Polycyclic aromatic hydrocarbon-DNA adducts in lung tissue from lung cancer patients.

In an attempt to probe for polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human subjects resulting from smoking (or other chronic environmental exposure), lung tissue and lung tumours were obtained from patients hospitalized for lung cancer. DNA was isolated from the tissue samples and examined both in an ELISA using a polyclonal antibody against (+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE)-DNA as well as by the nuclease P1-mediated modification of the 32P-post-labelling technique. The ELISA results showed BPDE-DNA antigenicity in lung DNA from 6 out of 21 patients, and adduct levels ranged from 2 to 134 adducts per 10(8) nucleotides. For all 21 patients, the autoradiographs of chromatograms of 32P-postlabelled digests of DNA from non-tumorous lung tissue showed a strong diagonal radioactive zone (DRZ). This DRZ was generally absent in tumorous tissue. DNA samples that were positive in the ELISA contained a dominant spot within the DRZ that co-chromatographed with the major BPDE-DNA adduct (BPDE-dG). The quantities of the BPDE-dG spots ranged from 2.1 to 42 adducts in 10(9) nucleotides. These values were lower than the levels found in the ELISA but correlated well with the ELISA results (Kendall W = 0.97; P = 0.00). The levels of the DRZ adducts ranged from 1.9 to 34 adducts in 10(8) nucleotides. Correlations between smoking and DNA adduct levels were poor because of the small number of current smokers (n = 13). However, smokers of filter cigarettes had significantly lower DNA adduct levels compared with smokers of cigarettes without a filter (P = 0.02 by Fischer's exact test).

A comparison of synaptophysin, chromogranin, and L-dopa decarboxylase as markers for neuroendocrine differentiation in lung cancer cell lines.

Synaptophysin is a Mr 38,000 integral membrane glycoprotein expressed by a variety of normal and neoplastic neuroendocrine cells. We studied synaptophysin as an immunocytochemical marker for neuroendocrine differentiation in lung cancer and compared it to the immunocytochemical expression of chromogranin A, a marker for dense core (endocrine) granules, and the biochemical activity of L-dopa decarboxylase (DDC), the key amine-handling enzyme. Of the 250 cell lines available to us, we selected examples representative of the following cell types: bronchial carcinoids (n = 4), small cell lung cancer (SCLC) (n = 7), extrapulmonary small cell carcinomas (n = 4), and non-small cell lung cancers (n = 18) whose neuroendocrine status had been previously determined on the basis of electron microscopy and DDC activity. We demonstrated (a) there was a higher incidence of synaptophysin than chromogranin A immunoreactivity in carcinoid (100 versus 75%), classic SCLC (70 versus 50%), and variant SCLC (57 versus 29%) cell lines; (b) 3 of the 4 (75%) extrapulmonary small cell lung cancer cell lines expressed synaptophysin and chromogranin A; (c) 5 of the 7 (71%) non-small cell lung cancer cell lines previously shown to express multiple neuroendocrine markers were positive for synaptophysin, chromogranin A, and DDC activity; (d) none of the other 11 non-small cell lung cancer cell lines expressed synaptophysin or chromogranin A; and (e) formalin fixation and paraffin embedding reduced synaptophysin immunoreactivity in 11 of 14 (79%) of the cell lines, as compared to freshly prepared specimens fixed in 95% ethanol. Western blot analysis using the synaptophysin antibody (SY38) demonstrated immunoreactive proteins ranging from Mr 43,000 to 45,000 in five representative cell lines. The concordance of expression of all three neuroendocrine markers was statistically significant when values for all cell lines were totalled. Synaptophysin was a more commonly expressed marker for variant SCLC cell lines, which rarely showed DDC activity. We conclude that synaptophysin may be a more sensitive and specific marker for neuroendocrine differentiation, when compared to chromogranin A and DDC in lung cancer cell lines which express only part of the neuroendocrine program.

Effects of tumor necrosis factor, alone or in combination with topoisomerase-II-targeted drugs, on human lung cancer cell lines.

