Cytologic and tissue culture characteristics of asbestos-induced mesothelioma in rats

Mesotheliomas were induced in rats by the intrapleural injection of Western Australia crocidolite asbestos. Over a two-year period, 10 of 18 animals in which implants were established developed mesotheliomas, for a 56% success rate. Histologically, most mesotheliomas were biphasic although predominantly spindle celled. Pleural fluid was examined in five of these malignant cases: three had a papillary epithelial picture, one had mainly anaplastic cells, and one contained predominantly spindle-shaped cells. Three types of cell aggregates occurred: classical collagen-containing papillary clusters, spindle-cell aggregates and cystlike spheres. These last structures corresponded to microcystic or adenomatoid growth present in four mesotheliomas. Two of the effusions were cultured successfully; the growth pattern was typically mesothelial, with in vitro production of collagen. Ultrastructurally, long, slender microvilli, cell junctions and intermediate filaments confirmed the mesothelial nature of these asbestos-induced rat malignancies.     

Morphological aspects of interactions between asbestos fibers and human mesothelial cell cytoskeleton

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

Cytopathology of malignant mesothelioma. Reappraisal of the diagnostic value of collagen cores

The identification of malignant mesothelial cells in cytological smears prepared from serous effusions is still hampered by the lack of features specific for mesothelial differentiation. We examined the diagnostic value of collagen cores within clusters of tumour cells in cytological smears prepared from effusions from 43 patients with malignant mesothelioma and of 62 cases of metastatic adenocarcinoma. In Giemsa-stained smears collagen cores were detected in 51% of the cases of malignant mesothelioma and in none of the smears with metastatic adenocarcinoma. Using the Azan stain, collagen cores were detected in 64% of the malignant mesotheliomas and 4% of the adenocarcinomas. Immunoelectron microscopy demonstrated that the collagen cores are largely composed of collagen type III fibrils and some elastin embedded in a homogenous extracellular matrix. It can be concluded that the presence of collagen cores within clusters of tumour cells is highly suggestive of mesothelial differentiation and a common finding in malignant mesothelioma.     

Ultrastructure of pleural mesothelioma and pulmonary adenocarcinoma in malignant effusions as compared with reactive mesothelial cells.

OBJECTIVE: To determine the ultrastructural features of diffuse malignant pleural mesothelioma cells in cytologic specimens from pleural effusions. STUDY DESIGN: We retrospectively studied 35 pleural effusions: 12 diffuse malignant pleural mesotheliomas (8 epithelial type, 4 biphasic type), 12 pulmonary adenocarcinomas and 11 cases of reactive mesothelial cells. RESULTS: In the cytoplasm, reactive and malignant mesothelial cells had more-abundant intermediate filaments (P < .05, P < .01) and fewer free ribosomes (P < .001, P < .001) than adenocarcinoma cells. Reactive mesothelial cells had fewer mitochondria than mesothelioma cells (P < .05). Mesothelioma cells had longer, thinner microvilli on the cell surfaces (P < .001); length/diameter ratios of microvilli were 19.1 +/- 7.0 (mesothelioma) vs. 9.1 +/- 2.2 (adenocarcinoma) and 9.2 +/- 2.4 (mesothelial cells). Giant intercellular junctions (desmosomes or desmosomelike structures > 1 micron in length) were found in eight cases of mesothelioma. Core filaments or rootlets in microvilli were present in two cases of adenocarcinoma. CONCLUSION: Because cytologic specimens from pleural effusions were easy to obtain, we think ultrastructural cytology is useful in distinguishing mesothelioma from adenocarcinoma and benign effusions.     

