Cells In Malignant Pleural Effusion Research Articles

Activation of Vα24NKT cells in malignant pleural effusion in patients with lung cancer

Valpha24NKT cells are lymphocytes expressing both T-cell antigen receptors and NK-cell antigen receptors on their cell surface and are involved in tumor immunity. They exert their antitumor effects after being activated by a specific ligand, alpha-galactosyl ceramide (alpha-GalCer). Malignant pleural effusion, a frequently occurring complication in patients with lung cancer, contains numerous lymphocytes. In the present study, we examined the presence and functions of Valpha24NKT cells in the lymphocytes in pleural effusion in vitro. The subjects were 13 untreated patients with primary lung cancer, who suffered malignant pleural effusion as a complication and who were treated between April 2004 and October 2007 at our hospital. Mononuclear cells were separated from the malignant pleural effusion and incubated with alpha-GalCer and IL-12. The production of IFN-gamma and IL-4 after incubation and the proportion of the Valpha24NKT cells before and after incubation were determined and compared. In the group cultured with alpha-GalCer alone, no significant increase in IFN-gamma production was observed in comparison with the control group. In the group cultured with alpha-GalCer+IL-12, IFN-gamma production increased significantly in comparison with the control group, and the proportion of Valpha24NKT cells increased after incubation. IL-4 production was very much lower than IFN-gamma production. Valpha24NKT cells were present in malignant pleural effusion in patients with lung cancer, but IFN-gamma production did not increase after addition of alpha-GalCer alone. The Valpha24NKT cells were activated by alpha-GalCer in the presence of IL-12. The Valpha24NKT cells in malignant pleural effusion were not activated by alpha-GalCer alone, suggesting that Valpha24NKT cell function is attenuated in malignant pleural effusion.

The Malignant Pleural Effusion as a Model to Investigate Intratumoral Heterogeneity in Lung Cancer

Malignant Pleural Effusions (MPE) may be useful as a model to study hierarchical progression of cancer and/or intratumoral heterogeneity. To strengthen the rationale for developing the MPE-model for these purposes, we set out to find evidence for the presence of cancer stem cells (CSC) in MPE and demonstrate an ability to sustain intratumoral heterogeneity in MPE-primary cultures. Our studies show that candidate lung CSC-expression signatures (PTEN, OCT4, hTERT, Bmi1, EZH2 and SUZ12) are evident in cell pellets isolated from MPE, and MPE-cytopathology also labels candidate-CSC (CD44, cMET, MDR-1, ALDH) subpopulations. Moreover, in primary cultures that use MPE as the source of both tumor cells and the tumor microenvironment (TME), candidate CSC are maintained over time. This allows us to live-sort candidate CSC-fractions from the MPE-tumor mix on the basis of surface markers (CD44, c-MET, uPAR, MDR-1) or differences in xenobiotic metabolism (ALDH). Thus, MPE-primary cultures provide an avenue to extract candidate CSC populations from individual (isogenic) MPE-tumors. This will allow us to test whether these cells can be discriminated in functional bioassays. Tumor heterogeneity in MPE-primary cultures is evidenced by variable immunolabeling, differences in colony-morphology, and differences in proliferation rates of cell subpopulations. Collectively, these data justify the ongoing development of the MPE-model for the investigation of intratumoral heterogeneity, tumor-TME interactions, and phenotypic validation of candidate lung CSC, in addition to providing direction for the pre-clinical development of rational therapeutics.

A Central Role for Tumor-derived Monocyte Chemoattractant Protein-1 in Malignant Pleural Effusion

