Cells In Pleural Effusion Research Articles

Lymphoma Cell Line (FL-18) and Epstein-Barr Virus–Carrying Cell Line (FL-18-EB) Obtained From a Patient With Follicular Lymphoma: Monoclonal Derivation and Different Properties

A novel cell line, FL-18, was established from the pleural effusion of a patient with follicular small cleaved cell lymphoma. At the same time, an Epstein-Barr virus (EBV) nuclear antigen (EBNA)-positive cell line, FL-18-EB, was established from the EBV-infected culture of the same pleural effusion cells. Both cell lines had the same monoclonal surface immunoglobulin (IgG kappa), and they had the same karyotype as that of the fresh pleural effusion cells in which a reciprocal translocation between the long arm of chromosomes 14 and 18 [t(14;18)(q32;q21)] was detected. Gene rearrangement analysis of immunoglobulin heavy-chain gene (JH) and kappa light-chain gene (J kappa) showed the same rearranged configuration in the two cell lines; however, some morphological and phenotypic differences were found. The FL-18-EB cells, which were morphologically similar to common EBNA-positive lymphoblastoid cell lines of normal B cell origin at the initial phase of culture, were larger than the FL-18 cells and contained multinucleated giant cells. The FL-18 cells lacked cytoplasmic immunoglobulin and were positive for common acute lymphoblastic leukemia antigen (CALLA), whereas the FL-18-EB cells had cytoplasmic immunoglobulin and were negative for CALLA. Thus, the phenotype of FL-18-EB seems to be a result of a shift by EBV infection to a more mature stage in the B cell differentiation pathway than that of FL-18. The paired availability of EBV-free and EBV-infected cell lines of a neoplastic clone is unique and valuable in considering EBV infectibility of neoplastic B cells and resultant phenotypic changes.

NK Cells in Carcinomatous and Tuberculous Pleurisy: Phenotypic and Functional Analyses of NK Cells in Peripheral Blood and Pleural Effusions

To compare local immunity with that in peripheral blood, we examined 14 patients with carcinomatous pleurisy and 22 patients with tuberculous pleurisy by phenotypic and functional analyses of NK cells. We calculated the proportions of NK cells by means of anti-Leu 7 and anti-Leu 11 mAbs and the rates of NK cell activity by means of K 562. The percentages of Leu 7+ and Leu 11+ cells in both carcinomatous and tuberculous pleural effusions were lower than those in peripheral blood; however, there was no difference between carcinomatous and tuberculous pleural effusions in the proportions of Leu 11+ cells. The NK cell activity in tuberculous pleural effusions was the same as that in peripheral blood, while in carcinomatous pleural effusions, NK cell activity was lower than in peripheral blood; however, NK cell activity was much higher in tuberculous than in carcinomatous pleural effusions. Our study offers some pathophysiologic insight into the responses of NK cells to cancer and to tuberculosis in relation to peripheral blood and pleural effusion.

Immunohistochemical analysis of human lung cancers with anti-ras p21 monoclonal antibodies.

The expression of ras oncogene product p21 in human malignant pleurisy and primary lung cancer was studied immunocyto-histochemically with monoclonal antibodies (MoAbs) rp-28 and rp-35 against ras p21. In pleural effusion cells, cancer cells revealed more intensively positive reaction with MoAb rp-35 than with MoAb rp-28, especially in the plasma membrane, and no positive reaction was obtained in any kind of inflammation cells with the exception of faintly positive reaction in the cytoplasm of macrophages. In primary lung cancers, well or moderately differentiated adenocarcinoma tissues showed higher reactivity with MoAb rp-28 than those of poorly differentiated adenocarcinoma or any other histological subtype of lung cancer. With MoAb rp-35, intensively positive reaction was obtained in most of cases with all different histological subtypes of lung cancer. The staining in cancer cells was usually localized intensively to the plasma membrane and weakly to the cytoplasm with both MoAbs. Normal bronchial epithelial and glandular tissues showed only cytoplasmic staining. These two MoAbs, especially MoAb rp-35, may be useful in clinicopathological applications for the diagnosis of malignant pleurisy and primary lung cancer.