Simian virus 40 and human tumors: It is time to study mechanisms

Several studies found simian virus 40 (SV40) in 47% to 83% of human mesotheliomas. Mesotheliomas are malignant tumors of the pleura and peritoneum, firmly associated with asbestos exposure. In this issue, Gazdar and colleagues ?Shivapurkar et al., 1999 found that SV40 is present only in the malignant cells and not in the surrounding stromal cells. Using the microdissection technique, they found SV40 in 54% of 93 mesotheliomas of the epithelial type. The surrounding reactive stromal cells, (20 lung cancers and 14 mesotheliomas of the sarcomatoid/fibrous type) did not contain SV40, confirming the specificity of their positive findings. Furthermore, SV40 was found in 14% of 14 non-malignant reactive mesothelial cell proliferations. In 12 cases of mesothelioma a noninvasive (or in situ) component was also identified. In all four cases in which SV40 sequences were present in the invasive component, sequences were also present in the accompanying noninvasive component. These data suggest that the virus resides in the mesothelial cells prior to tumor development. The data address the remaining concerns raised at an International Meeting organized by the NIH, FDA, and CDC in 1997 to definitively associate SV40 with human mesothelioma. It is time now to investigate the pathogenic mechanisms of this association, and if SV40-infected mesothelial cells are more susceptible to other carcinogens, such as asbestos. Furthermore, we must investigate the interaction between the host immune system and SV40-infected mesothelial cells, and study if the immunosuppressive activity of asbestos interferes with tumor rejection. These studies should lead to a better understanding of mesothelioma pathogenesis, and possibly to new therapeutic approaches aimed at interfering with the expression of the SV40 genome and/or at eliciting a strong immune response against SV40 infected mesothelial cells.     

Distinction between carcinoma cells and mesothelial cells in serous effusions. Usefulness of immunohistochemistry

The usefulness of an immunoperoxidase battery to distinguish carcinomatous from benign effusions was examined. Cell block sections from 90 previously diagnosed effusions were stained with antibodies to Leu-M1, B72.3, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA) and vimentin. The 90 cases comprised 69 carcinomas (23 mammary, 16 ovarian, 10 pulmonary, 7 gastrointestinal [GI] and 13 others), 2 malignant mesotheliomas and 19 cases with reactive mesothelial cells only. EMA and vimentin were the most useful markers for distinguishing carcinoma cells from reactive mesothelial cells. EMA reacted with 86% of the carcinomas while vimentin reacted with 90% of the reactive cases. Leu-M1, B72.3 and CEA, although generally less sensitive than EMA, were also helpful in this regard. Additionally, the use of Leu-M1 and CEA together may help to distinguish pulmonary from GI carcinomas.     

Utility of HBME-1 immunostaining in serous effusions

Utility of HBME-1 immunostaining in serous effusions
HBME-1 is an anti-mesothelial cell monoclonal antibody derived from human mesothelioma cells. We investigated 227 body cavity effusions to test its utility in differentiating mesothelioma from adenocarcinoma. HBME-1 outlined cell membranes in non-neoplastic mesothelial cells. Thick surface staining was observed on all mesotheliomas. HBME-1 reactivity was also detected in 24% of metastatic carcinomatous effusions. Most ovarian carcinomas (83%) reacted with this antibody, showing surface staining. Cytoplasmic HBME-1 immunoreactivity was observed in a small proportion of non-ovarian adenocarcinomas (14%). Despite its limited specificity, HBME-1 might be added to the battery of other markers of epithelial and/or mesothelial differentiation to be used in cases of suspected mesothelioma. Evaluation of suspicious cells should include careful study of the pattern of immunostaining.     

Distribution of silver-stained interphase nucleolar organizer regions as a parameter to distinguish neoplastic from nonneoplastic reactive cells in human effusions

The distribution of interphasic nucleolar organizer regions (NORs) was studied in cytologic preparations of human serous effusions in order to differentiate malignant cells from nonmalignant reactive cells. The study was carried out on 80 cases of metastatic adenocarcinoma, 10 cases of mesothelioma, 10 reactive pleural effusions and 5 peritoneal washings. Visualization of NORs at the light microscopic level was obtained using a silver-staining technique for acidic proteins selectively associated with NORs. The morphologic data were also statistically evaluated by means of an automated image analyzer. The quantity of silver-stained NORs was higher in cancer cells (both mesothelioma and adenocarcinoma) than in reactive mesothelial cells. Moreover, NORs were more irregularly distributed within the nucleoli and were more variably sized in cancer cells than in reactive mesothelial cells.     

