Development of a sensitive, specific reverse transcriptase polymerase chain reaction-based assay for epithelial tumour cells in effusions.
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We developed a sensitive and specific method for the detection of epithelial cancer cells in effusions with a two-stage molecular-based assay which combined enrichment for cancer cells by immunomagnetic bead selection and reverse transcriptase polymerase chain reaction (RT-PCR) detection of epithelial glycoprotein 2 (EGP-2) RNA. Preliminary experiments indicated that immunobead selection was essential to avoid occasional false-positive RT-PCR results, and this method detected ten breast cancer cells electively added to 10(7) cytologically negative effusion cells. We studied 110 cases of pleural (n = 68) and peritoneal (n = 42) effusions (30 from patients with known carcinoma and 80 from those without known carcinoma), and the results were compared with cytological findings. Of 18 effusions that were cytologically positive or suspicious for malignant cells, 17 (94%) were positive for EGP-2 RNA (the one negative sample was from a patient who recently received combination chemotherapy). Of 92 cytologically negative samples, 11 (12%) were positive for EGP-2, including six patients with a history of previous or current carcinoma. Our method appears to be highly specific and increases the sensitivity of detection of malignant cells; it may be a useful adjunct to routine cytopathological examination. (+info)
Differential diagnostic significance of the paucity of HLA-I antigens on metastatic breast carcinoma cells in effusions.
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Distinction between benign reactive mesothelial cells and metastatic breast adenocarcinoma cells in effusions from patients with a known prior history of breast cancer is not the easiest task in diagnostic pathology. Here, we report the usefulness of testing the expression of class I HLA antigens (HLA A, B, C) in this respect. Cytospins were prepared from effusions of patients without the history of breast cancer (5 cases) and from effusions of patients with infiltrating ductal carcinoma (11 cases). Three effusions from cancerous patients were not malignant cytologically. The expression of HLA-A, B, C, HLA-DR and beta2-microglobulin as well as the macrophage antigen, CD14, was evaluated by immunocytochemistry. In 10 of 11 effusions the cytologically malignant cells expressed very weak or undetectable HLA-A,B,C as compared to the mesothelial cells and macrophages. The paucity of expression of HLA-A, B, C was detectable in those 3 cases where a definitive cytological diagnosis of malignancy could not be established. In contrast, mesothelial cells and macrophages from all samples were uniformly and strongly positive for both HLA-A, B, C and beta2-microglobulin. We conclude that the paucity of HLA-I antigens provides a marker helpful in distinguishing metastatic breast carcinoma cells from reactive mesothelial cells in effusions. (+info)
Phase I study of intrapleural batimastat (BB-94), a matrix metalloproteinase inhibitor, in the treatment of malignant pleural effusions.
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Tumor cells and associated stromal cells secrete matrix metalloproteinases (MMPs), contributing to invasion, angiogenesis, and metastasis. Batimastat (BB-94) is a broad-spectrum MMP inhibitor that causes resolution of ascites and/or tumor growth delay in animal models of breast, ovarian, and colorectal cancer. We recruited 18 patients with cytologically positive malignant pleural effusions into a Phase I study of intrapleural BB-94. Three patients received single doses of BB-94 at each dose level: 15, 30, 60, 105, 135, and 300 mg/m2. Two patients were retreated with a second course at 60 and 105 mg/m2. BB-94 was detectable in plasma 1 h after intrapleural administration, and peak levels of 20-200 ng/ml occurred after 4 h to 1 week. BB-94 persisted in the plasma for up to 12 weeks, at levels exceeding the IC50s for target MMPs. Peak values were higher, and persistence in the plasma was longer after higher doses of BB-94. The treatment was well tolerated. Toxic effects included low-grade fever for 24-48 h (6 of 18 patients, 33%) and reversible asymptomatic elevation of liver enzymes (8 patients, 44%). Toxicity seemed unrelated to BB-94 dose or plasma levels. Sixteen patients evaluable for response required significantly fewer pleural aspirations in the 3 months after BB-94 compared with the 3 months before. Seven patients (44%) required no further pleural aspiration until death/last follow-up. After 1 month, patients treated with 60-300 mg/m2 BB-94 had significantly better dyspnea scores, indicating improved exercise tolerance, compared with baseline scores the day after BB-94. The maximum tolerated intrapleural dose remains to be defined, but it is clear that intrapleural BB-94 is well tolerated, with evidence of local activity. (+info)
High pleural fluid hyaluronan concentrations in rheumatoid arthritis.