Tumor cells in malignant pleural effusions (MPEs) are an important source of monocyte chemoattractant protein (MCP)-1. However, the role of tumor-derived MCP-1 in the pathogenesis and progression of MPE has not been determined. B16 mouse skin melanoma cells, which are deficient in MCP-1 expression, and mouse Lewis lung cancer (LLC) cells, which express high levels of MCP-1, were engineered to stably express MCP-1 and short hairpin RNAs (shRNAs) targeting the MCP-1 transcript, respectively. Cells were injected into the pleural cavities of syngeneic immunocompetent mice, and MPE volume and pleural tumors were quantified at necropsy (day 14). MCP-1 and other mediators were determined by cytometric bead array and enzyme-linked immunosorbent assay, and mononuclear and endothelial cells were identified by immunolabeling of F4/80 and factor VIII-related antigen respectively. Mouse survival was assessed using Kaplan-Meier analysis. Vascular permeability in mice with MPE was assessed using albumin-binding Evans blue. Statistical tests were two-sided. LLC cells expressing shRNA against MCP-1 elaborated less than 5% of the MCP-1 level in cells expressing nonspecific shRNA (control cells), and intrapleural delivery of these cells resulted in less MPE (mean MPE volume = 86 and 585 muL, respectively; difference = 499 muL; 95% confidence interval [CI] = 331 to 669 muL; P < .001), reduced MCP-1 levels in the pleural fluid, and lower mortality than when control cells were delivered. Overexpression of MCP-1 in intrapleurally injected B16 melanoma cells led to increased MPE and reduced survival. In mice with MPE, MCP-1 was a potent inducer of vascular permeability, mononuclear recruitment, and, in pleural tumors, of angiogenesis. MCP-1 produced by tumor cells is an important determinant of their capacity to induce the formation of MPE and may be a useful target for the treatment of malignant pleural disease.

Lysis of cancer cells by autologous T cells in breast cancer pleural effusates treated with anti-EpCAM BiTE antibody MT110

In the present study, the efficacy of a new drug, i.e. the bispecific single-chain antibody MT110 targeting the epithelial antigen EpCAM and the T-cell antigen CD3 was tested ex vivo in malignant pleural effusions (MPEs). EpCAM+ epithelial cells were found in 78% of the MPEs (n = 18). Ex vivo treatment of seven MPEs resulted in a dose-dependent specific lysis of 37 +/- 27% (+/- SD) EpCAM+ cells with 10 ng/ml (P = 0.03) and 57 +/- 29.5% EpCAM+ cells with 1,000 ng/ml MT110 (P = 0.016) after 72 h. As a prerequisite for redirected lysis, stimulation of the autologous CD4+ and CD8+ cells in MPE by 1,000 ng/ml MT110 resulted in 21 +/- 17% CD4+/CD25+ and 29.4 +/- 22% CD8+/CD25+ cells (P = 0.016, respectively) after 72 h. This was confirmed by a 22-fold release of TNF-alpha and 230-fold release of IFN-gamma (1,000 ng/ml, 48 h, P = 0.03, respectively). Thus, relapsed breast cancer patients resistant to standard treatment might benefit from targeted therapy using MT110.

Effects of the trifunctional antibody catumaxomab (anti-EpCAM x anti-CD3) on proliferation and cytokine secretion of immune cells in malignant pleural effusion

3046 Background: The trifunctional antibody catumaxomab specifically binds EpCAM+ tumor cells, CD3+ T lymphocytes and accessory cells via FcγR I/III. Thus the antibody induces tumor specific cell mediated cytotoxicity in vitro and in vivo. Following last years’ ASCO presentation on a phase I/II trial showing safety and efficacy of repetitive intrapleural administration of catumaxomab in patients with EpCAM positive, malignant pleural effusion (MPE), we now present data on the responsiveness of immune cells from pleural fluid to catumaxomab. Methods: Pleural fluid of patients with EpCAM-positive MPE treated with i.pl. catumaxomab was collected before treatment. Cells were harvested from the fluid and cultured ± 100 ng/ml catumaxomab using an in vitro proliferation assay. After 72 h of culture, proliferation of T cells (CD4+ and CD8+) and monocytes (CD11c+) was determined. In addition, cell supernatants after 24h incubation ± catumaxomab were analysed for their TH1/TH2 cytokine profile (IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF- a). Results: Incubation in presence of catumaxomab led to a pronounced increase of CD4+ CD8+ and CD11+ cell numbers indicating a proliferation of these cells, whereas cultures without catumaxomab showed no proliferation of immune cells. Analysis of supernatants after 24 h revealed levels of IL-2, IL-6, IFN-γ and TNF-a, from cells incubated with catumaxomab that were distinctly higher than in cultures without catumaxomab. Conclusions: The immunologic nature of catumaxomab-induced responses in patients with pleural effusion accompanied by a reduction of tumor cells, could be underlined impressively with in vitro data obtained from pleural cells showing catumaxomab-induced proliferation of T cells and accessory cells and TH1-directed cytokine secretion. The data are equivalent to results observed in the peritoneal fluid of ascites patients. [Table: see text]