Nuclear and cell area measurements in the cytological evaluation of pleural effusions: a study of subjective assessments and morphometric measurements.

Morphometric measurements of cellular area, nuclear area and nuclear:cytoplasmatic ratio were performed on single cells in pleural effusions from 15 patients with effusion caused by bronchial, ovarian, or mammary carcinoma. The results were compared with corresponding measurements on mesothelial cells in pleural effusions from 15 patients without malignant disease. Significant differences were found between the mean values from cellular area, nuclear area, and nuclear:cytoplasmatic ratios in mesothelial cells from benign effusion versus malignant cells from effusions associated with metastatic growth. Such morphometric measurements are, however, of little value in routine diagnostic work as more than 90% of the cells in the two groups showed similar values.

Monoclonal antibodies to human squamous cell carcinoma of the lung and their application to tumor diagnosis

Three immunoglobulin G1 monoclonal antibodies, LuCa2, LuCa3, and LuCa4, were produced by fusing murine myeloma NS1 cells with splenocytes obtained from a BALB/c mouse immunized with SK-MES1 cells derived from human squamous cell carcinoma of the lung. These three monoclonal antibodies were shown to recognize different protein antigens on SK-MES1 cells by indirect immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. While the pattern of cell line distribution of antigens recognized by these antibodies was not tumor type specific, their reactivity with tissue and pleural effusion was much more informative than with cell lines. The presence of target antigens in vivo was analyzed by immunoperoxidase staining of frozen tissue sections and immunofluorescence staining of tumor cells in pleural effusions. LuCa2 antibody was reactive with lung squamous carcinoma and adenocarcinoma tumor tissues and pleural effusions, but only infrequently with those of small cell carcinoma. This antibody was also reactive with many tumor tissues from other organs as well as with various normal tissues, including alveoli and bronchus. LuCa3 and LuCa4 antibodies reacted with lung squamous carcinoma in tissues and pleural effusions, but not with lung adenocarcinoma nor with small cell carcinoma. These two antibodies reacted only weakly with normal squamous tissues of the esophagus, skin, and cervix uteri, but not with various other normal tissues. Moreover, LuCa3 had weak reactivity with squamous cell carcinoma tissue of tongue and esophagus, whereas LuCa4 had no reactivity with nonpulmonary tumor tissues. LuCa3 and LuCa4 antibodies should be of clinical interest, because our data suggest that these antibodies may be potentially useful for the diagnosis of the histological type of lung tumor cells in both cancer tissue and pleural effusions.

Cytochemical, Immunologic, Chromosomal, and Molecular Genetic Analysis of a Novel Cell Line Derived From Hodgkin’s Disease

A novel cell line, KM-H2, was established from the pleural effusion of a patient with Hodgkin's disease of mixed cellular type. Multiple phenotypic studies were carried out with this cell line. Acid phosphatase and nonspecific esterase activities were detected. Rosette formation with T lymphocytes and the receptors for C3b and Fc portion of IgG were positive. Among the antigens tested with a total of 22 monoclonal antibodies defining hematopoietic cell subsets or lineages, Ki-1, Leu-M1, MCS1, HLA-DR, and OKT9 antigens were found to be positive. The other antigens reportedly specific for T cells, B cells, natural killer (NK) cells, monocytes, interdigitating reticulum (IR) cells and dendritic reticulum cells were negative. These phenotypic features were identical to those of the Sternberg-Reed (SR) and Hodgkin (H) cells in the fresh materials reported by other researchers. Moreover, the KM-H2 cells and the parental pleural effusion cells shared several structural chromosome anomalies. These findings indicated that the KM-H2 cells are derived from the SR and H cells. Molecular genetic analysis of the KM-H2 cells disclosed that the human immunoglobulin JH gene was rearranged but not the JK gene, and that the human T cell receptor beta chain gene was of the germline type. Based on these properties of the KM-H2 cells, Hodgkin's disease may be derived from a cell lineage other than T cell or B cell.