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Previous studies have shown that high pleural fluid (Pf) hyaluronan (HYA) concentrations may be due not only to malignant mesothelioma but also to inflammatory diseases. The objective of this study was to evaluate Pf-HYA in various nonmalignant inflammatory pleural disorders. A radiometric assay was used to determine HYA in Pf and serum (S) of 126 patients, 12 of whom had rheumatoid arthritis (RA), 22 tuberculosis, 22 pneumonia, 41 lung cancer, 10 malignant mesothelioma and 19 congestive heart failure. Pf-HYA values were correlated with values for Pf-tumour necrosis factor (TNF)-alpha and Pf-interleukin (IL)-1beta, as determined by radioimmunoassay. The highest median Pf-HYA (125.6 mg x L(-1), range 0.04-386.5 mg x L(-1)) occurred in patients with malignant mesothelioma. Among patients with nonmalignant inflammatory diseases, significantly higher median Pf-HYA were observed in those with rheumatoid arthritis (64.2 mg x L(-1), range 25.8-106.9 mg x L(-1)) than in those with tuberculosis (25.5 mg x L(-1), range 14.9-57.1 mg x L(-1), p<0.0005) or pneumonia (20.9 mg x L(-1), range 9.5-129.4 mg x L(-1), p<0.005). There was no correlation between Pf-HYA and S-HYA. Pf-HYA correlated positively with Pf-TNF-alpha (r=0.62) and Pf-IL-1beta (r=0.52). High pleural fluid hyaluronan occurs not only in malignant mesothelioma, but also in certain nonmalignant inflammatory diseases, especially rheumatoid arthritis. One explanation for the increase in pleural fluid hyaluronan may be local production of proinflammatory cytokines, such as tumour necrosis factor-alpha and interleukin-1beta. (+info)
Establishment of four new mesothelioma cell lines: characterization by ultrastructural and immunophenotypic analysis.
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The aim of this study was to assess the biological characteristics of four new malignant mesothelioma (MM) cell lines. Since simian virus (SV)40 sequences have been recently detected in MM, SV40 large T antigen (Tag) expression was also analysed. MM cell lines were characterized by morphological, ultrastructural and cytogenetic analysis. Expression of Tag and of relevant MM markers was studied by immunocytochemistry, surface antigens by indirect immunofluorescence and immunomodulating cytokines by enzyme-linked immunosorbent assay (ELISA). The four MM cell lines, established from pleural effusions, showed a slow proliferation rate and pleomorphic changes during culture. Cell lines expressed vimentin, cytokeratins 8 and 18, and the mesothelial antigen recognized by HBME-1 monoclonal antibody, but not carcinoembryonic antigen. Surface human leukocyte antigen (HLA)-class I and intercellular adhesion molecule (ICAM)-1 molecules were present on all the cell lines. While HLA class II and CD86 were constitutively undetectable, HLA-class II was present after interferon (IFN)-gamma stimulation. All cell lines displayed abnormal karyotypes with chromosome 6 abnormalities. Transforming growth factor (TGF)-beta2 and interleukin (IL)-6 were constitutively secreted, while tumour necrosis factor (TNF)-alpha was secreted only in response to lipopolysaccharide. Intranuclear Tag was expressed in two cell lines. The persistence of large T antigen with human leukocyte antigen class I and intercellular adhesion molecule-1 positivity may point to large T antigen as a target for cytotoxic T-lymphocyte-based immunotherapy in some malignant mesothelioma patients. (+info)
Overexpression of sialomucin complex, a rat homologue of MUC4, inhibits tumor killing by lymphokine-activated killer cells.
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Sialomucin complex (SMC) is a large heterodimeric glycoprotein complex composed of a mucin subunit ascites sialoglycoprotein-1 and a transmembrane subunit ascites sialoglycoprotein-2. It is a rat homologue of human mucin gene MUC4 and is abundantly expressed on the cell surface of highly metastatic ascites 13762 rat mammary adenocarcinoma cells. Because of their extended and rigid structures, mucin-type glycoproteins are suggested to have suppressing effects on cell-cell and cell-matrix interactions. During the metastatic process, these effects presumably cause tumor cell detachment from the primary tumor mass and facilitate escape of the tumor cells from immunosurveillance. Analyses of human breast cancer cells in solid tumors and tumor effusions showed that the more aggressive cells in effusions are stained with polyclonal antibodies against SMC more frequently than cells in solid tumors, suggesting a role for MUC4/SMC in tumor progression and metastasis. Previously, we generated recombinant cDNAs for SMC that vary in the number of mucin repeats to study the putative functions of SMC in tumor metastasis. These cDNAs were transfected into human cancer cell lines and tested for the effect of the expression of this gene. Here, using a tetracycline-responsive inducible expression system, we demonstrate that overexpression of SMC masks the surface antigens on target tumor cells and effectively suppresses tumor cell killing by cytotoxic lymphocytes. This effect results from the ability of SMC to block killer cell binding to the tumor cells and is dependent on both overexpression of the mucin and the number of mucin repeats in the expressed SMC. These results provide an explanation for the proposed role of SMC/MUC4 in tumor progression. (+info)
Multi-institutional randomized clinical study on the comparative effects of intracavital chemotherapy alone versus immunotherapy alone versus immunochemotherapy for malignant effusion.