CD4+CD25+Regulatory T Lymphocytes in Malignant Pleural Effusion

Active suppression by CD4(+)CD25(+) regulatory T lymphocytes plays an important role in the downregulation of T-cell responses to foreign and self-antigens. To analyze whether the CD4(+)CD25(+) regulatory T lymphocytes exist and function normally in malignant pleural effusion. The percentages of CD4(+)CD25(+) T lymphocytes in pleural effusion and peripheral blood from patients with lung cancer with malignant effusion, pleural lavage and peripheral blood from patients with lung cancer without effusion, and peripheral blood from healthy control subjects were determined by flow cytometry. The expressions of forkhead transcription factor Foxp3 and cytotoxic lymphocyte-associated antigen-4 were also examined. CD4(+)CD25(+) and CD4(+)CD25(-) T cells from pleural effusion and peripheral blood were isolated, and were cultured to observe the effects of CD4(+)CD25(+) cells on proliferation response of CD4(+)CD25(-) T cells in vitro. There were increased numbers of CD4(+)CD25(+) T cells in malignant pleural effusion from patients with lung cancer compared with pleural lavage from patients with lung cancer without pleural effusion, and that these cells have constitutive high-level expression of Foxp3 and cytotoxic lymphocyte-associated antigen-4. Furthermore, CD4(+)CD25(+) T cells mediate potent inhibition of proliferation response of CD4(+)CD25(-) T cells, and anticytotoxic lymphocyte-associated antigen-4 monoclonal antibody could reduce the inhibitory activity of CD4(+)CD25(+) T cells. The increased CD4(+)CD25(+) T cells found in malignant pleural effusion express high levels of Foxp3 transcription factor and potently suppress the proliferation of CD4(+)CD25(-) T cells, and cytotoxic lymphocyte-associated antigen-4 is involved in the suppressive activity of pleural CD4(+)CD25(+) T cells.

Microarray gene expression analysis of cells isolated from malignant pleural effusions (MPE) from patients with non-small cell lung cancer (NSCLC)

7369 Background: Many patients with advanced NSCLC are diagnosed via needle biopsy, providing little tissue for molecular profiling of their cancer. MPE may offer an easily accessible source of cancer cells in these patients. Methods: We collected pleural fluid from 14 patients with advanced NSCLC and MPE. Cells were isolated and treated as follows: 1) total RNA extraction (n=6); 2) positive selection of malignant cells using immunomagnetic beads coated with Ber-EP4 antibody followed by total RNA extraction (n=5); 3) both immunobead and non-immunobead methods (n=3). Cytologic examination was performed in all cases. RNA quality and quantity were assessed using the Agilent 2100 bioanalyzer system (Agilent Technologies, Palo Alto, CA). Cases with RNA suitable for gene expression analysis were assayed using oligonucleotide-based microarray (Affimetrix chips U133A and B). Results: All patients in the study had carcinoma cells by cytology, except one. The MPEs in 3 of 14 patients (21%) yielded RNA suitable for microarray analysis, including the patient whose MPE was cytologically negative for cancer. The presence of good quality RNA did not appear to correlate with specimen cellularity, the percentage of tumor cells versus other types of cells seen on cytologic examination, the time interval between collection and extraction, or whether or not immunobead separation was performed. Two cases that were cytologically positive for cancer had a much higher level of Mucin 5b (a specific tracheobronchial mucin) gene expression than the patient with cytologically negative MPE. Conclusion: RNA of sufficient quality for gene microarray expression analysis can be obtained from cells in MPE using a variety of techniques. More studies are required to determine whether the RNA signal obtained is cancer-specific. No significant financial relationships to disclose.

