the cell line. Integrin beta4 expression was low in early passages of the pleural effusion whereas the cell line exhibited high expression levels of beta4. HGF receptor (c-Met), EGF receptor, VEGF and VEGF receptor-2 (KDR) expression was detectable by semiquantitative RT-PCR and remained unchanged during cultivation. In contrast, VEGF receptor-1 (flt-1) expression showed lower expression after 4 years of cultivation. The cell line migrated towards HGF, but not towards VEGF. This study provides exemplary insight into the molecular metamorphosis tumor cells undergo in vivo or in vitro on their way from the primary tumor via an equivalent of the metastatic state and during the development of a clonal cell line.

Topics: Adult; Breast Neoplasms; Cell Line, Tumor; Female; Hepatocyte Growth Factor; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Integrin beta4; Keratins; Neoplasm Invasiveness; Neovascularization, Pathologic; Phenotype; Pleural Effusion; Vascular Endothelial Growth Factor A

The aim of this study was to evaluate the individual and combined diagnostic value of five tumour markers in the elderly patients with pleural effusions. Serum and pleural fluid levels of cytokeratin fragment 19 (CYFRA21-1), neuron-specific enolase (NSE), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 19-9 (CA19-9) and carbohydrate antigen 125 (CA125) were assayed in 32 elderly patients with malignant pleural effusions resulting from advanced lung cancer and in 30 elderly patients with benign pleural effusions by ELISA. Serum levels of CYFRA21-1, NSE, CA15-3, CA19-9 and CA125 in patients with malignant pleural effusions were 12.84 +/- 6.48 microg/l, 22.07 +/- 11.25 microg/l, 65.74 +/- 30.26 kU/l, 56.32 +/- 25.6 kU/l and 71.86 +/- 31.45 kU/l, respectively, and were significantly higher than those in patients with benign pleural effusions (p < 0.01). Pleural fluid levels of CYFRA21-1, CA15-3, CA19-9 and CA125 except NSE in patients with malignant pleural effusions were 18.64 +/- 8.15 microg/l, 59.31 +/- 27.35 kU/l, 48.24 +/- 21.56 kU/l and 62.16 +/- 27.79 kU/l, respectively, and were significantly higher than those in patients with benign pleural effusions (p < 0.01). The parallel combined testing of five tumour markers in serum increased the diagnostic sensitivity to 90.6%, and serial combined testing increased the diagnostic specificity to 93.3%. The sensitivity (%) and specificity (%) of these tumour markers in pleural fluid were as follows: CYFRA21-1, 84.4/90; CA15-3, 62.5/73.3; CA19-9, 37.5/66.7; CA125, 56.3/70; for differentiating malignant effusions from benign effusions. When CYFRA21-1 and CA15-3 combined, the sensitivity and specificity were increased (100% and 90% respectively). Serum and pleural fluid levels of the five tumour markers shows certain values in the diagnosis and differentiate diagnosis for malignant pleural effusions in the elderly patients from benign. The combined assay of five tumour markers in serum and the CYFRA21-1 combined with CA15-3 in pleural fluid were helpful and can increase the sensitivity and specificity in diagnosing malignant pleural effusions.

Topics: Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; CA-125 Antigen; CA-19-9 Antigen; Diagnosis, Differential; Female; Humans; Keratin-19; Keratins; Lung Neoplasms; Male; Middle Aged; Mucin-1; Phosphopyruvate Hydratase; Pleural Effusion; Sensitivity and Specificity

A 68-year-old man complaining of hoarseness and back pain, with no history of exposure to asbestos, was referred to our hospital in June 2002. He was admitted because his chest X-ray and CT scan showed atelectasis and a tumor-like region in the right lower lobe of the lung. Serum-CYFRA was 2.8 ng/ml, elevated slightly; however, no other tumor markers for lung cancer were elevated. A diagnosis of squamous cell lung cancer was made based on bronchial washing cytology. Persistent high fever and WBC count elevation did not respond to antibiotics, and reduced only after chemotherapy. Both serum G-CSF (217.0 pg/ml) and CYFRA in the pleural effusion (107.1 ng/ml) were elevated. The biopsy of the growing tumor in the right lateral abdominal wall revealed carcinoma with sarcomatous component or biphasic-type malignant pleural mesothelioma (MPM). In spite of chemotherapy and radiation therapy for the abdominal wall tumor, the tumor rapidly progressed and the patient died three months after admission. The findings at autopsy suggested the tumor was a sarcomatous MPM. However, immunohistochemical staining and tissue HABP staining revealed biphasic type MPM. Although CYFRA elevation in the serum and/or the pleural effusion in MPM patients has been previously reported, it has not been reported in any of the 5 MPM patients reported to have G-CSF elevation. Therefore, this is the first reported case of G-CSF-producing MPM with CYFRA elevation in both serum and the pleural effusion.

Topics: Aged; Antigens, Neoplasm; Biomarkers, Tumor; Granulocyte Colony-Stimulating Factor; Humans; Hyaluronan Receptors; Keratin-19; Keratins; Male; Mesothelioma; Pleural Effusion; Pleural Neoplasms; Staining and Labeling

Lung cancer, especially adenocarcinoma and large cell carcinoma, tends to spread to the pleural cavities. Once an effusion develops, the prognosis is generally dismal. Immunocytochemistry is frequently applied to effusions for diagnostic purposes, but the prognostic value of markers in malignant effusions have thus far attracted less attention. Dakopatts CK 5/6 antibody was applied to ethanol-fixed fresh cytospin preparations from malignant pleural effusions originating from 18 patients (11 men and 7 women) with a previously or later verified non-small cell lung carcinoma (NSCLC). In three cases, CK5/6 reactivity was found in part of the malignant population, whereas 10 cases showed reactivity in most tumor cells. The lack of reactivity in malignant cells was only seen in five effusions. Females showed significantly lower reactivity rates, with all negative effusions coming from female patients, whereas 9/10 effusions with reactivity in most malignant cells originated from males. CK5/6 reactivity was significantly correlated to survival, with a median survival time of 18 days for patients with CK5/6-negative tumors, and 212 days for those with positive tumors. The strong relationship between CK5/6 reactivity and survival, and the observed gender difference, warrants larger studies aimed at the clinical utility of CK5/6 as a prognostic marker in metastatic NSCLC, the possible functional role of CK5/6 in cell adhesion in advanced NSCLC and its possible hormonal control.

Topics: Aged; Aged, 80 and over; Antibodies; Carcinoma, Non-Small-Cell Lung; Female; Humans; Immunohistochemistry; Keratin-5; Keratin-6; Keratins; Lung Neoplasms; Male; Middle Aged; Pleural Effusion; Predictive Value of Tests; Prognosis; Survival Analysis

Levels of tumor markers in pleural effusions may help to establish the diagnosis of pleural malignancy, but the precise diagnostic value of each marker remains unclear. The aim of this study was to assess the diagnostic value of five common pleural fluid tumor markers, carcinoembryonic antigen (CEA), cytokeratin fragment (CYFRA) 21-1, cancer antigen (CA) 15-3, CA 19-9, and CA 125, and to review the literature from the past 15 years. Pleural fluid samples were collected prospectively from 116 patients and assayed for CEA, CYFRA 21-1, CA 15-3, CA 19-9, and CA 125 levels. A MEDLINE search of the English-language literature from the past 15 years was also done. Effusions were classified as benign or malignant on the basis of their definitive pathologic or cytologic diagnoses. The levels of all pleural tumor markers were statistically significantly higher in the malignant group than in the benign group. The marker with the highest accuracy was CEA (85.3%); CA 15-3, CYFRA 21-1, and CA 19-9 had similar accuracies (75.2%, 72.4%, and 71.5%, respectively), and CA 125 had the lowest accuracy (40.5%). On univariate analysis, tumor-marker combinations did not result in a greater accuracy than that of CEA alone. On multivariate logistic regression, CA 15-3 and CYFRA 21-1 were significant predictors of malignancy. Among the nine reports in the literature comparing 11 different tumor markers, CEA, CA 15-3, and CYFRA 21-1 yielded the best results. We conclude that pleural fluid analysis should include CEA for the diagnosis of malignancy. CA 15-3 and CYFRA 21-1 may serve as alternative options.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; CA-125 Antigen; CA-19-9 Antigen; Carcinoembryonic Antigen; Diagnosis, Differential; Female; Humans; Keratin-19; Keratins; Male; Middle Aged; Mucin-1; Pleural Effusion; Prospective Studies