Cytochemical, immunologic, chromosomal, and molecular genetic analysis of a novel cell line derived from Hodgkin's disease

A novel cell line, KM-H2, was established from the pleural effusion of a patient with Hodgkin's disease of mixed cellular type. Multiple phenotypic studies were carried out with this cell line. Acid phosphatase and nonspecific esterase activities were detected. Rosette formation with T lymphocytes and the receptors for C3b and Fc portion of IgG were positive. Among the antigens tested with a total of 22 monoclonal antibodies defining hematopoietic cell subsets or lineages, Ki-1, Leu-M1, MCS1, HLA-DR, and OKT9 antigens were found to be positive. The other antigens reportedly specific for T cells, B cells, natural killer (NK) cells, monocytes, interdigitating reticulum (IR) cells and dendritic reticulum cells were negative. These phenotypic features were identical to those of the Sternberg-Reed (SR) and Hodgkin (H) cells in the fresh materials reported by other researchers. Moreover, the KM-H2 cells and the parental pleural effusion cells shared several structural chromosome anomalies. These findings indicated that the KM-H2 cells are derived from the SR and H cells. Molecular genetic analysis of the KM-H2 cells disclosed that the human immunoglobulin JH gene was rearranged but not the JK gene, and that the human T cell receptor beta chain gene was of the germline type. Based on these properties of the KM-H2 cells, Hodgkin's disease may be derived from a cell lineage other than T cell or B cell.

Monoclonal Antibodies to Human Cytokeratins: Application to Various Epithelial and Mesothelial Cells

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.

Further cytogenetic evidence for a multistep pathogenesis of Ph-positive chronic myelogenous leukemia

A case of Ph-positive chronic myelogenous leukemia in blastic crisis was studied extensively by means of cytogenetic techniques. Karyotypic features, as well as growth patterns, kinetic data, and rates of sister chromatid exchange, were examined in bone marrow, blood, and pleural effusion cells. The data provide strong evidence for a multistep pathogenesis of the disease, the development of which appears to be linked to mechanisms of clonal selection and genetic imbalance in the malignant cell population.

Morphological damage induced in vivo by Lonidamine on human metastatic cancer cells.

Lonidamine was given to several patients affected with different types of neoplasias growing as metastases both in ascites and pleural effusion, and solid cutaneous metastases. The patients with tumor cells in ascites and pleural effusion were treated with Lonidamine per os or in loco injections. In cutaneous metastases, Lonidamine was administered by different routes: (1) per os; (2) local endoarterial; and (3) in association with hyperthermic perfusion with or without antiblastic drugs.

Immunocytochemical staining of cells in pleural and peritoneal effusions with a panel of monoclonal antibodies.

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.

The use of carcinoembryonic antigen for identification of human tumor cells in malignant effusions.

A specific anti-carcinoembryonic antigen (CEA) antiserum was used to identify CEA-positive tumor cells in peritoneal and pleural effusions obtained from patients with various malignant neoplasia. In 7 out of 10 fluids in which tumor cells were detected by cytological examinations, cytoplasmic CEA-positive cells were also detected by an indirect immunofluorescence test. In addition, out of 11 fluid samples cytologically negative for tumor cells, CEA-positive cells were found in 8 cases. When both staining for cytoplasmic CEA and soluble fluid CEA levels were considered in combination, 81% of the samples were found to be positive by either one or both of these markers, whereas only 54% were positive by using cytological criteria. The data suggest that CEA marker may be used to identify tumor cells and to assess malignancy in pleural and peritoneal effusions in patients with certain types of cancer. The CEA marker was also used for identifying tumor cells grown in tissue cultures and for separating viable CEA-positive and CEA-negative cells from a mixed cell population using the fluorescence-activated cell sorter.

A NEW MARKER FOR HUMAN CANCER CELLS. 3. IMMUNOCYTOCHEMICAL DETECTION OF MALIGNANT CELLS IN SEROUS FLUIDS WITH THE Cal ANTIBODY

The Cal antibody has been used in an immunocytochemical procedure on smears of cells recovered from 50 effusions from the pericardial, peritoneal, and pleural cavities. In 25 samples containing malignant cells, as assessed by conventional morphological criteria, this antibody distinguished malignant from non-malignant cells in 21 samples. The exceptions were 2 lymphomas and 2 out of 6 carcinomas of the lung. The Cal antibody also reacted with malignant mesothelial cells from 2 mesotheliomas, but not with reactive mesothelial cells. This antibody thus provides a new method for the identification of malignant cells in pericardial, peritoneal, and pleural effusions.

