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.     

Role of multipotent fibroblasts in the healing colonic mucosa of rabbits. Ultrastructural and immunocytochemical study.

Light- and electron microscopy and immunocytochemistry were used to study the healing colonic mucosa of rabbits after experimental excision. Between 3 and 5 days, abundant young fibroblasts which retained many features of mesenchymal cells invaded the growing capillaries into the loose connective tissue of the healing colonic mucosa. Our electron microscopy revealed the transformation of these young fibroblasts into smooth muscle cells, into histiocyte-like cells involved in phagocytotic activity, and into vasoformative cells incorporated into the growing capillaries. The mitotic proliferation of pre-existing smooth muscle cells at the ulcer margin did not seem to be the major reason for re-establishment of the muscular tissue. The present immunocytochemistry revealed an active production of fibronectin in rough endoplasmic reticulum in the young fibroblasts. This may mean that this glycoprotein is involved in the re-establishment of both connective and muscular tissues by enhancement of adhesion and chemoattractant activities of such cells. In addition, the immunoreaction of endothelial cells of the growing capillaries suggests a role of this glycoprotein in the acceleration of the neocapillarization.     

Surface configuration of mesothelial cells in effusions. A comparative light microscopic and scanning electron microscopic study.

Surface configuration of mesothelial cells identified by light microscopy (LM) has been studied by scanning electron microscopy (SEM). It has been shown that mesothelial cells may have a variable SEM appearance. The surfaces of a small proportion of mesothelial cells are covered by regular microvilli (MV) and show openings of the pinocytotic vesicles. The surfaces of the majority of these cells are covered by vesicles or blebs. An intermediate population of mesothelial cells, i.e., cells displaying side-by-side blebs and MV, has also been observed. The latter cells no longer display pinocytotic vesicles. Occasional mesothelial cells have smooth surfaces. It has been shown by LM and transmission electron microscopy that cells with blebs are viable and capable of mitotic activity. It is concluded that mesothelial cells, detached from their epithelial setting, lose microvilli and pinocytotic vesicles and acquire surface blebs. The possible relationship between mesothelial cells and macrophages based on surface features has been discussed.     

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.     

Ultrastructure of the lymphatic capillaries of the diaphragm and their interrelationship with peritoneal mesothelium

In 28 mature rats, ultrastructure of the diaphragmal lymphatic capillaries and mesothelial tegmen was studied. Interrelation of their cellular elements and possible ways of metabolic absorbtion from the abdomen were clarified. The diaphragmal lymphatic capillaries were demonstrated to situate under mesothelial cells and they are separated by a thin layer of longitudial collagenic fibers. As cross section of the lymphatic capillary demonstrates, the capillary wall consists of 3--6 endothelial cells with a set of organelles, among which are: mitochondria, Golgi's complex, some pinocytotic vesicles.     

An unusual cytologic presentation of mesothelioma in serous effusions

Three cases of diffuse malignant mesothelioma in which samples of pleural fluid showed an unusual cytologic picture are presented. Instead of cells of an obvious mesothelial type forming organized clusters, the smears were dominated by foamy macrophage-like cells, with or without certain nuclear features suggesting malignancy. It is suggested that these cells were derived from neoplastic mesothelial cells by a process of differentiation.     

Ultrastructural studies of cells in body cavity effusions

Transmission electron microscopic examination of benign (16 cases) and malignant (2 cases) mesothelial cells and metastatic carcinoma (10 cases) was performed. These studies showed long, slender, branching and bushy microvilli with high length-to-diameter ratios to be the most important distinguishing features of the mesothelial cells. Cytoplasmic intermediate filaments were present in all mesothelial cells as well as in carcinoma cells. The mesothelial cells showed an absence of mucin vacuoles, intracellular lumens and luminal tight junctions, which are seen in adenocarcinoma cells. The pinocytotic vesicles were found to be more numerous in the mesothelial cells. Lipid vacuoles, lysosomes, Golgi apparatus and intercellular lumens appeared to be variably present in all mesothelial and carcinoma cells. The methodology is discussed and pertinent literature reviewed.     

Expression of epithelial membrane antigen on malignant mesothelioma cells. An immunocytochemical and immunoelectron microscopic study

The presence of epithelial membrane antigen (EMA) on malignant mesothelial cells found in pleural and ascitic fluids was demonstrated immunocytochemically using a monoclonal anti-EMA antibody. Serous fluids of 25 patients with malignant mesotheliomas were investigated. In 23 cases, varying numbers of EMA-positive tumor cells were present; in 2 cases, no such cells were found. Immunoelectron microscopy was performed both on Lowicryl-embedded sediments of serous fluids and by application of preembedding techniques using the immunogold method. Expression of EMA by the immunogold method was found selectively on the villi of the malignant mesothelioma cells whereas the nonvillous, flat surfaces were largely EMA-negative. The results indicate that immunoelectron microscopy may offer a useful adjunct in the diagnosis of malignant mesothelioma in serous fluids.     