Immunohistochemical study of lysozyme, alpha 1-anti-chymotrypsin, tissue polypeptide antigen, keratin and carcinoembryonic antigen in effusion sediments

The cellular sediments of 42 malignant and 16 benign effusions (58 cases) were studied using the immunoperoxidase technique. Serial sections of formalin-fixed, paraffin-embedded residual sediments of effusions, sent for routine cytologic examination, were studied by commercially available polyclonal antisera against lysozyme, alpha 1-anti-trypsin, alpha 1-anti-chymotrypsin, tissue polypeptide antigen (TPA), a wide-spectrum anti-keratin, carcinoembryonic antigen (CEA) and, in single cases, thyroglobulin and prostate-specific antigen. A final definite diagnosis from histologic study of biopsy or autopsy specimens was known in all cases. All carcinomas, the mesotheliomas and the reactive mesothelial cells showed a positive reaction for TPA and, partly, the wide-spectrum keratin. Lysozyme could be demonstrated in the cells of the one proven malignant fibrous histiocytoma; all malignant epithelial cells were negative. Alpha 1-anti-chymotrypsin and alpha 1-anti-trypsin showed similar reactions: they were often positive in carcinoma cells of the breast, the bronchial system and the pancreas, in contrast to a mostly negative reaction in carcinomas of the stomach and ovary. CEA showed considerable differences; it was always negative in benign and malignant mesothelial proliferations but mostly positive in carcinomas of the stomach, pancreas and bronchial system. It was only positive in less than 20% of the carcinomas of the breast and always negative in the proven malignant effusions of primary carcinomas of the ovary and prostate. Studying a combination of several tumor markers is possible in serial paraffin-embedded sections and may be a valuable criterion in the cytologic diagnosis of effusions.     

Early diagnosis of mesothelioma in serous effusions using AgNOR analysis

OBJECTIVE: To compare the results of conventional cytology, DNA image cytometry, immunocytochemistry and argyrophilic nucleolar organizer region (AgNOR) analysis for the diagnosis of malignant cells in serous effusions. STUDY DESIGN: One hundred twenty effusions, 40 with carcinoses, 40 with malignant mesotheliomas and 40 without tumor cells on follow-up were studied by conventional cytology and three adjunctive methods. RESULTS: Unequivocal tumor cells were detected in 92.5% of effusions due to carcinoses and in 45% due to mesotheliomas. Applying immunocytochemistry with BerEP-4 positivity and DNA image cytometry with aneuploidy as a marker revealed 100% of carcinoses and 71.7% of mesotheliomas. Applying the experimentally found thresholds of 2.5 AgNORs as "satellites" and 4.5 AgNORs as "satellites and clusters" together as mean values per nucleus resulted in a 95% correct rate of mesothelioma and 100% rate of carcinoma cell identification without false positive diagnoses. CONCLUSION: AgNOR analysis may be a useful adjunct to other methods in the routine diagnosis of malignant serous effusions. It seems to be the most sensitive method in early cytologic diagnosis of mesotheliomas in effusions. Seventy-three percent of malignant mesotheliomas were diagnosed cytologically at first on effusions. Forty-seven percent of patients with malignant mesotheliomas were identified at the early tumor stage T1 N0 M0.     

Cellular and molecular parameters of mesothelioma

Malignant mesotheliomas (MM) are neoplasms arising from mesothelial cells that line the body cavities, most commonly the pleural and peritoneal cavities. Although traditionally recognized as associated with occupational asbestos exposures, MMs can appear in individuals with no documented exposures to asbestos fibers, and emerging data suggest that genetic susceptibility and simian virus 40 (SV40) infections also facilitate the development of MMs. Both asbestos exposure and transfection of human mesothelial cells with SV40 large and small antigens (Tag, tag) cause genetic modifications and cell signaling events, most notably the induction of cell survival pathways and activation of receptors, and other proteins that favor the growth and establishment of MMs as well as their resistance to chemotherapy. Recent advances in high-throughput technologies documenting gene and protein expression in patients and animal models of MMs can now be validated in human MM tissue arrays. These have revealed expression profiles that allow more accurate diagnosis and prognosis of MMs. More importantly, serum proteomics has revealed two new candidates (osteopontin and serum mesothelin-related protein or SMRP) potentially useful in screening individuals for MMs. These mechanistic approaches offer new hope for early detection and treatment of these devastating tumors.     