Tumor necrosis factor alpha (TNF) exhibits cytotoxic activity on some solid tumors and has been reported to be synergistic with topoisomerase-II-targeted antineoplastic agents. A wide range of TNF concentrations (from 10 to 10,000 U/ml) was tested in 9 human lung cancer cell lines (5 small-cell and 4 non-small-cell carcinomas) using a semi-automated MTT assay. TNF was not cytotoxic in 8 cell lines, while an adenocarcinoma cell line was marginally sensitive to the cytokine. Using 125I-TNF we were able to show the presence of specific binding sites for TNF in 4/9 human lung cancer cell lines. Scatchard analysis of the marginally sensitive cell line showed high-affinity, saturable binding. With 5 cell lines we also tested whether TNF affected the cytotoxicity of doxorubicin and etoposide, 2 topoisomerase II-targeted drugs which are widely used in the therapy of lung cancer. No significant increase in cytotoxicity was seen when TNF was added to the 2 anti-neoplastic agents. In contrast to certain other human and mouse lines, human lung cancer cell lines appear to be resistant to TNF, despite the presence of the receptor in some of them; moreover, no synergistic effect of TNF and 2 topoisomerase-II-targeted drugs was evident in these human cell lines.

Voltage-operated calcium channels in small cell lung carcinoma cell lines: pharmacological, functional, and immunological properties.

Different subtypes of voltage-operated calcium channels (VOCCs) are expressed in different tissues and can be distinguished by functional and pharmacological criteria. One type of high voltage-activated calcium channel, specifically recognized by the peptide neurotoxin omega-conotoxin (omega CTx), is expressed only in neurons. Seven different human small cell lung carcinoma (SCC) cell lines were also found to bind 125I-omega CTx. The binding was specific, saturable, and of high affinity. 125I-omega CTx binding was not antagonized by the calcium channel ligands verapamil, nitrendipine, and diltiazem. There was a correlation between the amount of toxin binding and the detection of depolarization-induced calcium fluxes studied with the fluorimetric probe Fura2. Fura2 experiments also demonstrated that, in addition to omega CTx-sensitive calcium channels, SCC cell lines also expressed omega CTx-insensitive calcium channels, which were antagonized by nitrendipine and verapamil. 125I-omega CTx-labeled VOCCs from SCC cells were, furthermore, precipitated by anti-VOCC autoantibodies obtained from patients affected by the Lambert-Eaton myasthenic syndrome, a neuromuscular disease often associated with SCC. The present findings further indicate the presence of neuronal molecules with important biological function on SCC plasma membrane and add new insights into the pathogenetic mechanism of autoimmune neurological paraneoplastic diseases, like Lambert-Eaton myasthenic syndrome.

Phase I clinical comparative study of monoclonal antibody KS1/4 and KS1/4-methotrexate immunconjugate in patients with non-small cell lung carcinoma.

A Phase Ia clinical trial was undertaken to evaluate and compare murine monoclonal antibody KS1/4 and KS1/4-methotrexate immunoconjugate in patients with Stage IIIB or IV non-small cell carcinoma of the lung. Six patients received KS1/4 alone and five patients received KS1/4-methotrexate conjugate. The maximal total dose received per patient in both groups was 1661 mg. Mild to moderate side effects in both groups included fever, chills, anorexia, nausea, vomiting, diarrhea, anemia, and brief transaminasemia. One patient who received antibody alone had an apparent acute immune complex-mediated reaction. Ten of 11 patients had a human anti-mouse response. Posttreatment carcinoma biopsies revealed binding of monoclonal antibody KS1/4 and deposition of C3d and C4c complement fragments. Monoclonal antibody binding and complement deposition correlated with increasing doses of infused antibody. There was one possible clinical response.

NCAM: a surface marker for human small cell lung cancer cells.