Microarray gene expression analysis of cells isolated from malignant pleural effusions (MPE) from patients with non-small cell lung cancer (NSCLC)

7369 Background: Many patients with advanced NSCLC are diagnosed via needle biopsy, providing little tissue for molecular profiling of their cancer. MPE may offer an easily accessible source of cancer cells in these patients. Methods: We collected pleural fluid from 14 patients with advanced NSCLC and MPE. Cells were isolated and treated as follows: 1) total RNA extraction (n=6); 2) positive selection of malignant cells using immunomagnetic beads coated with Ber-EP4 antibody followed by total RNA extraction (n=5); 3) both immunobead and non-immunobead methods (n=3). Cytologic examination was performed in all cases. RNA quality and quantity were assessed using the Agilent 2100 bioanalyzer system (Agilent Technologies, Palo Alto, CA). Cases with RNA suitable for gene expression analysis were assayed using oligonucleotide-based microarray (Affimetrix chips U133A and B). Results: All patients in the study had carcinoma cells by cytology, except one. The MPEs in 3 of 14 patients (21%) yielded RNA suitable for microarray analysis, including the patient whose MPE was cytologically negative for cancer. The presence of good quality RNA did not appear to correlate with specimen cellularity, the percentage of tumor cells versus other types of cells seen on cytologic examination, the time interval between collection and extraction, or whether or not immunobead separation was performed. Two cases that were cytologically positive for cancer had a much higher level of Mucin 5b (a specific tracheobronchial mucin) gene expression than the patient with cytologically negative MPE. Conclusion: RNA of sufficient quality for gene microarray expression analysis can be obtained from cells in MPE using a variety of techniques. More studies are required to determine whether the RNA signal obtained is cancer-specific. No significant financial relationships to disclose.

Immunosuppressive mechanisms in the microenvironment of malignant pleural effusions

Malignant effusions in serous cavities constitute a unique milieu for direct contact of tumor cells with host lymphoid cells in a fluid phase. The aim of this study was to depict agents responsible for suppression of lymphoid cells with putative anti-tumor potential. Pleural effusions drawn from 44 (18 non-malignant and 26 malignant) patients were tested for selected cytokines—interleukin-10 (IL-10), transforming growth factor beta (TGF-β1) and soluble Fas ligand (sFasL) and nuclear membrane proteins (NMPs) content by ELISA. TCR-ζ expression of T cells and TUNEL reaction for apoptosis were evaluated by three color flow cytometry. Both cytokine concentrations were found to be significantly elevated in malignant pleural effusions (MPE) as compared to non-malignant ones. It was also true for sFasL content. Moreover, NMPs corresponding to decoy cell fragments, were also heightened in MPE. Concentrations of NMPs correlated with the percent of apoptotic (TUNEL+) T CD3+ lymphocytes and inversely correlated with the percent of T cells. The low expression of TCR-ζ chain on T cells corresponded to high concentration of sFasL in MPE. In conclusion, the above data suggest that out of three suppression agents tested, only sFasL appears to show correlation with the downregulation of T cells in MPE.

Stromal-derived factor-1alpha/CXCL12-CXCR 4 axis is involved in the dissemination of NSCLC cells into pleural space.

Malignant pleural effusion (PE) is one of the poor prognostic factors in non-small cell lung cancer (NSCLC), and the detailed mechanism of the malignant PE formation is not fully elucidated. Recently, CXCR4, a receptor for chemokine stromal-derived factor-1alpha (SDF-1alpha) that can induce chemotaxis of cells, was reported to be expressed on NSCLC. In this study, we hypothesized that the SDF-1alpha/CXCR4 axis may be involved in the dissemination of malignant cells into pleural space, and investigated its expression, function, and signaling pathway using NSCLC cell lines and clinical samples from 43 patients with NSCLC with malignant PE. We found functional expression of CXCR4 on NSCLC cell lines, and also found that SDF-1alpha could induce migration via phosphatidylinositol 3 (PI-3) kinase- and p44/42 mitogen-activated protein kinase-dependent manner. The SDF-1alpha levels in malignant PE were significantly higher than those in transudate PE and showed a significant positive correlation with PE volumes. The sensitivity and specificity for prediction of recurrence of malignant PE was 61.5% and 83.3%, respectively (cutoff SDF-1alpha = 2,500 ng/ml), and better than those using pH of PE. Cancer cells in malignant PE expressed CXCR4, and mesothelial cells of the pleura stained positive for SDF-1alpha. The SDF-1alpha/CXCR4 axis is involved in the dissemination of NSCLC cells into pleural space.