To assess the diagnostic values of carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and cytokeratin 19 fragments (CYFRA 21-1) as markers of pleurisy in primary lung cancer.. Prospective case-control study.. A tertiary university hospital.. Thirty-four patients with lung cancer and 16 patients with tuberculous pleurisy.. Levels of CEA, NSE, and CYFRA 21-1 were measured by immunoassay in the serum and pleural fluid of patients with lung cancer and of patients with tuberculous pleurisy. Patients with lung cancer were found to have significantly higher serum and pleural fluid levels of CEA and CYFRA 21-1 than patients with tuberculous pleurisy. Using cutoff values of 5 ng/mL, 20 ng/mL, and 3.3 ng/mL for serum CEA, NSE, and CYFRA 21-1, respectively, the sensitivities and specificities of these tumor markers were as follows for differentiating malignant effusion from benign: CEA, 68% and 93%; NSE, 34% and 93%; and CYFRA 21-1, 45% and 100%. Using cutoff values of 5 ng/mL, 20 ng/mL, and 45 ng/mL for pleural fluid, the sensitivities and specificities were as follows: CEA, 82% and 94%; NSE, 36% and 94%; and CYFRA 21-1, 61% and 81%. A combination of pleural fluid CEA and NSE increased sensitivity and specificity.. In the diagnosis of malignant effusion associated with lung cancer, the determinations of CEA and NSE in pleural fluid could enhance diagnostic yield better than those of all three tumor markers.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Carcinoembryonic Antigen; Case-Control Studies; Humans; Keratin-19; Keratins; Lung Neoplasms; Peptide Fragments; Phosphopyruvate Hydratase; Pleural Effusion; Sensitivity and Specificity

Human mesothelial cells obtained from benign effusions retain their proliferative capacity and grow uniformly either with a fibroblastic or epithelioid morphology in vitro. These cultures therefore provide a model for the process of mesothelial differentiation in vivo. To study this differentiation, we isolated differentially expressed genes obtained by suppression subtractive hybridization. Of the nine genes found to be overexpressed in fibroblastic mesothelial cells, three are matrix-associated (integrin alpha5, collagen binding protein 2, human cartilage glycoprotein 39), whereas the others are associated with a proliferative cell type (14-3-3 epsilon, plexin B2, N33, and three genes encoding ribosomal elements). Seven of the eight genes upregulated in the epithelioid phenotype are related rather to specialized functions, such as metabolism (aldose reductase, lecithin:cholesterol acyltransferase, ATPase 6), cytoskeletal composition (cytokeratins 7 and 8), and regulation of differentiation (granulin, annexin II). Immunohistochemistry with available antibodies to six of the differentially expressed gene products confirmed the differences also in pleural tissues, where submesothelial cells displayed the fibroblastic markers, whereas surface cells displayed the epithelioid markers. In summary, this approach revealed a pattern of genes coordinately regulated during mesothelial differentiation and suggests that mesothelium may regenerate also by recruiting cells from the submesothelial layer. Some of the gene products may also be useful markers for differentiation and activation in serosal tissues.

Topics: 14-3-3 Proteins; Adipokines; Aldehyde Reductase; Blotting, Southern; Carrier Proteins; Cell Differentiation; Cell Division; Cells, Cultured; Chitinase-3-Like Protein 1; Epithelial Cells; Fibroblasts; Gene Expression; Glycoproteins; Humans; In Situ Hybridization; Integrin alpha5; Intercellular Signaling Peptides and Proteins; Keratin-7; Keratins; Lectins; Nerve Tissue Proteins; Pleural Effusion; Progranulins; Receptors, Cell Surface; Tyrosine 3-Monooxygenase

The diagnostic value of tumor markers in pleural fluid is subject to debate. The aim of this study was to evaluate the diagnostic performance of several tumor markers in common use for detecting malignant pleural disease.. Blinded comparison of four tumor markers in pleural fluid with a confirmatory diagnosis of malignancy by pleural cytology or thoracoscopic biopsy.. Two teaching hospitals in Spain.. A total of 416 patients (166 with definite malignant effusions, 77 with probable malignant effusions, and 173 with benign effusions) were enrolled. Among them, there were 42 patients recruited from one of the participant centers with thoracoscopic facilities, who had false-negative fluid cytology findings and malignancy confirmed by medical thoracoscopy. Tumor markers in pleural fluid were determined either by electrochemiluminescence immunoassay (carcinoembryonic antigen [CEA], carbohydrate antigen 15-3 [CA 15-3], cytokeratin 19 fragments [CYFRA 21-1]) or microparticle enzyme immunoassay (cancer antigen 125 [CA 125]) technologies. Cutoff points that yielded 100% specificity (ie, all patients with benign effusions had levels below this cutoff) were selected for each marker.. Malignant pleural effusions (PEs) had higher levels of pleural fluid markers than did effusions due to benign conditions. At 100% specificity, a pleural CEA > 50 ng/mL, CA 125 > 2,800 U/mL, CA 15-3 > 75 U/mL, and CYFRA 21-1 > 175 ng/mL had 29%, 17%, 30%, and 22% overall sensitivities, respectively. The combination of the four tumor markers reached 54% sensitivity, whereas the combined use of the cytology and the tumor marker panel increased the diagnostic yield of the former by 18% (95% confidence interval, 13 to 23%). More than one third of cytology-negative malignant PEs could be identified by at least one marker of the panel.. No single pleural fluid marker seems to be accurate enough as to be introduced in the routine workup of PE diagnosis. However, a tumor marker panel may represent a helpful adjunct to cytology in order to rule in malignancy as a probable diagnosis, thus guiding the selection of patients who might benefit from further invasive procedures.