Immunocytochemical and immunochemical techniques were used to study the expression of the neural cell adhesion molecule (NCAM) by human lung cancer cell lines. Intense surface staining for NCAM was found at light and electron microscopic levels on small cell lung cancer cells. The NCAM polypeptide of Mr 140,000 (NCAM 140) was detected by immunoblotting in all of 7 small cell lung cancer cell lines examined and in one out of two of the closely related large cell cancer cell lines: it was not detected in cell lines obtained from one patient with a mesothelioma, in two cases of adenocarcinoma, nor in two cases of squamous cell cancer. In contrast, neuron-specific enolase was found by immunoblotting in all the lung cancer cell lines tested and synaptophysin in all but the adenocarcinoma cell lines. These antigens were localized intracellularly. The specific expression of NCAM 140 by human small and large cell lung carcinomas suggests its potential as a diagnostic marker.

[Analysis of cellular DNA-RNA content using flow cytometry between primary and metastatic foci in lung carcinoma].

By flow cytometry, cellular DNA-RNA content was analyzed with Acridine Orange staining for primary tumor and the metastatic foci in 12 cases of primary lung carcinoma. Five (42%) of the primary and metastatic foci showed heterogeneous cellular DNA content, with high cellular DNA content of metastatic foci in all cases. Cellular RNA content was significantly higher in metastatic foci than in primary foci, indicating possible higher proliferative activity of the former foci. Three different metastatic foci in one patient showed essentially the same cellular DNA content. From the above it is evident that primary and metastatic foci of lung carcinoma show considerable heterogeneity not only in cellular DNA content but RNA content as well, thus indicating proliferative activity of the metastatic focus. It is consequently advisable to use a combination of various supplementary medications differing in cytocidal effect. Cases with higher cellular DNA content may likely have higher metastatic potential.

A study of human small-cell lung carcinoma (hSCLC) cell lines with different sensitivities to detect relevant mechanisms of cisplatin (CDDP) resistance.

The cisplatin(CDDP)-resistant cell line GLC4-CDDP shows a variety of differences from the parent line GLC4. The aim of this study was to determine which of the observed changes correlated with the degree of resistance and was therefore relevant to the phenomenon of CDDP resistance. For these experiments we used cells of the sensitive hSCLC cell line GLC4 and the in vitro-acquired CDDP-resistant sublines GLC4-CDDP3 and GLC4-CDDP11, with a resistance factor (RF) of 3 and 11 respectively for CDDP and of 1.8 and 7.4 respectively for carboplatin. Carboplatin was used, in addition to CDDP in seeking relevant mechanisms. No consistency was found between the RF and the growth pattern or antigen expression, cellular volume, doubling time, cellular or nuclear platinum (Pt) content or the level of Pt-non-histone chromatin protein (NHCP) binding. A correlation was found between the RF and the level of glutathione (GSH), and a trend was found for the level of Pt-DNA binding, Pt-GG adduct content and the amount of interstrand cross-links (ISC). These changes might therefore be relevant for the development of resistance. These findings are compatible with a GSH-induced reduction of the amount of reactive Pt in the resistant cell, resulting in a lower net platination and toxic Pt-DNA adduct formation.

Trace metal lung disease: in vitro interaction of hard metals with human lung and plasma components.

Hard metal pneumoconiosis is an occupational pulmonary disease caused by long-term exposure to dust produced in the hard metal industry. In vitro experiments have been carried out to study the solubility and metabolic behaviour in human lung tissue and plasma of hard metal alloy constituents such as cobalt, tungsten, tantalum, titanium and niobium. The experiments were carried out using 60Co, 187W, 182Ta, 44Ti and 95Nb radiotracers in combination with neutron activation, radio-release tests and gel filtration techniques. Leaching experiments from neutron-irradiated hard metal dust showed that cobalt was highly soluble, especially in the lung cytosol and plasma, in comparison with tantalum and tungsten. The gel filtration experiments showed three biochemical pools of cobalt in both lung and plasma components, in accordance with the hypothesis that cobalt represents the allergic factor in the development of hard metal disease. High affinity for proteins was observed for Nb, Ta and Ti, but not for W, in agreement with the dissimilar biological half-lives of these elements in the body. The different ability of the metals to interact with biochemical components and to be solubilized in biological media may explain the various degrees of retention in the lung, which would influence the metabolic pathways. This would explain the presence of Co, Ta and W in body fluids, as well as in the public hair and toenails of hard metal workers.