Spontaneously immortalized mouse mesothelial cells display characteristics of malignant transformation

Abstract. Objectives: Mesotheliomas occur in occult serous cavities after chronic exposure of mesothelial cells to asbestos fibres. Molecular events that contribute to the development of this cancer are therefore not readily accessible for study. We have used in vitro culture systems to study and compare induced and spontaneous transformation events in primary mouse mesothelial cells. Materials and methods: Mouse mesothelial cells were cultivated until small populations of proliferating cells emerged from senescing cultures. Spontaneously transformed cultures of cells were characterized and compared to malignantly transformed cells. Results: Human mesothelial cells had a finite lifespan of 10–15 population doublings when cultured in vitro; mouse mesothelial cells typically exhibit this same pattern. Here, we show that mouse mesothelial cells can be cultured for extended periods and that these cells can transform spontaneously. Lines of spontaneously transformed cells generated in this study are immortal and growth factor‐independent. They display the salient characteristic features of transformation, including increased proliferation rate, lack of contact inhibition, aneuploidy and ability to grow in anchorage‐independent conditions. A subset of these cell lines developed into tumours in syngeneic mice. Comparative gene expression analysis demonstrated that spontaneously transformed cell lines were more closely related to neoplastic cells than to primary cells. Conclusion: These findings have implications for interpretation of in vitro transformation studies, demonstrating broad similarity between spontaneous and induced genetic changes.     

Quantitative ultrastructural study of nuclei from exfoliated benign and malignant mesothelial cells and metastatic adenocarcinoma cells

Various approaches, including morphometric image analysis, are currently being used to improve the distinction between diffuse mesothelioma and metastatic adenocarcinoma of the serous membranes. Since exfoliated cells of malignant mesotheliomas were thought to have nuclear profile contours with greater irregularity than the similar profiles in metastatic adenocarcinoma cells in pleural effusions, this and other nuclear parameters were measured in ultrastructurally examined preparations from three cases of reactive mesothelial hyperplasia, seven examples of diffuse mesothelioma and three cases of metastatic adenocarcinoma (with primaries in the ovary, esophagus and prostate). Contrary to the subjective impression, the nuclei in metastatic adenocarcinomas actually had a mean nuclear contour index greater than that found in diffuse mesotheliomas; statistically, the difference was not significant. Likewise, such other nuclear parameters as nuclear area, condensed chromatin area and contour index, percentage of condensed chromatin and number of condensed chromatin clumps per nuclear profile did not discriminate between malignant mesotheliomas and adenocarcinomas metastatic to pleural surfaces. These morphometric results quantitate the similarities in nuclear size, nuclear shape and condensed chromatin arrangement in these two types of tumor and explain why the cytopathologist has such great difficulty in distinguishing between exfoliated mesothelioma and adenocarcinoma cells in most cases.     

Lower proliferation rate in metastatic effusion mesothelial cells than in benign effusions

OBJECTIVE: To determine the proliferation rates of mesothelial cells in metastatic and benign effusions. STUDY DESIGN: Immunohistochemistry was performed on formalin-fixed pellets from 16 malignant and 9 benign clinical effusions. Dual staining with antibodies against Ki-67 (MIB-1) and desmin was applied to all effusions to differentiate between benign mesothelial cells and malignant cells, and the proportions of desmin+/Ki-67+ and desmin+/Ki-67- cells were calculated. RESULTS: In 7 malignant effusions no proliferating mesothelial cells were found, whereas some rate of proliferation could always be demonstrated in mesothelial cells in the benign effusions. Further, the median proportions of proliferating cells, malignant 2% vs. benign 11%, differed significantly. CONCLUSIONS: To our knowledge this finding has not been previously described, and it may have implications for both cytologic diagnosis and the understanding of tumor biology and the interaction between tumor cells and mesothelial cells.     