Topics: Aged; Antigens, Neoplasm; Biomarkers, Tumor; CA-125 Antigen; Carcinoembryonic Antigen; Cytodiagnosis; Diagnosis, Differential; Female; Humans; Keratin-19; Keratins; Male; Mucin-1; Pleural Effusion; Pleural Effusion, Malignant; ROC Curve; Sensitivity and Specificity

Lung adenocarcinoma presenting as malignant pleural effusion (MPE) is common in Taiwan. Microscopically, the involved pleurae are infiltrated by numerous tumor foci, which suggests that the cancer cells are highly invasive. Overexpression of HER-2/neu has been related to proliferation, antiapoptosis, and the high invasiveness of various cancer cells. We therefore were interested in studying the role of HER-2/neu in MPE-associated adenocarcinoma cell lung cancer (ADCLC).. The expression of HER-2/neu in pleural effusion was measured by ELISA. The HER-2/neu protein expression on tumor cells was evaluated by immunohistochemical (IHC) staining, and gene amplification was assayed by fluorescence in situ hybridization.. The mean value of HER-2/neu in pleural effusions of patients with ADCLC and other nonmalignant lung diseases was 9.9 and 2.7 ng/ml, respectively. The difference is statistically significant (P < 0.001). Compared with cytokeratin 19 fragment CYFRA 21-1, the performance of HER-2/neu as a tumor marker in pleural effusion diagnosis was better. Overexpression of HER-2/neu in tumor tissues was found in 70% (23 of 32) of patients with MPE-associated ADCLC, 30% (13 of 43) with stage I/II non-small cell lung cancer (NSCLC), and 44% (14 of 32) with stage III NSCLC. The incidence of HER-2/neu overexpression in tumor tissues of patients with MPE-associated ADCLC was significantly higher than that of patients with stage I-III NSCLC without MPE. HER-2/neu gene amplification was uncommon (1.9%). The correlation between the IHC H-score in tumor samples and the pleural effusion level of HER-2/neu was significant (P < 0.01). A higher incidence of HER-2/neu expression beyond the cutoff point (5.5 ng/ml) in pleural effusions was also found in patients whose IHC H-scores were >50.. These findings indicate that HER-2/neu is important in the pathogenesis of MPE-associated ADCLC and is a potential tumor marker for a diagnosis of pleural effusion.

Topics: Adenocarcinoma; Antigens, Neoplasm; Apoptosis; Biomarkers, Tumor; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Keratin-19; Keratins; Lung Neoplasms; Neoplasm Metastasis; Pleural Effusion; Receptor, ErbB-2

The diagnostic value of tumor markers Cyfra 21-1 and neuron-specific enolase in blood serum and pleural fluid in differential diagnosis of pleural exudation in cancer patients and patients with nontumor pleurisy was evaluated. The most pronounced changes were characteristic of Cyfra 21-1. In patients with pleurisy caused by malignant tumors the degree and incidence of increased Cyfra 21-1 concentrations in the serum and pleural fluid were higher than in patients with pleural exudation of nontumor origin. These data attest to high diagnostic sensitivity and specificity of Cyfra 21-1. Complex measurements of the marker in the serum and pleural fluid will improve diagnosis of pleural exudation of tumorous etiology.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Case-Control Studies; Diagnosis, Differential; Exudates and Transudates; Female; Humans; Keratin-19; Keratins; Male; Middle Aged; Neoplasms; Neurons; Phosphopyruvate Hydratase; Pleural Effusion; Sensitivity and Specificity

In cytological preparations, reactive mesothelial cells (RMC) in serous effusions are sometimes difficult to distinguish from adenocarcinoma cells (AC). RMC and AC can be distinguished by lectin-binding patterns, but the pattern of binding of lectins to normal mesothelium is not well defined. We investigated the expression of cytoskeletal filaments, cytokeratin (CK) and vimentin (VM), and the cell surface binding pattern of 10 lectins (HPA, SBA, ABA, DSA, PNA, RCA-I, UEA-I, LTA, WGA and ConA) in the serosa of 48 adenocarcinoma specimens. We also investigated the usefulness of six lectins (HPA, SBA, RCA-I, UEA-I, LTA and WGA) in identification of RMC and AC in 16 serous effusions. DSA reactivity was significantly higher (P < 0.05) in static mesothelial cells (SMC) than in RMC. Reactivity for LTA and ConA was significantly lower (P < 0.05) in SMC than in RMC. Anti-CK and anti-VM immunoreactivity was always positive in RMC and almost negative in SMC. In serous effusions, HPA, SBA and UEA-I binding was evident in 100, 88 and 81% of AC, respectively. Little to no binding of HPA, SBA or UEA-I was detected in RMC. Our results suggest that the morphological differences between SMC and RMC are likely to be due to differences in cytoskeletal composition, with accompanying changes in cell-surface lectin-binding patterns. HPA, SBA and UEA-I are likely to be useful markers for identification of RMC and AC in cytology.

Topics: Adenocarcinoma; Ascites; Binding Sites; Biomarkers; Cytoskeleton; Epithelium; Gastrointestinal Neoplasms; Humans; Immunohistochemistry; Keratins; Lectins; Lung Neoplasms; Pleural Effusion; Serous Membrane; Vimentin

The aim of the study was to assess diagnosis value of tumor markers for differential diagnosis between mesothelioma and other pleural tumors.. Prospective study of 85 patients attending our hospital with malignant pleural effusion. The diagnostic approach involved routine pleurocentesis followed by pleural needle. When precise diagnosis was not achieved, thoracoscopy with pleural biopsies was performed. Carcinoembryonic antigen (CEA), hyaluronic acid, tissue polypeptide antigen and cyfra 21 to 1 were measured in serum and pleural fluid.. By using receiver operating characteristics curves and area under curves, the best diagnostic characteristics were obtained with pleural and serum CEA concentrations. The area under the curve was larger for pleural ACE than for serum ACE. The sensitivity and specificity of a pleural CEA level exceeding 3 ng/mL for ruling out the diagnosis of mesothelioma were 100% and 77%, respectively.. A CEA level above 3 ng/mL in pleural fluid eliminated the diagnosis of mesothelioma, whereas the other markers were not sufficiently discriminant. However, despite a negative predictive value of 100% at a cutoff of 3 ng/mL, CEA assay in pleural fluid only avoids a small number of diagnostic thoracoscopies.

Topics: Adenocarcinoma; Aged; Antigens, Neoplasm; Area Under Curve; Biomarkers, Tumor; Carcinoembryonic Antigen; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Diagnosis, Differential; Female; Humans; Hyaluronic Acid; Keratin-19; Keratins; Lung Neoplasms; Male; Mesothelioma; Middle Aged; Pleural Effusion; Pleural Neoplasms; Prospective Studies; ROC Curve; Sensitivity and Specificity; Tissue Polypeptide Antigen

Interphase cytogenetics by fluorescence in situ hybridization (FISH) can be used to detect malignant cells characterized by chromosomal aneuploidy. However, apparent aneusomy in normal "control" tissues has to be considered when using FISH as diagnostic tool. In effusions as model tissue exposed to metastasis, the definition of cut-off levels for background aneusomy by FISH was aimed in this study. Using centromeric probes representing chromosomes 7, 8, 11, 12, 17 and 18, extensive chromosome copy number enumeration by single-color FISH analysis was performed in pleural and ascitic effusions derived from 15 patients with various, non-malignant diseases. In all effusions, cells with gain of hybridization signals for several or all chromosomes tested were found (in up to 1.94% of cells). A consistent finding was high grade hyperdiploidy (>4 centromeric signals). Mesothelial elements mainly contributed to hyperdiploidy in effusions, as demonstrated by a combined analysis of FISH and immunocytochemistry with staining for cytokeratin. Dual-color FISH analysis showed that hyperdiploidy was predominantly corresponding to polyploidization; however, there were always minor cell populations classified as aneuploid by dual-color FISH. In conclusion, stringent criteria have to be applied to distinguish malignancy-related aneuploidy from background aneusomy by FISH.