Cytogenetic Study in Suspicious Cases of Malignant Pleural Effusion.

This study was performed to detect malignant cells in suspicious cases of malignant pleural effusion by cytogenetic analysis. MATERIALS AND METGODS: Eleven cases with pleural effusion were included in this study. Cells in pleural effusion were treated by direct, or short term, culture to prepare chromosomes. To analyze chromosomes, the G-banding method was used. Chromosome preparations succeeded in 10 cases. 5 cases had normal karyotypes, but in 2 of these cases malignant cells were detected on cytological examination. The other 5 cases had abnormal chromosomes, but on cytological examination showed normal cell appearances. Cytogenetic analysis of pleural effusions is not used routinely, but is more sensitive than the cytological examination of malignant pleural effusions. So, chromosome analysis is a good diagnostic tool, when chromosomal abnormalities are detected in an effusion. If a combination of cytology and cytogenetic study are used, the chance of detecting malignant cells in pleural effusion will be higher, and then more invasive diagnostic procedures, such as thoracoscopy or thoracotomy, could be avoided.

Retroviral Gene Transfer Is Inhibited by Chondroitin Sulfate Proteoglycans/Glycosaminoglycans in Malignant Pleural Effusions

Gene therapy may be an important adjuvant for treating cancer in the pleural space. The initial results of retroviral gene transfer to cancer cells in malignant pleural effusions revealed that transduction was markedly inhibited, and studies to characterize the inhibitory factor(s) were performed. The inhibition was contained within the soluble, rather than cellular, components of the effusions and was demonstrated with amphotropic, gibbon ape leukemia virus, and vesicular stomatitis virus-glycoprotein pseudotyped retroviral vectors. After excluding complement proteins, a series of studies identified chondroitin sulfates (CSs) as the inhibitory substances. First, treatment of the effusions with mammalian hyaluronidase or chondroitinases, but not Streptomyces hyaluronidase, abolished the inhibitory activity. Second, addition of exogenous CS glycosaminoglycans mimicked the inhibition observed with pleural effusions. Third, immunoassays and biochemical analyses of malignant pleural effusion specimens revealed CS in relevant concentrations within pleural fluid. Fourth, proteoglycans/glycosaminoglycans isolated from the effusions inhibited retroviral gene transfer. Analyses of the mechanism of inhibition indicate that the chondroitin sulfates interact with vector in solution rather than at the target cell surface. These results suggest that drainage of the malignant pleural effusion, and perhaps enzymatic pretreatment of the pleural cavity, will be necessary for efficient retroviral vector mediated gene delivery to pleural metastases.

Detection of Hyperdiploid Malignant Cells in Pleural Effusions With Chromosome-Specific Probes and Fluorescence In Situ Hybridization

To compare the efficacy of fluorescence in situ hybridization (FISH) by using chromosome-specific probes with standard cytology and cytogenetics for detection of hyperdiploid malignant cells in pleural effusions. A blind study was done on 26 pleural effusions from 25 patients who had undergone thoracentesis (14 with and 11 without a malignant condition). Cytology, cytogenetics, and FISH with probes specific for chromosomes 7, 8, 12, 18, X, and Y were done on each pleural effusion. For FISH studies, malignant specimens were defined as having 8% or more of cells with hyperdiploidy. Results of cytology and FISH were both normal in each of the 11 patients with benign pleural effusions. Cytogenetic studies were successful in six of these patients: five were chromosomally normal, but one male patient had an abnormal clone that lacked a Y chromosome. Among the 14 patients with malignant pleural effusions, cytology and FISH were abnormal in 8 and 6, respectively. Cytogenetic studies were successful in 11 of these patients, and an abnormal clone was found in 5. FISH can detect hyperdiploid malignant cells in pleural effusions and can be useful in the work-up of patients suspected of having malignant pleural effusions.