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The current prospective randomized study was designed to compare the effects of intracavitary (i.c.) chemotherapy vs immunotherapy vs immunochemotherapy for malignant effusion. Between 1992 and 1995, a total of 42 patients with malignant effusion were registered, and 41 patients were eligible for statistical analysis. The primary diseases of the eligible patients included 27 gastric, four colorectal, four pancreatic, three lung, two liver and one oesophageal cancers. The patients with malignant effusion were randomly assigned into one of three i.c. therapeutic regimens: chemotherapy alone with weekly injection of anticancer agents (ACAs: cisplatin, mitomycin-C, adriamycin, etc.) (Group A, n = 13); immunotherapy alone with weekly injection of streptococcal preparation OK-432 (Group B, n = 14); or immunochemotherapy with ACAs and OK-432 (Group C, n = 14). The response of the effusion, patient survival and the kinetics of cytokines in the effusion were compared. There were no differences in the patients' backgrounds. The side-effects of the regimens included pain, anorexia, fever, leucopenia and anaemia and there were no differences in their incidence among the three groups. One patient died after cisplatin (CDDP) administration in Group A. Cytologic examination revealed that tumour cells in the effusion disappeared in 23% of Group A cases, 36% of Group B cases and 36% of Group C cases. The malignant effusion did not disappear in any of the Group A cases; however, the effusion disappeared in 29% of Group B cases and 43% of Group C cases (P = 0.03, Group A vs Group C). Furthermore, the 50% survival period was 1.6 months for Group A, 2.4 months for Group B and 3.5 months for Group C. The 6-month survival rate was 7% for Group A, 6% for Group B and 34% for Group C, and the 1-year survival rate was 0%, 0% and 17% respectively (P = 0.048, Group A vs Group C by the log-rank test). The analysis of the cytokine kinetics revealed a prominent increase in the level of interleukin-6 in the effusion in Group C. These results suggest that i.c. immunochemotherapy with OK-432 and ACAs may be more beneficial than i.c. chemotherapy alone or immunotherapy alone. (+info)
Vascular endothelial growth factor (VEGF) in inflammatory and malignant pleural effusions.
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BACKGROUND: Investigation and management of pleural effusions is an important clinical problem yet the pathogenesis of pleural fluid accumulation is poorly understood. Vascular endothelial growth factor (VEGF) is a potent inducer of capillary permeability that is produced by both malignant and inflammatory cells. A study was undertaken to determine whether VEGF has a potential pathogenic role in the development of pleural effusions and whether VEGF receptors are present on human pleural mesothelial cells. METHODS: Normal and inflamed pleura were examined immunohistochemically for the presence of FLT-1 (the fms-like tyrosine kinase receptor of VEGF). VEGF levels were measured by ELISA in 78 consecutive patients presenting with undiagnosed unilateral pleural effusions and the levels were correlated with the aetiology of the effusions. RESULTS: Immunohistochemical staining of normal and diseased pleura demonstrated the presence of the FLT-1 VEGF receptor on human mesothelial cells. Median VEGF levels were 2500 pg/ml in the malignant group and 305 pg/ml in the non-malignant group (median difference 1397.5 pg/ml (95% CI 851 to 2693), p<0.005). Median VEGF levels varied according to tumour histology. VEGF levels were also significantly raised compared with transudates (median 36.5 pg/ml) in empyema (4651 pg/ml (95% CI 833 to 10 000), p<0.001) and parainfectious effusions (360 pg/ml (95% CI 46 to 597), p<0.005). CONCLUSIONS: This first report of VEGF receptors on pleural mesothelial cells has indicated a potential mechanism for the biological activity of VEGF on pleural tissue. VEGF levels are raised in the majority of exudative effusions, implying a pathogenic role for this molecule in the development of pleural effusions. (+info)