Topics: Adult; Aged; Aged, 80 and over; Aneuploidy; Ascitic Fluid; Epithelium; Female; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Keratins; Male; Middle Aged; Pleural Effusion; Polyploidy

To provide cytospins as a means of external quality assurance (EQA), while maintaining antigen expression integrity and achieving uniformity of material for all participating laboratories.. Cells were collected from two adenocarcinoma and one reactive pleural effusion specimens. Lymphoid cells were also collected through aspiration of a resected tonsil specimen. All cells were collected in Hanks balanced salt solution (HBSS); cytospins were made and fixed in methanol or acetone alone or protected using a layer of 10% polyethylene glycol (PEG) in 50% methanol. Two laboratories participated (RGHT and UCL).. Cytokeratin expression detected using either CAM5.2 or AE1/AE3 antibodies was sensitive to temperature. Without PEG, unacceptable or negative staining was seen within six weeks of preparation. LCA was not sensitive to temperature, with good staining scores being achieved after eight weeks following preparation.. It is possible to send alcohol fixed, air dried cytospins to laboratories participating as part of an EQA scheme for immunocytology. Some antigens will require protection from temperature variations during transit. A layer of 10% PEG in 50% methanol, allowed to air dry, is suitable for this purpose. Participating laboratories will only have to remove this layer using methanol before their localisation technique for assessment.

Topics: Adenocarcinoma; Cell Separation; Cytodiagnosis; Ethanol; Humans; Immunoenzyme Techniques; Keratins; Neoplasm Proteins; Pleural Effusion; Polyethylene Glycols; Quality Assurance, Health Care; Tissue Preservation

To the authors' knowledge the role of tumor marker determination in the differential diagnosis of pleural effusions has not been established definitively. The current article reports the results of a study of CYFRA 21-1, carcinoembryonic antigen (CEA), cancer antigen 125 (CA 125), squamous cell antigen (SCC), and neuron specific enolase (NSE) in the serum and pleural fluid of patients with pleural effusions of diverse etiologies.. One hundred forty-six patients with pleural effusions (43 malignant, 47 tuberculous, 32 miscellaneous benign, and 24 paramalignant) were studied prospectively. Levels of CYFRA 21-1, CA 125, CEA, NSE, and SCC were measured by radioimmunoassay in the pleural fluid in all patients and in the serum in 118 patients.. There were no significant differences between the serum and pleural fluid levels of tumor markers with the exception of CA 125, which was higher in the pleural fluid. With maximum specificity, the highest sensitivity in the diagnosis of pleural malignancy was obtained with a combination of CYFRA 21-1 (with a cutoff value of 150 U/L), CEA (with a cutoff value of 40 ng/mL), and CA 125 (with a cutoff value of 1000 ng/mL) in pleural fluid. NSE and SCC added no diagnostic value. The simultaneous use of tumor markers and cytology in pleural fluid increased the sensitivity from 55.8% to 81%.. These findings suggest that a combination of CYFRA 21-1, CEA, and CA 125 in the pleural fluid can be a useful addition to pleural cytology in the diagnosis of malignant pleural effusion.

Topics: Adult; Aged; Antigens, Neoplasm; Biomarkers, Tumor; CA-125 Antigen; Carcinoembryonic Antigen; Exudates and Transudates; Female; Humans; Immunoradiometric Assay; Keratin-19; Keratins; Male; Middle Aged; Neoplasms; Phosphopyruvate Hydratase; Pleural Effusion; Pleural Effusion, Malignant; Prospective Studies; Serpins; Tuberculosis

We examined changes in cytokeratin 19 fragment (CYFRA 21-1) levels in relation to the T factor in 64 non-small cell N2M0 lung cancer patients. Although a correlation between the levels and T factor was found (rho=0.627, p<0.0001), there was no difference between the levels in T3 and T4. Serum CYFRA 21-1 levels increased in the order of the following groups: the limited tumor group (T1+T2: n=28), the group with tumor extending to the pleura or chest wall (T3: n=13), and the group with tumor invading into the mediastinum (T4: n=12). The level was lower in the group with malignant pleural effusion (T4: n=11) than in the group with tumor invading into the mediastinum (6.7+/-4.7 ng/ml vs. 12.2+/-8.1 ng/ml, p=0.0046). We suspect that the presence of malignant pleural effusion is not directly related to the three-dimensional expansion of the tumor and this is a reason why CYFRA 21-1 levels in T4 are not higher than those in T3.

Topics: Antigens, Neoplasm; Carcinoma, Non-Small-Cell Lung; Humans; Keratin-19; Keratins; Lung Neoplasms; Pleural Effusion; Radiometry

Mesothelial cells are of mesenchymal origin, although they also have epithelial characteristics. Such cells obtained from benign effusions are not terminally differentiated and can be kept in short-term cultures. These cultures grow with an either epithelial or fibroblast-like phenotype, a pattern which is stable through the early passages. Several factors have been associated with mesothelial differentiation. The Wilms' tumour susceptibility gene 1 (WT1) is expressed during transitions of mesenchyme to epithelial tissues, as in the embryonic kidney, and it has been suggested as a marker for the mesothelial lineage. The proteoglycans (PGs) and hyaluronan are also differentially synthesised by epithelial and fibroblastic malignant mesothelioma cells and the cell surface PGs seem to indicate phenotypic differentiation even in epithelial tumours. To investigate how the epithelial and fibroblast-like differentiation of benign mesothelial cells was correlated to WT1, PGs and hyaluronan synthase, we studied their expression by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analyses. The expressions of these genes were all associated with a variation in phenotypic differentiation. Cell lines with epithelial morphology expressed more mRNA coding for WT1 and cell surface PGs than did the fibroblastic ones, the difference being greatest for syndecan-4 and glypican. The increase in WT1-associated mRNA was about as great as that of syndecans. Fibroblast-like cells, on the other hand, expressed substantially more of the matrix PGs versican and biglycan, while decorin expression was detected in only trace amounts in both morphological phenotypes. Hyaluronan synthase varied individually between the cell lines, although epithelial cells often expressed higher levels. The results indicate that the regulation of mesothelial differentiation is multifactorial and also involves WT1 and several PGs.

Topics: Actins; Calbindin 2; Cell Differentiation; Cells, Cultured; DNA-Binding Proteins; Epithelial Cells; Gene Expression; Glucuronosyltransferase; Glycosyltransferases; Humans; Hyaluronan Synthases; Immunohistochemistry; Keratins; Membrane Proteins; Microscopy, Electron, Scanning; Pleural Effusion; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; S100 Calcium Binding Protein G; Transcription Factors; Transferases; Vimentin; WT1 Proteins; Xenopus Proteins

The diagnostic value of tumour markers in pleural effusion is not yet clearly defined. CEA (Carcinoembryonic Antigen), CYFRA 21-1 (Cytokeratin 19-Fragment) and TPA-M, a new monoclonal-based radioimmunoassay for TPA (Tissue Polypeptide Antigen), were measured in pleural fluid and sera of 125 consecutive patients who underwent medical thoracoscopy. The group consisted of 79 patients with malignant and 45 with non-malignant pleural effusion and 1 patient without definitive diagnosis, and hence 124 patients were available for assessing the diagnostic value. In pleural fluid based on a specificity of 90% versus benign diseases the sensitivity for CEA was 52.5%; with the maximum achievable specificity of 80% for CYFRA 21-1 the sensitivity was 68% and for TPA-M with 67% the sensitivity was 67%. Based on the cut-off values for these specificities the combined use of the three tumour markers resulted in a sensitivity of 85.7% but with a lower specificity of 59.1%. There is only a limited value for tumour markers in the diagnosis of pleural effusion.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Carcinoembryonic Antigen; Female; Humans; Keratin-19; Keratins; Lung Neoplasms; Male; Middle Aged; Pleural Effusion; Pleural Effusion, Malignant; Radioimmunoassay; Sensitivity and Specificity; Thoracoscopy; Tissue Polypeptide Antigen