Detection of hyperdiploid malignant cells in pleural effusions with chromosome-specific probes and fluorescence in situ hybridization.

Detection of hyperdiploid malignant cells in pleural effusions with chromosome-specific probes and fluorescence in situ hybridization.

P53 gene product in pleural effusions. Practical use in distinguishing benign from malignant cells.

To determine the utility of positive p53 immunostaining as an adjunct in the diagnosis of malignancy in pleural effusions, we reviewed 103 effusions representing the typical range of diagnoses encountered in the evaluation of pleural fluid cytology. Immunohistochemistry was performed on paraffin-embedded cell blocks using a monoclonal antibody to the p53 suppressor gene product clone BP53-12 and a standard avidin-biotin complex technique with a citrate buffer antigen retrieval solution. Forty-one of 75 effusions with an unequivocal cytologic diagnosis of malignancy were immunopositive for p53 protein (55%). One of nine effusions cytologically interpreted as showing reactive mesothelial cells showed immunopositivity; that case was subsequently diagnosed as a mesothelioma on pleural biopsy. Nineteen cases were interpreted as suspicious for malignancy. Of these, 16 were negative, and 3 were positive for p53 protein. Of the three positive cases, two showed the presence of non-small cell and poorly differentiated large cell carcinoma. These findings suggest that p53 protein immunostaining is relatively sensitive and highly specific in differentiating benign mesothelial cells from malignant cells in pleural effusions. While negative p53 protein immunostaining does not exclude malignancy, positive staining in reactive or suspicious cells warrants further diagnostic evaluation of the patient.

Value of DNA analysis in addition to cytological testing in the diagnosis of malignant pleural effusions.

Aneuploidy appears to be a highly specific marker for cancer, and measurement of cellular DNA content by flow cytometry is rapid and reliable. This study was undertaken to determine if the addition of DNA analysis improved the sensitivity of cytological diagnosis of malignancy in pleural fluid. Pleural effusions from 92 patients were studied by cytological examination and flow cytometry. In 41 patients the final diagnosis was malignancy, there were 40 cases of benign effusions including 22 with pleural tuberculosis, and in the remaining 11 patients with biopsy proven cancer the presence of malignant cells was not found by cytological and histological means in the pleural fluid. Aneuploidy and cytological malignancy were found in 14 samples. There were seven cases with abnormal flow cytometry and negative cytological results. In 12 patients the cytological test results were positive but DNA analysis was normal. Thirty six samples of fluid were both diploid and cytologically negative. Of the 22 tuberculous effusions seven contained aneuploid cells. The sensitivity of DNA and cytological analysis was 51.2% and 63.4%, respectively. The specificity of DNA analysis was 74.5%. DNA analysis of cells in malignant pleural effusions is both less sensitive and specific than the cytological diagnosis. Flow cytometric analysis is not recommended for routine use in the diagnosis of pleural effusions.

Characterization of a new IL-6-dependent human B-lymphoma cell line in long term culture

We have established a cell line (DS-1) of B-cell lineage in long-term culture. It was derived from an immunodeficient patient with intestinal lymphangiectasia and lymphoma by culturing malignant pleural effusion cells with IL-6 in vitro. The cell surface phenotype was: PCA-1, HLA Class II(+); CD25, CD19, CD20, CD30, CD38(−). Cell proliferation was poor in medium and exhibited an eight-fold, dose-dependent increase of proliferation in response to rIL-6 of human but not murine origin. The secretion of IgG into culture supernatants by DS-1 was not enhanced by rIL-6. While constitutive production of IL-6 was not detected by bioassay using murine B9 hybridoma cells or by ELISA, the presence of IL-6 message was detected in polyA+ selected mRNA by Northern analysis. Spontaneous proliferation of DS-1 cells was inhibited by neutralizing polyclonal antibodies to IL-6 (37%) and mAb to IL-6 (54%) and IL-6R (53%). DS-1 expressed both high and low affinity IL-6 receptors ( K d 1.2 × 10 −11 and 6.7 × 10 −10, respectively) by radiolabelled binding and Scatchard analysis. Thus, DS-1 represents an autocrine IL-6-producing cell line of B-cell lineage which resembles lymphoid malignancies arising in patients with AIDS and other immunodeficiency diseases. Despite constitutive IL-6 production, the in vitro growth of DS-1 is dependent upon exogenous IL-6. DS-1 may thus be useful as a model of IL-6 dependency. This cell line may also facilitate development of strategies for diagnosis and treatment of B-cell lymphomas in immunocompromised patients.