PPD-specific proliferative response in humans. I. Analysis of PPD-specific proliferative cells from tuberculous pleurisy patients and healthy controls with monoclonal antibodies specific for human T subsets.

Peripheral mononuclear cells (PBL) from tuberculin reaction (TR)-negative tuberculous pleurisy patients proliferated poorly with PPD, while the cells of pleural effusion from these patients showed a proliferative response to PPD as well as did the healthy control PBL. Surface antigens of peripheral blood and pleural effusion were examined by using monoclonal antibodies. The Leu 1-positive cell population can be divided into four groups, namely (1) Leu 1+, Leu2a+, Leu 3a+, (2) Leu 1+, Leu 2a+, Leu 3a-, (3) Leu 1+, Leu 2a-, Leu3a+, and (4) Leu 1+, Leu 2a-, Leu 3a- cell populations. Results of analysis of surface antigens of PPD-specific proliferative cells in peripheral blood and pleural effusion from tuberculous pleurisy patients as well as healthy controls indicate that the PPD-specific proliferative response is mediated by Leu 1+, Leu 2a-, Leu 3a+ cells and Leu 1+, Leu 2a-, Leu3a- cells.

Natural killer cells in carcinomatous pleural effusions

Effector cells in carcinomatous pleural effusions of patients with primary or secondary lung cancer were examined for natural killer (NK) activity against K562 cells in a 4-h chromium release assay, and for mitogenic responses and lymphocyte subpopulation constitutions. NK activity of lymphocyte-rich mononuclear cells isolated from carcinomatous pleural effusions by centrifugation on a discontinuous gradient of Ficoll-Hypaque was markedly low in seven of 40 patients studied, and absent in the other 33 cases. NK activity of peripheral blood mononuclear cells from the patients was lower than that of cells from normal donors, but always higher than that of effusion cells from the same patients. NK cells in the peripheral blood and in pleural effusions had some characteristics in common, in that they lacked a capacity to bind sheep erythrocytes, were nonadherent to Sephadex G-10 beads and nylon wool, and belonged to large granular lymphocytes. On the other hand, nonmalignant effusions of patients with congestive heart failure had significant NK activity. The effector cells in the effusions included a higher frequency of T cells than those in the peripheral blood of the same patients. Proliferative responses to phytohemagglutinin and concanavalin A of effusion cells were comparable to those of normal blood cells and were higher than those of blood cells from the same patients. The reason for low NK activity and high mitogenic response in carcinomatous pleural effusions is as yet undefined.

Concanavalin A-inducible suppressor cells in pleural effusions and peripheral blood of cancer patients

Carcinomatous pleural effusions of 18 of 20 patients with lung cancer contained suppressor cell precursors that could be activated by concanavalin A (Con A) to suppress the proliferative responses of autologous and allogeneic lymphocytes to phytohemagglutinin and Con A. However, pleural effusion cells showed no suppressor function without prior activation by Con A. In contrast, the peripheral blood of the cancer patients exhibiting impaired mitogenic response contained nonspecific spontaneous suppressor cells capable of inhibiting the lymphoproliferative response to mitogens without prior activation by Con A, but these cells were not able to show further suppressor function even after activation by Con A. The maximum suppression was observed after 48-h treatment of lymphocytes with optimally mitogenic doses of Con A. The Con A-inducible suppressor cells of the pleural effusion and spontaneous suppressor cells of the peripheral blood of cancer patients had the same characteristics with regard to the capacity to suppress the mitogenic responses of autologous and allogeneic lymphocytes, belonging to the group of nylon wool-nonadherent T cells and being sensitive to in vitro culture and resistant to treatment with mitomycin C.