Cytokinetic investigation of lung tumors using the anti-bromodeoxyuridine (BUdR) monoclonal antibody method: comparison with DNA flow cytometric data.

In order to investigate the cytokinetics of malignant tumors and non-malignant lesions of the lung, tissue samples from 57 patients affected by non-small-cell carcinoma (NSCLC), small-cell carcinoma (SCLC), and benign and inflammatory lesions have been analyzed using the BUdR monoclonal antibody (MAb) method. This method is based on the preparation, at the time of surgery, of viable monocellular suspensions (using collagenase and DNase treatment) and the concomitant administration of BudR. The percentage of BudR-labelled cells was monitored by fluorescent microscopy using an FITC-labelled second antibody. In NSCLC, each histological group showed a wide range of labelling index (LI) values. On the contrary, SCLC exhibited a more homogeneous kinetic behaviour as evidenced by a narrowly distributed, higher LI. Tumors shown to be diploid by flow cytometry did not show a lower LI than aneuploid tumors. Furthermore, differences were constantly observed between the S-phase percent calculated using BUdR and that calculated using the DNA flow cytometric (FC) histogram, the latter always showing higher S-phase values. In an attempt to study the intra-tumor proliferative heterogeneity, multiple-site sampling was performed. Proliferative heterogeneity seemed to be higher inter-tumor than intra-tumor. Finally, a positive correlation (p less than 0.05) was found between LI and the actual doubling time (DT) of the primary tumor mass, evaluated using sequential radiographs. In conclusion, the present BUdR method can be considered a useful source of relevant information on in vivo cell growth, in parallel to other clinical (DT) and biological (DNA content) approaches.

Distinct patterns of chromogranin A-related species can be demonstrated in endocrine cells.

We have studied the pattern of chromogranin A (CgA)-related species in different human endocrine cells that produce CgA and also express the calcitonin gene. Antibodies against CgA peptides that span its linear sequence were used in Western analysis of cell lines derived from medullary thyroid carcinoma (MTC), small cell lung cancers (SCLC), epidermoid cell lung cancer (ECLC) and a pulmonary carcinoid tumor (CRND). Each of the cell lines demonstrated a distinct pattern of CgA-related species. Gel filtration studies also revealed multiple and different forms of immunoreactive CgA in the cell lines. Although proteolysis may contribute to our results, these observations suggest that native CgA is processed to smaller species in a tissue-specific pattern by different endocrine cells. More conclusive studies, however, are necessary to establish that cell processing leads to the specific CgA moieties that we have observed.

[Immunohistological coexpression of keratin and vimentin in the epithelial neoplastic cells].

Filed formalin-fixed paraffin blocks of 128 cases of epithelial neoplasms were selected for immunohistochemical study of keratin and vimentin expression. The results showed that 35.1% (45/128) of different carcinomas expressed vimentin. The immuno-positivity of vimentin in thyroid carcinomas, ovarian carcinomas, prostatic adenocarcinomas, pulmonary carcinomas and malignant mesotheliomas were 81.8%, 42.8%, 66.7%, 30.5% and 53.4%, respectively. Carcinomas of breast, kidneys, salivary glands, adrenal glands and nasopharyngeal carcinomas also showed various degrees of positive reaction. The results suggest that an immunohistochemical positive vimentin reaction does not exclude histopathological diagnosis of carcinomas. The significance and noticeable aspects of immunohistochemical methods in histopathological diagnosis are also discussed.

[Evaluation of double cancers in relation to previous asbestos exposure].

Ten cases of double cancers (a lung cancer and a stomach cancer) were evaluated in relation to previous asbestos exposure. Ten cases involved male more than 69 years old. Five cases had developed their two cancers simultaneously and other 5 cases had developed their lung cancer after stomach cancer surgery. The lung cancer was their main disease. Four cases had early stage stomach cancers and 5 cases with a stomach cancer had no relapse after surgery. Eight cases had occupational histories of asbestos exposure. Significantly high numbers of asbestos bodies were detected in the autopsied lung in almost all cases. According to the X-ray analysis, almost all asbestos fibers detected in the lung were chrysotile. Additionally, the Brinkman Index (B.I.) of these 10 cases ranged between 700 and 2,000. The combination of asbestos exposure and smoking is thought to be an important factor in the development of such double cancers.