Differentiation between malignant and benign pleural effusions is often difficult. Serum level of Cyfra 21-1, a marker of cytokeratin 19 fragments, has been used in the diagnosis and monitoring of epithelial tumours, especially bronchogenic carcinomas.. This study is designed to establish the usefulness of effusion Cyfra 21-1 level in differentiating malignant from benign effusions.. Forty-eight malignant effusion aspirates (proven by cytology or pleural biopsy) and 34 benign samples were compared. Cyfra 21-1 concentration was measured by a solid phase sandwich radioimmunoassay (Centocur, USA).. Cyfra 21-1 level was significantly higher in malignant effusions (geometric mean 123.6 ng/mL, 95% confidence interval [CI] 76.6-199.4) than in benign ones (geometric mean 14.3 ng/mL, 95% CI 8.5-23.9), p<0.00005. By Receiver Operating Characteristics curve analysis, the sensitivity is 77% for a specificity of 79% if the cut-off is set at 32 ng/mL. No significant difference was observed (p=0.1) in Cyfra 21-1 concentration between adenocarcinoma and mesothelioma effusions. Cyfra 21-1 level was not influenced by the effusion protein concentration (r=0.29), or by renal function as measured by serum creatinine (r=0.1). There was no significant difference between Cyfra 21-1 levels in benign exudate and transudate effusions, p=0.28.. Cyfra 21-1 is a useful adjunct in the workup of effusions but should not replace conventional investigations as there is considerable overlap in levels between benign and malignant groups. It is unable to differentiate between subgroups of malignancies.

Topics: Biomarkers, Tumor; Diagnosis, Differential; Humans; Keratins; Pleural Effusion; Pleural Effusion, Malignant; Radioimmunoassay; ROC Curve; Sensitivity and Specificity

CYFRA 21-1 assay, measuring cytokeratin 19 fragments, was compared with carcinoembryonic antigen (CEA) assay, as an addition to cytological analysis for the diagnosis of malignant effusions. Both markers were determined with commercial enzyme immunoassays in pleural fluid from 196 patients. Cytological analysis and/or pleural biopsy confirmed the malignant origin of the effusion in 99 patients (76 carcinomas, nine pleural mesotheliomas and 14 non-epithelial malignancies). Effusions were confirmed as benign in 97 patients (33 cardiac failures, 39 infectious diseases--including 12 tuberculosis-- and 25 miscellaneous effusions). Both markers were significantly higher in malignant than in benign effusions. All the patients with non-epithelial malignancies presented CYFRA and CEA values lower than the 95% diagnostic specificity thresholds (100 and 6 ng ml(-1) respectively). The diagnostic sensitivity in the group of carcinomas and mesotheliomas was similar for CYFRA (58.8%) and CEA (64.7%). However, CEA had a significantly higher sensitivity in carcinomas (72.4% vs 55.3%), while CYFRA had a clearly higher sensitivity in mesotheliomas (89.9% vs 0%). Interestingly, 12 out of the 16 malignant effusions with a negative cytology were CEA and/or CYFRA positive. Regarding their high diagnostic sensitivity and their complementarity, CEA and CYFRA appear to be very useful for the diagnosis of malignant pleural effusions when cytology is negative.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Biomarkers, Tumor; Carcinoembryonic Antigen; Child; Female; Humans; Keratin-19; Keratins; Male; Middle Aged; Neoplasms; Pleural Effusion; Retrospective Studies; Sensitivity and Specificity

Fifty-two specimens of body cavity fluids from 52 patients were analyzed with conventional cytology, immunocytochemistry, and flow cytometric DNA ploidy methods to evaluate the most appropriate way of applying and interpreting immunocytochemistry and to evaluate the contribution of DNA ploidy analysis to conventional cytology in the diagnosis of body cavity fluids. The results suggest that conventional cytology still has an important role in the diagnosis of body cavity fluids. MOC 31 is the most sensitive monoclonal antibody for distinguishing benign mesothelial cells from malignant epithelial cells. Immunocytochemistry with the combination of cytokeratin, desmin, and MOC 31 with or without epithelial membrane antigen is suggested as a helpful ancillary method for the differential diagnosis of body cavity fluids. Flow cytometric DNA ploidy analysis also provides additional information in some difficult cases. Appropriate integration of clinical information and results of conventional cytology, immunocytochemistry, and flow cytometry are necessary to achieve the most accurate diagnosis in patients with effusion involving a body cavity.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Ascitic Fluid; Body Fluids; Cytodiagnosis; Desmin; DNA; Female; Flow Cytometry; Humans; Immunohistochemistry; Keratins; Male; Middle Aged; Mucin-1; Pleural Effusion; Ploidies; Prospective Studies

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Humans; Keratin-19; Keratins; Lung Neoplasms; Male; Mesothelioma; Middle Aged; Neoplastic Cells, Circulating; Pleural Effusion; Pleural Neoplasms

The role of tumour marker assays in differentiating malignant from benign pleural effusions is not yet clear. This study was designed to prospectively assess the individual and combined diagnostic utility of three tumour markers in patients with pleural effusion. Pleural and serum levels of carcinoembryonic antigen (CEA), carbohydrate antigen 15-3 (CA 15-3) and cytokeratin 19 fragment (CYFRA 21-1) were determined in 115 patients with pleural effusions (42 malignant and 73 benign). The diagnostic utility of each tumour marker was assessed using accuracy to determine the optimal cut-off point, whilst a logistic regression model was used to obtain the optimal combined test. In serum, every marker showed an individual high specificity (over 97%) for malignancy. The sensitivity of CEA, CA 15-3 and CYFRA 21-1 was 36, 48 and 31%, respectively. In patients without renal failure, the sensitivity of CYFRA 21-1 rose to 53%, while those of CEA and CA 15-3 remained almost unchanged. In pleural fluid, CYFRA 21-1 showed low sensitivity (32%) and specificity (82%), while CEA showed the highest sensitivity (57%). Excluding patients with renal failure, the combined determination in serum of CEA, CA 15-3 and CYFRA 21-1 has a high accuracy (88%), similar to that for CEA plus CA 15-3 in pleural fluid (87%). We conclude that CYFRA 21-1 is useless in pleural fluid and should not be used in serum for patients with renal failure. The combined determination of CEA, CA 15-3 and CYFRA 21-1 in serum may obviate its determination in pleural fluid.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoembryonic Antigen; Diagnosis, Differential; Exudates and Transudates; Female; Humans; Keratins; Male; Middle Aged; Mucin-1; Pleural Effusion; Pleural Effusion, Malignant; Renal Insufficiency; Sensitivity and Specificity

We have performed immunocytochemical, immunoelectron microscopy, Western blot, and culture techniques using monoclonal antibodies against cytokeratin, vimentin, and desmin on 17 benign and 20 malignant effusions of pleural and ascitic origin. Triple coexpression of these three antigens was observed in benign reactive mesothelial cells as well as in one case of mesothelioma. All metastatic adenocarcinoma cells were consistently negative to desmin and positive to cytokeratin and vimentin. Present results were helpful to distinguish reactive and malignant mesothelioma from metastatic carcinoma cells in effusions.

Topics: Adenocarcinoma; Ascitic Fluid; Blotting, Western; Cells, Cultured; Desmin; Diagnosis, Differential; Epithelium; Female; Follow-Up Studies; Humans; Immunoenzyme Techniques; Keratins; Mesothelioma; Models, Biological; Pleural Effusion; Vimentin

The nuclear DNA contents of atypical mesothelial cells from five patients who had an eosinophilic pleural effusion (EPE) were studied by the use of DAPI (4',6-diamidino-2-phenylindole dihydrochloride) DNA staining. Analysis of the nuclear DNA content revealed a polyploid pattern, with a major peak in the tetraploid region. Using an immunocytochemical technique, the atypical mesothelial cells showed a positive reaction for cytokeratin. In contrast carcinoembryonic antigen (CEA) was always negative in these cells. It is suggested that the atypical mesothelial cells with EPE had a higher rate of proliferation than did the normal mesothelial cells.