Density distribution and immunological reactivity of tumor cells from peritoneal effusions of patients with ovarian neoplasms.

Ascitic samples from 19 patients with primary ovarian non-mucinous carcinomas, three with Krukenberg tumors and eight with noncancerous peritoneal effusions were studied by conventional cytology and immunocytochemical staining. Density gradient centrifugation was applied to fractionate ascitic fluid cells. The enrichment of cell types by this method facilitated their cytomorphological characterization and identification of neoplastic cell subpopulations existing in peritoneal effusions. Immunophenotypic studies of cells were made using monoclonal antibodies (mAbs) against ovarian carcinoma-associated antigens (OC 125, 10B, 8C) and carcino-embryonic antigen (CEA). Non-specific cross-reacting antigen (NCA) was applied as a marker for granulocytes which often accompany peritoneal effusions. Our results indicated that immunofluorescence (IF) staining contributed to the distinction between the primary and secondary ovarian carcinomas. Density gradient centrifugation appeared to be a useful method for separation of mesothelial cells.     

Cytogenetic response to asbestos fibers in cultured human primary mesothelial cells from 10 different donors

The ability of amosite asbestos fibers to induce chromosomal aberrations in human primary mesothelial cells obtained from pleural effusions of 10 noncancerous patients was investigated. The glutathione S-transferase M1 (GSTM1) genotypes of the patients were determined, since the GSTM1 null genotype has been associated with increased susceptibility to lung cancer and chemically induced cytogenetic damage. Four of the patients represented the GSTM1 null genotype, and six the GSTM1 positive genotype. Successful chromosome aberration analyses were obtained from six cases, three of them with the GSTM1 null genotype. The level of aberrant cells in unexposed cultures ranged from 2.0% to 7.5%. Statistically significant increases (2.3–3.0-fold compared to controls) in the number of aberrant cells were observed in two cases only: in one case treated with 1 μg/cm² of amosite, and in another treated with 2 μg/cm² of amosite. Cell cultures from four individuals showed minor or no increases in the numbers of aberrant cells in the doses tested (1 and 2 μg/cm²). Chromosome breaks were the major type of aberration. The amosite exposed cells with significantly increased aberrations were from patients with GSTM1 positive genotypes. Two cases that showed no cytogenetic response to asbestos fibers were of the GSTM1 null genotype. Thus, our results suggest that the lack of the GSTM1 gene does not render human mesothelial cells more susceptible to chromosomal damage induced by asbestos. GSTM1 null cells appeared, however, to be more sensitive to the growth inhibitory effects of asbestos than did GSTM1 positive cells. Variation in the cytogenetic response of human primary mesothelial cells to asbestos fibers was observed to exist, but the fibers do not appear to be potent inducers of structural chromosomal aberrations in these cells. It remains to be established whether individual sensitivity to asbestos fibers, due to specific genetic traits, exists.     

Cytophotometric determination of DNA in mesotheliomas and reactive mesothelial cells

Cytophotometry was used to study the nuclear DNA content of cells in Feulgen-stained effusion specimens from 18 patients with mesothelioma and 14 patients with reactive mesothelial proliferations. The mean DNA content (MDNA) of mesothelioma cells was significantly higher than that of reactive mesothelial cells (P less than .001). Other parameters reflecting the DNA content also differed significantly between the two kinds of cells, including (1) the ratio of mean mesothelial DNA to mean lymphocyte DNA, (2) the percentage of mesothelial cells with DNA content exceeding three times the lymphocyte MDNA and (3) the coefficient of variation of the DNA content. Since these parameters were highly correlated, only one was accepted in a stepwise linear discriminant model for distinguishing reactive from mesotheliomatous effusions. The model correctly classified all of the reactive effusions studied and 89% of the mesotheliomatous effusions. These results indicate that DNA analysis, using the Feulgen stain and cytophotometry, yields criteria that may be useful in distinguishing benign reactive mesothelial cells from malignant mesothelioma in effusions when used in conjunction with other traditional parameters.     