Advantages of the Mucopolysaccharide Stains in Differential Diagnosis of Adenocarcinoma Cells in Pleural and Peritoneal Effusions

In this report, the authors introduce an advantage of the mucopolysaccharide stains in differential diagnosis of adenocarcinoma cells in pleural and peritoneal effusions. Periodic acid-Shiff (PAS) positive materials are almost always detected in the cytoplasm of adenocarcinoma cells. There is, however, some differences in size, amount and distributions of PAS-positive materials among adenocarcinoma cells originated from different organs. They show reticular or granular pattern in papillary adenocarcinoma and accumulative pattern in tubular and poorly differentiated adenocarcinoma of the stomach, granular pattern in serous cystadenocarcinoma and diffuse, accumulative or reticular pattern in pseudomucinous cystadenocarcinoma of the ovary, reticular or granular pattern in papillotubular and medullary tubular carcinoma of the breast reticular pattern in adenocarcinoma of the lung, and reticular or granular pattern in adenocarcinoma of the gallbladder, colon and uterine cervix. Alcian blue stain findings are also described.The authors considered the polysaccharide stains as helpful and useful methods, not only for differentiation of carcinoma cells from other benign cells, but also for detection of a primary lesion and of a histological subtype of adenocarcinoma, in combination with Papanicolaou and Giemsa stains.

14q translocations, having a break point at 14q13, in lymphoid malignancy

In three patients with non-Hodgkin lymphomas and in one cell line (HPL-Hod) derived from pleural effusion cells of a patient with Hodgkin's disease, rearrangements of the long arm of chromosome No. 14(14q) were observed. These rearrangements appeared to be consistently associated with a 14q translocation, suggestive of occurrence of a break at 14q13. The translocation in an individual case could occur with 1p, 2q, 4q, and another 14q. A 14q13 translocation may be comparable with a 14q32 translocation, which has often been observed in various types of lymphoid malignancy.

Establishment and characterization of human B‐lymphocytic lymphoma cell lines (balm‐3, ‐4 and ‐5): Intraclonal variation in the B‐cell differentiation stage

This study describes the establishment of three non-Burkitt B-lymphoma cell lines (BALM-3, BALM-4 and BALM-5) originating from the pleural effusion of a patient with a poorly differentiated diffuse lymphocytic lymphoma. The cells of BALM-3, -4 and -5 exhibited a number of properties which distinguish them from the usual B-cell type lymphoblastoid cell lines. Thus, they lacked the Epstein-Barr virus genome and had abnormal chromosome constitutions including a 14q+ marker. The presence of the identical surface immunoglobulin isotypes (gamma and chi chain determinants), and Ia-like B-cell-associated antigen in the cultured cells and in the "fresh" lymphoma cells in vivo was demonstrated. These findings strongly suggested that these cell lines have B-cell characteristics and were derived from the original tumor cell population. BALM-5 cells, however, showed somewhat different growth, cell surface marker profile and functional characteristics compared to those of BALM-3, and -4 cells. These variations suggest that the BALM-5 cells were probably at different stages of B-cell maturation than those of BALM-3 and -4, even though all three cell lines (established in three separate flasks) originated from the cells of the same pleural effusion of a lymphoma with monoclonal B-cell characteristics.

Neoplastic Multinucleated Giant Cells in Pleural Effusion of Lung Cancer

The patient, a 64-year-old man, complained of pain of the right chest. Lung cancer was suspected by X-ray examination and was confirmed by cytological study of the sputum (Class V, adenocarcinoma).He was treated by Linac X-ray irradiation and with anti-cancer drugs, After 2-week hospitalization, he was pointed out pleural effusion in the right chest. A yellowish-brown exsudate was aspirated and with the first materials was revealed an adenocarcinoma with multinucleated giant cells measuring 15-60 μin diameter. With the second materials, it was difficult to differentiate multinucleated giant cells from giant cell carcinoma or malignant mesothelioma because of a large amount of giant and multinucleated neoplastic cells.The tumor cells were PAS and alcian-blue positive.Electron microscopically, the tumor cells were found to have mucus-forming vacuoles, intracytoplasmic microcysts and cytophagocytosis.Autopsy revealed carcinoma of the upper lobe of the right lung with extensive metastasis to lymph nodes. The right pleura was markedly fibrosed and thickened with tumor cell infiltrations. Microscopic diagnosis was bronchial adenocarcinoma with mucin formation.In this case, cancer cells were observed to become multinucleate and gigantic due to treatment, which made diagnosis difficult. Further, as the therapeutic effect differed between free cells and filling proliferating ones, this cytologically interesting problem and others were discussed.