Topics: Adult; Aged; Biomarkers; Biomarkers, Tumor; Carcinoembryonic Antigen; Carcinoma, Squamous Cell; Cell Division; Cell Nucleus; Crohn Disease; DNA; Eosinophilia; Epithelium; Female; Humans; Indoles; Keratins; Lung Neoplasms; Male; Mediastinal Cyst; Membrane Glycoproteins; Mucin-1; Pleural Effusion; Pneumonia; Pneumothorax; Polyploidy

We examined the feasibility of performing non-radioactive in situ hybridization (ISH) in flow cytometrically sorted (tumor) cells with a chromosome #1 specific centromere probe. The study was performed in a model system of HL60 cells mixed with different quantities of HeLa cells. These latter cells were sorted directly onto poly-l-lysine coated glass slides on the basis of their keratin content, a cytoskeletal component not present in HL60 cells. Overall morphology of the separated HeLa cells was excellent and, after the ISH procedure, the appropriate number of ISH spots was observed in more than 85% of the sorted cells. This percentage did not differ significantly in cell mixtures with different percentages of HeLa cells (down to 1%). Sorting of HeLa cells in different phases of the cell cycle, and subsequent ISH, revealed the same spot number for chromosome #1 in all cell cycle stages, including mitosis. In the latter phase of the cell cycle we did not find a duplication of the chromosome #1 centromere, not even after sorting of the mitotic cells on the basis of specific labeling with an antibody to mitotin. The early G2 mitotin negative fraction, however, showed a significant percentage of cells with a duplicate spot number, most likely representing a tetraploid cell fraction in this HeLa cell culture. The protocol that evolved from these model studies was applied to cell suspensions of malignant body cavity effusions as well as solid bladder carcinomas. In several of these cases numerical chromosome aberrations could be detected by ISH more evidently after sorting on the basis of keratin labeling.

Topics: Antibodies, Monoclonal; Cell Cycle; Cell Cycle Proteins; Cell Separation; Centromere; Chromosomes, Human; Chromosomes, Human, Pair 1; DNA Probes; Flow Cytometry; HeLa Cells; Humans; Interphase; Keratins; Leukemia, Promyelocytic, Acute; Minichromosome Maintenance Complex Component 2; Neoplasms; Nuclear Proteins; Nucleic Acid Hybridization; Pleural Effusion; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Malignant mesothelioma (MM) is an aggressive tumour of the serosal cavities which is associated with exposure to asbestos. Studies of this tumour have been limited by a paucity of well-characterized human MM cell lines. In this study, 5 human MM cell lines were established from pleural effusions of patients with this malignancy. All 5 patients were males with known crocidolite asbestos exposure, who had received no treatment for their disease and in whom the diagnosis was confirmed by cytology, histology and electron microscopy (EM). These lines have been in culture from 11 to 25 months, and all of them for more than 18 passages. The appearance of the cells in culture was extremely varied; in 3 of the lines they were spindle-shaped with few vacuoles (JU77, LO68 and ONE58); in 1 line they had a thick, stellate shape with vacuoles (NO36) and in 1 they were very pleomorphic in both shape and size with irregular membranes and numerous vacuoles [DeH128 (M)]. Upon reaching confluence, cells in 3 of the 5 lines assumed the cobblestone-like pattern characteristic of epithelial-type cells, whereas in the other 2 (LO68 and ONE58) they remained spindle-shaped. All 5 lines demonstrated a loss of contact inhibition (i.e., piling) at confluence. Minimum doubling times varied significantly from 18 hr (JU77) to more than 30 hr [DeH128 (M)]. Cytological examination showed characteristic mesothelial/mesothelioma morphology, and epithelial membrane antigen (EMA) and cytokeratin were demonstrated in cells from all 5 lines. These cells lacked CEA and epithelial mucin. The presence of cell junctions, glycogen and numerous long, thin, branching microvilli was readily demonstrable by EM. All lines had abnormal karyotypes, with the modal chromosome number varying from 40 to 80. Variable chromosome numbers, numerous structural rearrangements and unrecognizable marker chromosomes were readily observed; however, the only consistent change seen was del 6q21 in 4 of the 5 lines. The establishment of these 5 cultured human MM cell lines now provides an opportunity for comparative study of several aspects of the biology of MM in vitro as well as screening new treatment modalities.

Topics: Adult; Asbestos; Carcinoembryonic Antigen; Cell Division; Cytoplasm; Glycogen; Humans; Karyotyping; Keratins; Male; Membrane Glycoproteins; Mesothelioma; Microscopy, Electron; Middle Aged; Mucin-1; Pleural Effusion; Polymorphism, Restriction Fragment Length; Tumor Cells, Cultured; Vacuoles

In order to assess the value of immunocytochemical staining as a method of discriminating between benign reactive mesothelial cells and malignant epithelial cells in serous effusions, we have studied the reactions of a panel of commercially available antibodies on cells harvested from 83 pleural and peritoneal fluids and compared the results with the clinical and cytological diagnoses. The antibodies used were raised against cytokeratin (PKK1), epithelial membrane antigen (EMA), carcino-embryonic antigen (CEA), pregnancy specific B1-glycoprotein (SP1) and leucocyte common antigen (LCA). Anti-CEA was positive in 16 of 39 effusions (41%) containing carcinoma cells. Pregnancy specific B1-glycoprotein (SP1) was positive in 33% of the same samples. Mesothelial cells did not stain with these antibodies. Thus anti-CEA and SP1 can be used to discriminate between benign mesothelial and malignant epithelial cells in effusions. Anti-PKK1 stained both benign reactive mesothelial cells and malignant epithelial cells and cannot be used to discriminate between these two cell types. Strong positive staining of malignant cells was noted with anti-EMA. However, as occasional weak staining of mesothelial cells was also noted, strong staining with this antibody may be regarded as suspicious but not conclusive of malignancy.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Ascitic Fluid; Carcinoembryonic Antigen; Cytodiagnosis; Humans; Immunoenzyme Techniques; Keratins; Membrane Glycoproteins; Mucin-1; Pleural Effusion; Pleural Effusion, Malignant

Topics: Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Ascites; Biomarkers, Tumor; Carcinoembryonic Antigen; Diagnosis, Differential; Humans; Immunoenzyme Techniques; Keratins; Membrane Glycoproteins; Mucin-1; Neoplasms; Pericardial Effusion; Pleural Effusion

The immunohistochemical profile (i.e., carcinoembryonic antigen, keratin proteins, epithelial membrane antigen, human milk fat globule-derived antigen, and mucin) of paraffin-embedded cell blocks of 20 malignant effusions from patients with malignant mesothelioma was compared with that of 39 malignant effusions from patients with metastatic adenocarcinoma to determine whether these markers distinguished between these tumor types. Twenty-three adenocarcinomas (59 per cent) stained for mucin. Immunoreactivity for carcinoembryonic antigen (CEA) was observed in 28 adenocarcinomas (72 per cent). All were immunoreactive for keratin proteins, and 29 adenocarcinomas (74 per cent), including seven that were mucin and CEA negative and exhibited a "peripheral predominant" staining pattern for keratin proteins. By contrast, none of the mesotheliomas stained for mucin or for CEA, and, although all were immunoreactive for keratin proteins, none demonstrated a peripheral predominant pattern of staining. Epithelial membrane antigen and milk fat globule-derived antigen were identified in the majority of both mesotheliomas and adenocarcinomas. Neither staining intensity nor pattern of reactivity of these markers clearly distinguished the tumors. This study of cell blocks of serous effusions suggests that staining for mucin, immunoreactivity for carcinoembryonic antigen, and a peripheral predominant pattern of reactivity for keratin proteins represent highly characteristic markers of adenocarcinomas, which identify the majority of these tumors (38 of 39) and allow their distinction from malignant mesotheliomas.