Monoclonal antibodies in the cytodiagnosis of serous effusions

In order to assess the value of immunocytochemical staining as a method of discriminating between benign reactive mesothelial cells and malignant epithelial cells in serous effusions, we have studied the reactions of a panel of commercially available antibodies on cells harvested from 83 pleural and peritoneal fluids and compared the results with the clinical and cytological diagnoses. The antibodies used were raised against cytokeratin (PKK1), epithelial membrane antigen (EMA), carcino-embryonic antigen (CEA), pregnancy specific B1-glycoprotein (SP1) and leucocyte common antigen (LCA). Anti-CEA was positive in 16 of 39 effusions (41%) containing carcinoma cells. Pregnancy specific B1-glycoprotein (SP1) was positive in 33% of the same samples. Mesothelial cells did not stain with these antibodies. Thus anti-CEA and SP1 can be used to discriminate between benign mesothelial and malignant epithelial cells in effusions. Anti-PKK1 stained both benign reactive mesothelial cells and malignant epithelial cells and cannot be used to discriminate between these two cell types. Strong positive staining of malignant cells was noted with anti-EMA. However, as occasional weak staining of mesothelial cells was also noted, strong staining with this antibody may be regarded as suspicious but not conclusive of malignancy.     

Origin of coronary endothelial cells from epicardial mesothelium in avian embryos

It has been established that coronary vessels develop through self-assembly of mesenchymal vascular progenitors in the subepicardium. Mesenchymal precursors of vascular smooth muscle cells and fibroblasts are known to originate from an epithelial-to-mesenchymal transformation of the epicardial mesothelium, but the origin of the coronary endothelium is still obscure. We herein report that at least part of the population of the precursors of the coronary endothelium are epicardially-derived cells (EPDCs). We have performed an EPDC lineage study through retroviral and fluorescent labelling of the proepicardial and epicardial mesothelium of avian embryos. In all the experiments onlythe surface mesothelium was labelled after 3 h of reincubation. However, endothelial cells from subepicardial vessels were labelled after 24-48 h and endothelial cells of intramyocardial vessels were also labelled after 48-96 h of reincubation. In addition, the development of the coronary vessels was studied in quail-chick chimeras, obtaining results which also support a mesothelial origin for endothelial and smooth muscle cells. Finally, quail proepicardial explants cultured on Matrigel showed colocalization of cytokeratin and QH1 (mesothelial and endothelial markers, respectively) after 24 h. These results, taken together, suggest that EPDC show similar competence to that displayed by bipotential vascular progenitor cells [Yamashita et al., Nature 408: 92-96 (2000)] which are able to differentiate into endothelium or smooth muscle depending on their exposure to VEGF or PDGF-BB. It is conceivable that the earliest EPDC differentiate into endothelial cells in response to myocardially-secreted VEGF, while further EPDC would be recruited by the nascent capillaries via PDGFR-beta signalling, giving rise to mural cells.     

Cytology of mesothelioma of the tunica vaginalis metastatic to the lung

The case of a 63-year-old man with malignant mesothelioma of the tunica vaginalis testis confirmed by electron microscopy is presented. The tumor recurred in the scrotal skin and subsequently metastasized to the inguinal and retroperitoneal lymph nodes. Four and a half years after the original diagnosis, pulmonary metastases were discovered by cytologic examination of bronchial washings and confirmed by transbronchial lung biopsy. The cytologic features of the metastatic malignant mesothelial cells are described and compared to those of mesothelioma cells in body cavity fluids. The role of cytology in the early detection of such tumors is discussed.     

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.     

Immunolocalization of caveolin-1 in rat and human mesothelium.

Flask-shaped vesicles have been described as caveolae in mesothelial cells in a number of animal species based on morphological criteria only. Using an antibody against caveolin-1, said to be a biochemical marker of caveolae, immunoelectron microscopy suggests that many but not all such vesicles in mesothelial cells are caveolae. Mesothelial cells from different anatomical sites showed obvious variations in both the population density and distribution of these flask-shaped vesicles and in their density of immunostaining. Lung and pericardial sac had the highest staining density. In some sites (e.g., lung, bladder, colon) caveolae were equally distributed between apical and basolateral surfaces, whereas in others (e.g., spleen, liver), they were predominantly apical. Additional immunopositive sites in the peritoneal membrane were identified, including the epineurium of peripheral nerves and the endothelium of lymphatic vessels. We further suggest that variations in the number of mesothelial cell caveolae and the density of their immunolabeling may have implications for our understanding of certain diseases such as malignant mesothelioma, especially in view of the recent hypothesis that it may be caused by SV40, a virus that appears to enter cells via caveolae.     