Lectin-resistant variants of mouse Lewis lung carcinoma cells. II. Altered glycosylation of membrane glycoproteins.

Lectin-resistant variants of mouse Lewis lung carcinoma LL2 cell line, selected with wheat germ agglutinin (WGAR), Ricinus communis agglutinin II (RCA IIR) and Aleuria aurantia agglutinin (AAAR) were studied. Total cellular glycopeptides of the parent LL2 line and of the five lectin-resistant variants were analyzed by gel filtration and affinity chromatography on immobilized concanavalin A and Lens culinaris agglutinin. The results revealed that low-metastatic WGAR and RCA IIR variants possessed less highly branched tri- and tetra-antennary N-acetyllactosaminic type glycans with a simultaneous increase in biantennary N-acetyllactosaminic type, oligomannosidic type or hybrid type glycans, as compared to the parent metastasizing LL2 cell line. These findings imply that cell surface carbohydrate changes may possibly be relevant for metastasis. However, the AAAR variant, which possessed reduced spontaneous metastatic ability after s.c. administration, but increased experimental metastatic ability after i.v. inoculation, exhibited apparently the same glycan pattern than the parent LL2 line. This particular variant is under investigation in order to find specific modification(s) of glycan(s) which could play a specific role in the metastatic process.

Confirmation of lymphomatous pulmonary involvement by immunophenotypic and gene rearrangement analysis of bronchoalveolar lavage fluid.

Pathologic diagnosis of pulmonary involvement with lymphoma has traditionally necessitated open-lung biopsy in most cases. Specimens large enough to allow recognition of characteristic cytologic and architectural changes are usually not obtained bronchoscopically. Even when adequate biopsy specimens are available, however, unequivocal differentiation of lymphoma from benign inflammatory proliferative lesions (for example, pseudolymphoma or lymphocytic interstitial pneumonitis) is not possible on the basis of light microscopic findings alone. Pathologists have relied on immunohistologic examinations in which antibodies directed against B-cell and T-cell surface antigens are used to help distinguish benign from malignant lymphoid proliferations. Unfortunately, even immunohistologic findings may be nondiagnostic, particularly in T-cell lymphomas and in cases in which lymphoma is surrounded by a benign reactive lymphocytosis. Recent development of molecular biologic techniques (for example, Southern blotting) has provided the ability to detect a monoclonal population of cells based on detection of rearrangements of the genes that encode either B-cell immunoglobulin proteins or T-cell antigen receptor proteins. This technique is capable of detecting a clone of cells even when they constitute as little as 5% of a lymphoid infiltrate. Bronchoalveolar lavage can provide samples of sufficient size to facilitate Southern blotting. We present the first case wherein pulmonary involvement with a B-cell lymphoma was confirmed by both immunohistologic and molecular biologic studies of bronchoalveolar lavage fluid.

Atypical endocrine granules in atypical endocrine tumor (AET) of the lung. An immunoelectron microscopic study.

Highly dense granules are a hallmark for recognizing atypical endocrine tumor (AET) of the lung. We report a case of AET with many atypical neurosecretory-type granules: moderately dense granules (mean size 373.7 nm) and "target" granules with a central dense core (425.1 nm), both apparently larger than the highly dense granules (223.3 nm). Immunoelectron microscopical studies demonstrated that all three types of granule were positive for gastrin-releasing peptide (GRP), human chorionic gonadotropin alpha-subunit (hCG alpha), calcitonin or serotonin. Although the size profiles of positive granules were similar for calcitonin and hCG alpha, they were different from those of GRP or serotonin granules. The presence of atypical granules and the different size profiles of hormonal products in AET indicate that caution is required in ultrastructural evaluation of granules in lung carcinomas.

Differentiation-related expression of epidermal growth factor receptors in human lung carcinoma demonstrated histochemically by biotinylated epidermal growth factor.