Topics: Adenocarcinoma; Antigens; Carcinoembryonic Antigen; Female; Humans; Immunoenzyme Techniques; Keratins; Male; Membrane Proteins; Mesothelioma; Middle Aged; Mucin-1; Mucins; Pleural Effusion; Pleural Neoplasms

The usefulness of tumor marker assay in pleural effusions for differential diagnosis is still debated. From the observation of common antigens on tissue polypeptide antigen (TPA) and keratins 8, 18 and 19 and vimentin, all substances contained in normal and neoplastic mesothelium, we felt it opportune to evaluate the use of TPA assay in 105 pleural effusions (46 benign and 59 malignant). The values were much higher than those found in blood. In hydrothorax the median value was 454 U/l (range, 59-1923), in exudative effusions 846 U/l (range, 258-4485), in metastatic pleural effusions 1277 U/l (range, 58-32352) and in mesotheliomas 7705 (range, 759-16000). The maximum value found in nonmalignant effusions was 4485 U/l; this value was taken as a cutoff level, so only 29.9% of the tumors were positive to the test. Our results showed this assay to be not very important for a differential diagnosis of malignant and nonmalignant pleural effusions. Nevertheless, the different TPA patterns in mesotheliomas (66.6% positive) and metastatic pleural effusions (15.9%) suggest that further studies are warranted.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Neoplasm; Diagnosis, Differential; Female; Humans; Keratins; Male; Mesothelioma; Middle Aged; Neoplasm Proteins; Peptides; Pleural Diseases; Pleural Effusion; Pleural Neoplasms; Tissue Polypeptide Antigen; Vimentin

The diagnostic value of an immunoperoxidase panel composed of antisera to carcinoembryonic antigen (CEA), epithelial membrane antigens (EMA), and high- and low-molecular-weight cytokeratins was evaluated on 39 consecutive pleural and peritoneal fluid specimens and correlated with routine cytologic and histochemical studies. The cases were classified into two groups: malignant (epithelial and small-cell undifferentiated carcinomas) and benign effusions. We found that the CEA and EMA antisera identified 91% and 100% of the epithelial malignancies, respectively. Mucin stains were positive in only 41% (mucicarmine) and 59% (Alcian blue) of these cases. The battery of cytokeratin markers identified malignant cells from 45%-100% of the cases but showed considerable positive staining of mesothelial cells. A selective review of the literature is presented along with recommendations for technical improvement in immunoperoxidase studies. We believe that an immunoperoxidase panel can be of considerable value in the cytologic diagnosis of epithelial malignancies in effusions. The panel composed of CEA and EMA can prove helpful in a routine cytology practice. Although the cytokeratin marker identified malignant cells, the concomitant immunostaining of mesothelial cells limits its utility. The commercially available panel can be a potential aid in improving the accuracy of serous fluid cytologic examination by decreasing both false-positive and false-negative diagnoses and thereby helping to prevent delays in diagnosis and treatment.

Topics: Ascitic Fluid; Carcinoembryonic Antigen; Cytodiagnosis; Female; Humans; Immunoenzyme Techniques; Keratins; Membrane Proteins; Molecular Weight; Mucin-1; Peritoneal Neoplasms; Pleural Effusion; Pleural Neoplasms

In serous effusions the distinction between reactive mesothelial cells and malignant cells (especially adenocarcinoma cells and malignant mesothelial cells) is frequently a cause of diagnostic difficulty. The present paper describes the immunocytochemical staining of cells in 76 effusions from 71 patients with different malignancies. In 91% of the effusions obtained from patients with adenocarcinomas, the cells stained positive for anti-EMA, 94% for anti-CEA, 64% for anti-LeuM1-antigen and 75% for anti-keratins. In more than 90% of the cases the reactive mesothelial cells stained positive for anti-keratins, but not for anti-EMA, anti-CEA or anti-LeuM1-antigen. It is concluded that a panel of the antibodies against EMA, CEA, LeuM1-antigen and keratins is valuable in the distinction between adenocarcinoma cells, malignant mesothelial cells and reactive mesothelial cells in serous effusions.

Topics: Adenocarcinoma; Antibodies; Antigens, Differentiation, T-Lymphocyte; Antigens, Surface; Ascitic Fluid; Carcinoembryonic Antigen; Histocompatibility Antigens; Humans; Keratins; Leukocyte Common Antigens; Membrane Proteins; Mesothelioma; Mucin-1; Pleural Effusion

Immunocytochemical methods were evaluated in order to find a practical one for cell identification on cytologic preparation of body fluids. The effects of fixatives and Triton X-100 treatment on the preservation of cell-type-specific antigens and cell morphologic characteristics were also examined. The method using the alkaline phosphatase-antialkaline phosphatase (APAAP) complex as the indicator is recommended because of its high specificity and sensitivity. With this method, currently available and potentially useful monoclonal antibodies were examined, and the antibodies that were useful for the identification of normal and neoplastic cells commonly present in body fluids were selected for practical applications.

Topics: Antibodies, Monoclonal; Ascitic Fluid; Cerebrospinal Fluid; Desmin; Exudates and Transudates; Glial Fibrillary Acidic Protein; Humans; Immunoenzyme Techniques; Intermediate Filaments; Keratins; Octoxynol; Pleural Effusion; Polyethylene Glycols; Preservation, Biological; Staining and Labeling

The interaction of chrysotile and amphibole asbestos fibers with the cytoskeleton of cultured human mesothelial cells from nontumoral pleural effusions was studied using scanning electron and immunofluorescence microscopy. Asbestos-exposed mesothelial cells show a massive annular condensation of cytokeratin filaments, forming a concentric ring enveloping the nucleus and the phagocytosed asbestos fibers. By detergent extraction of the cells it could be shown that the asbestos fibers are in close contact with the nuclear membrane and associated with the cytoskeletal framework of the cells. An association of cytokeratin filaments with the asbestos could be observed during phagocytosis of the fibers. The disturbance of the cell cytoskeleton and the close morphologic contact between asbestos fibers and the nuclear membrane may have some relevance in explaining the well-recognized carcinogenic effects of asbestos mineral fibers.

Topics: Antibodies, Monoclonal; Asbestos; Cells, Cultured; Cytoskeleton; Fibroblasts; Humans; Keratins; Microscopy, Electron, Scanning; Nuclear Envelope; Phagocytosis; Pleural Effusion

The method described below combines an immunoreaction with Papanicolaou's stain on cytological smears. For the immunoreaction, the avidin-biotin-complex (ABC) method was used. The method was tested on various cytological material with the monoclonal antibody lu-5 and two polyclonal antibodies (anti-keratin and anti-chymotrypsin). Wet fixation of the smears with a modified Delaunay's solution is recommended. Drying of the material impairs immunoreactivity. The main advantages of the technique are the clear-cut permanent immunostaining and the preservation of the nuclear structure, permitting a combined immuncytological characterization of cellular products and conventional cyto-diagnosis.