Immunocytochemical Staining of Serous Effusions With the Monoclonal Antibody Ber-Ep4

Cytospin preparations were made from 102 serous effusions for immunocytochemical staining using a panel of monoclonal antibodies including a new monoclonal antibody Ber-EP4. On cytological examination, 32 fluids were reported to contain tumour cells consistent with metastatic adenocarcinoma; 66 contained benign cells only and three were reported to contain cells suspicious of malignancy. One effusion contained tumour cells consistent with malignant mesothelioma. Positive staining of the tumour cells with Ber-EP4 was observed in the 32 effusions (100%) which contained adenocarcinoma cells. No staining of the mesothelial cells in these 32 specimens was observed. Carcinoembryonic antigen, epithelial membrane antigen Ca2 and CD15 staining of tumour cells was noted in 53%, 50%, 50% and 9% of these cases, respectively. None of the mesothelial cells in the benign effusions stained with Ber-EP4. Nor did the malignant mesothelial cells in the only case of malignant mesothelioma. These findings suggest that Ber-EP4 is a valuable addition to antibodies available for the differential diagnosis of mesothelial cells and adenocarcinoma cells in serous effusions.     

Vitronectin adsorption to chrysotile asbestos increases fiber phagocytosis and toxicity for mesothelial cells

Biological modification of asbestos fibers can alter their interaction with target cells. We have shown that vitronectin (VN), a major adhesive protein in serum, adsorbs to crocidolite asbestos and increases fiber phagocytosis by mesothelial cells via integrins. Because chrysotile asbestos differs significantly from crocidolite in charge and shape, we asked whether VN would also adsorb to chrysotile asbestos and increase its toxicity for mesothelial cells. We found that VN, either from purified solutions or from serum, adsorbed to chrysotile but at a lower amount per surface area than to crocidolite. Nevertheless, VN coating increased the phagocytosis of chrysotile as well as of crocidolite asbestos. VN coating of both chrysotile and crocidolite, but not of glass beads, increased intracellular oxidation and apoptosis of mesothelial cells. The additional apoptosis could be blocked by integrin-ligand blockade with arginine-glycine-aspartic acid peptides, confirming a role for integrins in the fiber-induced toxicity. We conclude that VN increases the phagocytosis of chrysotile as well as of crocidolite asbestos and that phagocytosis is important in fiber-induced toxicity for mesothelial cells.     

Integrated nuclear fluorescence and expression of hormone-binding sites in malignant pleural effusions

OBJECTIVE: To investigate potential disease- and prognosis-associated nuclear and cellular features from cell properties in a prospective study on malignant pleural effusions. STUDY DESIGN: Integrated nuclear fluorescence and the expression of binding capacities of carrier-immobilized estradiol, progesterone and testosterone; and of labeled sarcolectin; and the presence of calcyclin were measured in 50 cases with proven malignant pleural effusions (10 mesotheliomas, 40 metastasizing tumors). A double fluorescence technique using the fluorochrome DAPI and a Texas Red-based avidin-biotin detection system were applied. Detailed clinical data, including the follow-up for up to 40 months, were included. RESULTS: Pleural effusions in all patients with mesotheliomas occurred prior to (9/10) or at the time of histologic confirmation. Mesotheliomas had the highest tumor cell fraction (12.4%) in S phase and breast carcinomas the lowest (10.7%). More than 80% of malignant cells expressed binding capacities for the applied probes. A statistically significant correlation was noted between the S-phase-related tumor cell fraction and the expression of progesterone receptors. Survival was associated with tumor origin, treatment by pleurodesis, and certain cytometric and histochemical features. CONCLUSION: The immunofluorescence double-staining technique can be applied successfully in malignant effusions to combine DNA measurements with those of immunohistochemical and ligand histochemical reactivity.