Induction of killer cells in malignant pleural effusions obtained from lung cancer patients: [formula omitted] Dep. Int. Med., Sec. 3, Sapporo Med. Coll., Japan

Induction of killer cells in malignant pleural effusions obtained from lung cancer patients: [formula omitted] Dep. Int. Med., Sec. 3, Sapporo Med. Coll., Japan

An scanning electron microscopic study of adenocarcinoma cells in pleural effusions

Cytodiagnosis of pleural effusions is very important in the clinical assessment of respiratory diseases. The existence of cancerous cells in effusions is a good evidence of indicating malignant nature of the disease process, and thus determines the treatment and prognosis of the patients. Observation of the cells derived from pleural effusion with light microscopy or transmission electron microscopy can perform detailed analysis of the cytoplasm and the nucleus. It is expected that other useful morphologic data is still contained on the plasmic membrane or cell surface. However, this can only be explored by scanning electron microscopy. In order to understand more comprehensively the surface structure of malignant cells in pleural effusion, specimens obtained from 69 patients including 26 men and 43 women were examined with scanning electron microscope in this study. The clinical diagnosis was primary adenocarcinoma of the lung in 46 patients, breast cancer in 20 patients and ovarian cancer in 3 patients. Pleural metastasis occurred in all of the patients and their chest X-ray films showed pleural effusion.

Somatic changes in B-lymphoproliferative disorders (B-LPD) detected by DNA-fingerprinting.

Specific genomic alterations may be involved in the transformation of a normal cell to a malignant one (Nowell, 1986). Characteristic translocations, often related to cellular oncogenes, are frequent in B-non-Hodgkin's lymphoma (B-NHL). In Burkitt's lymphoma, a translocation t(8; 14) activates the c-myc oncogene and in 85% of follicular lymphomas a translocation t(14; 18) is detected, involving the putative oncogene bcl-2 (Dalla Favera et al., 1982; Tsujimoto et al., 1985). The clonal evolution of B-NHL is marked by successive chromosomal changes (Yunis et al., 1987), some of which correlate with histologic signs of progression and a more malignant course of the disease (Koduru et al., 1987). Karyotype analysis, however, is technically difficult in solid tumours and impossible in cases where only frozen tissue is available. By detecting a large amount of chromosomal loci dispersed throughout the genome, minisatellite probes may serve as probes for chromosomal changes (Jeffreys et al., 1985). Thein et al. used this approach to screen the DNA of 35 patients with a variety of cancers and found differences between the constitutional DNA and tumour DNA in ten cases (Thein et al., 1987). The changes were interpreted as either unequal sister-chromatid exchanges producing new fragments, or variations in fragment intensity and hence chromosomal copy number. Using minisatellite probe 33.15, we studied two EBV-negative cell lines, HH2 and HH3, derived from pleural effusion cells of a single B-immunoblastic NHL. In parallel, karyotype analysis was performed to relate the changes seen in DNA-fingerprint directly to cytogenetic differences. Cell line HH2 and the original malignant pleural effusion cells showed 48XY + 7 +dell2(q24) t(14; 18)(q32; q21). Cell line HH3 showed the same karyotype except that no partial trisomy 12 was present. On hybridizing to the minisatellite probe 33.15, the fingerprints of cell lines HH2 and HH3 and of the original tumour were indistinguishable, but for 5.1 and 2.7kb fragments present in HH2 and in the original tumour and absent in HH3 (Figure 1). This finding suggests the location of