Topics: Antibodies, Monoclonal; Avidin; Biotin; Chymotrypsin; Cytodiagnosis; Cytological Techniques; Epithelium; Histocytochemistry; Humans; Immunologic Techniques; Keratins; Neoplasms; Pleural Effusion; Staining and Labeling

Immunohistological analysis of human tissue using monoclonal antibodies against cytokeratins, which are confined to cells of epithelial origin, is a valuable technique. Using human epidermal keratins as antigen, we prepared monoclonal antibodies against cytokeratins (ZK1, ZK7, ZK61 and ZK99) and against a desmosomal protein (ZK31). Immunohistochemical staining of human skin sections using these antibodies showed a specific reaction with the epidermis: ZK1 stained the entire epidermis, ZK7 only the basal layer, ZK61 and ZK99 the suprabasal layers, and ZK31 the cellular interfaces. In order to test for antibody specificity, immunoblots with human epidermal and amnion epithelial cytokeratin polypeptides, as well as immunofluorescence microscopy of simple epithelia (glandular and simple columnar epithelia) were performed. ZK1, ZK61 and ZK99 reacted preferentially with cytokeratin polypeptides of stratified squamous epithelia and ZK7 recognized cytokeratins of stratified and simple epithelia. When the ZK antibodies were tested on mesothelial cells in pleural effusions, only ZK7 reacted with these cells. Biochemical analysis of cytokeratin accumulation in cells of primary and long-term cultures indicated that the cytokeratin pattern of mesothelial cells was quite unstable, while that of amnion epithelial cells showed only minor quantitative changes. The use of these antibodies to determine the epithelial origin of cells present in pleural effusions is proposed.

Topics: Amnion; Antibodies, Monoclonal; Breast; Cells, Cultured; Cytoskeleton; Electrophoresis, Polyacrylamide Gel; Epidermal Cells; Epithelial Cells; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Intermediate Filaments; Keratins; Molecular Weight; Pleural Effusion

A method developed for the initiation and maintenance in primary culture of human normal mammary epithelial cells was adopted for the growth of epithelial cells from 45 primary human breast tumors. The cells were grown on a naturally produced extracellular matrix (ECM) or on regular tissue culture plastic in a serum-free medium containing growth supplements and high-density lipoprotein (HDL). Successful enzymatic dissociation of the tumor biopsy into organoid structures and cell aggregates was crucial for subsequent cell attachment and growth. Fifty-five percent of the biopsy specimens were successfully dissociated and 87% of these gave rise to actively dividing epithelial cells forming monolayer cultures. In contrast, only 21% of the biopsies which were not optimally dissociated yielded growing cultures. Variations in sample size, duration of enzymatic digestion, and tumor composition affected the outcome of tumor dissociation. Omission of serum from the culture medium prevented the growth of fibroblasts, while plating on ECM greatly improved and in some cases was essential for cell attachment and subsequent outgrowth. The epithelial nature of the cells was verified by their cuboidal and closely apposed morphology and positive staining with antikeratin antibodies. The growth and subculture requirements and the expression of the B38.1 tumor marker were compared in human mammary epithelial cells derived from solid tumors, pleural effusion and normal breast tissue.

Topics: Antibodies, Neoplasm; Biopsy; Blood; Breast Neoplasms; Cell Division; Cells, Cultured; Cytological Techniques; Epithelial Cells; Extracellular Matrix; Female; Humans; Keratins; Microscopy, Phase-Contrast; Pleural Effusion

An enzyme-linked immunosorbent assay (ELISA) for anti-keratin antibodies was prepared by coating microplates with epidermal keratin purified from the stratum corneum from human foot. Naturally occurring auto-antibodies bind keratin via their f(ab')2 fragment. They were assayed in the serum from 65 healthy people. The serum titer increased significantly with aging. Auto-antibodies stained all the layers of a normal human epidermis whereas an immune serum, prepared by injecting a rabbit with pure human keratin proteins, stained more efficiently the outer layers of the human skin; pre-immune rabbit serum did not stain human skin at all. The lowest anti-keratin activities were observed in serum from patients with squamous cell lung carcinoma and with mesothelioma. The activity was lower in pleural fluid from patients with pleural mesothelioma than in pleural fluid from other types of cancer. This is possibly due to the fixation of autoantibodies onto the pleural tumor or on cell debris arising from the tumor.

Topics: Animals; Antibodies; Autoantibodies; Carcinoma, Squamous Cell; Enzyme-Linked Immunosorbent Assay; Histocytochemistry; Humans; Immunoenzyme Techniques; Keratins; Lung Diseases; Lung Neoplasms; Mesothelioma; Pleural Effusion; Rabbits

A panel of seven monoclonal antibodies was applied to smears of cell deposit from 70 pleural and peritoneal fluids, using an immunoalkaline phosphatase (IAP) procedure. The cases were chosen to show typical cytological patterns, both benign and malignant, and in this way the diagnostic value of the method could be assessed. The antibodies used were 2D1 (anti-leucocyte), Ca 1, HMFG-2 (anti-milk fat globule membrane), LE61 and M73 (both anti-intermediate filament antibodies), anti-CEA, and K92 (anti-keratin). The anti-leucocyte antibody was found useful for distinguishing lymphoma from carcinoma. Anti-CEA gave positive reactions in 80% of carcinoma cases and was not seen to react with any other cell types. Ca 1 was positive with some cells in 95% of carcinoma cases, but mesothelial cells reacted with it in two cases. A strong reaction with the anti-milk fat globule membrane antibody was very constant in carcinoma but was also seen in mesothelial cells in 30% of benign effusions. The anti-keratin reacted with malignant cells in only a small proportion of cases. The antibodies against epithelial intermediate filaments reacted equally strongly with benign mesothelial cells and carcinoma cells, but gave negative reactions with lymphoma cells. It is concluded that a suitably chosen panel of monoclonal antibodies can be of great value in identifying neoplastic cells in serous effusions.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Ascitic Fluid; Carcinoembryonic Antigen; Cytoskeleton; Female; Humans; Immunoenzyme Techniques; Keratins; Leukocytes; Male; Membrane Proteins; Mucin-1; Neoplasms; Pleural Effusion; Staining and Labeling

An immunoperoxidase technique employing antibody to prekeratin was used to study distribution and pattern of staining of prekeratin filaments in cytological smears obtained from 42 specimens of pleural and peritoneal effusions (27 benign, 15 malignant). The smears were either air-dried or ethanol-fixed. Both benign and malignant mesothelial cells showed distinctive peripheral or perinuclear staining patterns which differed from the characteristic arborizing pattern in adenocarcinoma cells. The ultrastructure of these 2 cell types studied in 27 body fluids (12 benign, 15 malignant) and in 13 malignant tumors (3 mesotheliomas, 10 adenocarcinomas) showed a distinctive localizaton of intermediate filaments which corresponded to and could explain the pattern of staining obtained using the immunoperoxidase technique. The immunohistochemical and ultrastructural findings appeared characteristic for benign and malignant mesothelial cells as well as for adenocarcinoma cells, and could be used as markers to differentiate mesothelial tumors and reactive mesothelial cells from adenocarcinomas.

Topics: Adenocarcinoma; Ascitic Fluid; Diagnosis, Differential; Humans; Immunoenzyme Techniques; Keratins; Lung Neoplasms; Mesothelioma; Pleural Effusion; Protein Precursors

Topics: Amino Acids; Animals; Biopsy; Cattle; Chondroitin; Chromatography; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis; Glycosaminoglycans; Heparin; Humans; Hyaluronic Acid; Hyaluronoglucosaminidase; Hydrolysis; Keratins; Male; Mesothelioma; Neoplasm Metastasis; Neoplasm Proteins; Oxidation-Reduction; Periodic Acid; Pleural Effusion; Pleural Neoplasms; Proteins; Staining and Labeling; Sulfuric Acids; Testis; Vitreous Body