Stress fibers in the mesenteric mesothelial cells of the large intestine of the bullfrog,Rana catesbeiana

Summary Actin-containing cytoplasmic fibers were visualized in the mesenteric mesothelial cells of the large intestine of bullfrog tadpoles by rhodamine-phalloidin staining of en face preparations of mesothelial cells. These fibers were concurrently stained by immunofluorescence using antibodies to myosin or -actinin. Electron microscopy showed the presence of bundles of microfilaments in the basal cytoplasm of the cells. Such fibers in the mesothelial cells may be comparable to the stress fibers present in cultured cells. The mesothelial cells initially formed axially oriented stress fibers when they changed from a rhombic to a slender spindle-like shape. On the other hand, stress fibers disappeared as cells transformed from elongated to polygonal shapes during the period of metamorphic climax. Expression of stress fibers in these cells appears to be related to the degree of tension loaded on the mesentery, which may be generated by mesenteric winding. These stress fibers in the mesothelial cells may serve to regulate cellular transformation. They may also help to maintain cellular integrity by strengthening the cellular attachment to subepithelial tissue against tensile stress exerted on the mesentery.     

A new epithelial membrane antigen (Calam 27) as a marker of carcinoma in serous effusions

A new monoclonal antibody (Calam 27) that reacts with a membrane antigen present on cells of epithelial origin, but not on cells of mesothelial origin, was investigated as a means of distinguishing between mesothelial cells and malignant cells in cytologic smears of serous effusions from patients with carcinoma. Immunofluorescence staining of cells in 151 effusions from 109 patients with different diseases showed a good correlation between the cytologic diagnosis on routine preparations and the staining with Calam 27. Calam 27 was also used to study the ploidy and cell cycle kinetics of carcinoma cells versus reactive mesothelial cells and normal cells by flow cytometry; these experiments confirmed that Calam 27 is not reactive with mesothelial cells. In conclusion, Calam 27 staining can help to confirm the cytodiagnoses in cases with carcinomatous effusions.     

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.     

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.     

Cytology of benign multicystic peritoneal mesothelioma in peritoneal washings

Objective:  To describe the cytological aspect of peritoneal washings in benign multicystic peritoneal mesothelioma (BMPM).
Methods:  Three peritoneal washing specimens stained by standard cytological and histological procedures and analysed by light microscopy.
Results:  The specimens showed an abundance of monomorphous mesothelial cells devoid of atypia or mitoses. The mesothelial cells were calretinin positive. They also showed numerous squamous metaplastic cells arranged in flat sheets or isolated cells. The background contained some inflammatory cells.
Conclusion:  The combination of cytology of the peritoneal washing, histology (cell block and surgical specimen) and clinical history allow differentiation of BMPM from other cystic lesions (cystic lymphangioma and malignant mesothelioma).     

HGF/SF Induces Mesothelial Cell Migration and Proliferation by Autocrine and Paracrine Pathways

Mesothelial repair differs from that of other epithelial-like surfaces as healing does not occur solely by centripetal in-growth of cells as a sheet from the wound margins. Mesothelial cells lose their cell-cell junctions, divide, and adopt a fibroblast-like morphology while scattering across and covering the wound surface. These features are consistent with a cellular response to hepatocyte growth factor/scatter factor (HGF/SF). In this study, we examined the ability of mesothelial cells to secrete HGF/SF and investigated its possible role as an autocrine regulator of mesothelial cell motility and proliferation. We found that human primary mesothelial cells expressed HGF/SF mRNA and secreted active HGF/SF into conditioned medium as determined by ELISA and in a scattering bioassay. These cells also expressed the HGF/SF receptor, Met, as shown by RT-PCR and by Western blot analysis and immunofluorescence. Incubation of mesothelial cells with neutralizing antibodies to HGF/SF decreased cell migration to 25% of controls, whereas addition of HGF/SF disrupted cell-cell junctions and induced scattering and enhanced mesothelial cell migration. Furthermore, HGF/SF showed a small but significant mitogenic effect on all mesothelial cell lines examined. In conclusion, HGF/SF is produced by mesothelial cells and induces both motility and proliferation of these cells. These data are consistent with HGF/SF playing an autocrine role in mesothelial healing.     

Peritoneal washings in ovarian tumors. Potential sources of error in cytologic diagnosis

Peritoneal washings were performed on 48 patients with suspected or known ovarian carcinoma. The procedure was part of the initial surgical staging in 27 patients with presumed stage I and II ovarian cancer and was performed during second-look operations in 21 other cases with proven ovarian malignancy. This paper presents the microscopic features of the washings, with particular emphasis on the cytologic differentiation between benign and malignant findings outside of the ovary. Thirty-four cases showed benign or reactive mesothelial cells and no evidence of peritoneal disease. The washings of six patient showed malignant cells, which were confirmed histologically. Notable atypia that mimicked ovarian carcinoma was found in eight patients who had benign or borderline lesions. These findings included papillary and glandlike epithelial structures, with varying degrees of cellular atypia and psammoma bodies. The histologic counterparts of these atypicalities were Müllerian inclusions, mesothelial proliferations and borderline serous tumors. The differential diagnosis between these entities is essential because false-positive cytologic diagnoses may alter postoperative treatment in some patients.