Biotin-labeled epidermal growth factor (EGF) was applied to routinely processed sections of 64 cases of human lung carcinoma as a histochemical tool for demonstrating EGF-specific receptors. Formalin-fixed, paraffin-embedded tissue sections were deparaffinized and incubated with the labeled EGF (10 micrograms/ml) for 60 min at room temperature. The specific binding of the growth factor to its receptor was visualized by the avidin-biotin complex (ABC) technique. Positive binding capacities were obtained for the following cases: 15/16 epidermoid carcinoma; 13/15 adenocarcinoma; 2/11 large cell anaplastic carcinoma; 12/20 small cell anaplastic carcinoma; 0/11 normal lung tissue; 0/6 main bronchi; 0/1 hamartoma; 0/1 primary fibrosarcoma of lung. In addition, a strong positive reaction was seen for neutrophilic granulocytes present within the tumorous tissue. Data indicate that EGF receptors are frequently expressed in more differentiated carcinoma in comparison with anaplastic carcinoma of lung.

Prognostic significance of laminin in adenocarcinoma of the lung.

The distribution of laminin in tumor-associated basement membrane was immunohistochemically investigated in 115 cases of adenocarcinoma of the lung. The distribution of laminin was classified into continuous and discontinuous staining patterns. The incidence of the discontinuous pattern was less in early-stage disease than that in advanced stages (P less than 0.01). In patients with stage I, the incidence of discontinuous patterns was greater in short-term survivors than in long-term survivors (P less than 0.05). By contrast, in patients with stage III, the discontinuous pattern of laminin was frequently seen in both long-term survivors and short-term survivors, with no difference between the two groups. These data suggest that the discontinuous pattern of laminin in tumor-associated basement membrane reflects the spread and dissemination of tumor, hence a close relationship to the prognosis.

Small-cell-type poorly differentiated squamous cell carcinoma of the lung. Cytologic, immunohistochemical and nuclear DNA content analysis.

The relationship between the nuclear DNA content, the immunohistochemical findings, the clinical characteristics (tumor volume doubling time and survival) and the cytomorphologic features of small cell poorly differentiated squamous cell carcinoma of the lung was studied in ten cases. There were no significant correlations between the immunohistochemical stainings for neuron-specific enolase and keratin and the clinical characteristics in these cases. The DNA histogram patterns were classified as type I or type II, depending on the degree of dispersion of values. There was no relationship between the immunohistochemical findings and the DNA histogram patterns. Only the DNA histogram patterns were related to some of the clinical characteristics: patients with type II histograms had significantly shorter tumor volume doubling times than did patients with type I histograms. Such information may aid in distinguishing the small cell type of poorly differentiated squamous carcinoma from classic small cell carcinoma of the lung, with which it may be confused.

Urokinase receptors in lung cancer and normal lung.

Cell membranes from ten non-small cell lung cancers and four specimens of adjacent lung tissue were assessed for the presence of urokinase type plasminogen activator (uPA) receptors. Displacement binding studies using 125I labelled urokinase showed specific binding on lung cancer and lung membrane preparations. Scatchard analysis showed that the dissociation constant of high affinity sites on tumour membranes was 2.9 x 10(-11) M/1 and on lung membranes was 2 x 10(-9) M/1. The concentration of high affinity binding sites on tumour membrane was 54 fmol/mg of membrane protein and on normal lung membrane was 170 fmol/mg protein. Two-point binding assays showed specific binding of urokinase on five of eight tumour membranes and one of three normal lung membranes. There was no correlation between the amount of urokinase bound and tumour subtype or extent of disease. Because of interactions between uPA and epidermal growth factor receptors (EGFr) in cell culture and because lung cancers express increased EGFr we studied the association of uPA receptors and EGFr. Seven tumours expressed EGFr at 6.8-67.6 fmol/mg of protein of EGFr and four normal lung membranes had EGFr at 5.2-15.6 fmol/mg protein EGFr. There was no correlation between uPA receptors and EGFr in this series. We conclude that non-small cell lung cancers carry receptors for urokinase and this provides a novel mechanism for control of local proteolysis.