Cytopathology of Pleural Mesotheliomas

Cytopathology of Pleural Mesotheliomas Gia-Khanh Nguyen, MD Key Words: Pleural mesothelioma; Epithelial mesothelioma; Sarcomatous mesothelioma; Mixed mesothelioma; Exfoliative cytology; Fineneedle aspiration cytology; Immunocytochemistry; Electron microscopy; Diagnostic accuracy; Diagnostic pitfalls Abstract Pleural mesothelioma is a rare and highly malignant tumor that has shown increasing incidence. In approximately 90% of patients the tumor manifests initially as recurrent, unilateral, bloody pleural effusions; in the remaining 10% of patients the tumor is detected by chest radiography. As a result, pleural mesotheliomas can be diagnosed by cytologic examination of pleural effusions or transthoracic fineneedle aspirates; the cytologic manifestations of such an examination are described in detail. Additionally, the handling of cytologic specimens, diagnostic problems, and the value and limitations of immunocytochemical and electron microscopic studies and other ancillary techniques in the cytologic identification of pleural mesotheliomas are discussed. Malignant mesothelioma, commonly known as mesothelioma, is a rare neoplasm arising from the lining cells of a serosal cavity. 1-3 It is about 4 times more common in the pleural cavity than in the peritoneal cavity, and it rarely occurs in the pericardium and tunica vaginalis. 1,2 The incidence of pleural mesothelioma varies among the populations surveyed. 3 Incidence rates of 0.65 to 21.4 cases in men and 0.35 to 1.9 cases in women per million population per year have been reported from different countries and from different states in the United States. 3 In recent years, the incidence has been increasing steadily, and the projected estimates of the number of cases in the United States for the period 1980 to 2008 range from 19,000 to 79,000. 4 Epidemiologic studies have linked occupational exposure to asbestos to the development of pleural mesotheliomas in 70% to 90% of the cases, and the average latent period is 35 years. 1 Other causative factors include exposure to erionite, therapeutic radiation, chronic infection, and some carcinogenic chemicals. 1-3 The tumor occurs mainly during the fifth and sixth decades of life and rarely occurs in children. 1 Of the reported cases, 75% occur in males. 1-3 Almost all patients with the disease die within 6 to 12 months after the diagnosis. 1 It may manifest as a diffuse or localized growth; the diffuse form accounts for about 75% of all cases. 1 In about 90% of patients, the disease manifests initially with recurrent, unilateral, bloody pleural effusions. In fewer than 10% of the cases, pleural tumors without pleural effusions are detected by chest radiography. 1 Thus, pleural mesotheliomas may be diagnosed by cytologic examination of effusions or materials obtained by transthoracic fine-needle aspiration biopsy (FNAB). This article reviews the cytopathologic features of pleural mesotheliomas in associated effusions and in materials obtained by transthoracic FNAB. S68 Am J Clin Pathol 2000;114(Suppl 1):S68-S81 American Society of Clinical Pathologists

Collection and Preparation of Cell Samples Proper collection and preparation of cytologic specimens are critical for an accurate cytologic diagnosis. The preparation techniques vary with the types of cell samples. Pleural Effusion The diagnosis of mesotheliomas by effusion cytology depends largely on the number of tumor cells present in the specimen. For optimal results, the whole effusion sample should be submitted for cytologic evaluation, and no fixative is needed if the specimen is prepared without delay. In effusion samples without added fixatives that are refrigerated at 4 C, the morphologic features of the cells will be preserved for cytologic study for several days. 5 The minimum amount of a sample that commonly yields adequate cells for cytologic diagnosis is about 200 ml. 6 Depending on the amount of blood present, the techniques for fluid sample preparation are different. For a nonbloody sample, 4 to 6 cytologic preparations and a cell block are prepared by the routine cytocentrifuge technique. To prepare a cell block, a portion of the sediment is suspended in an equal volume of plasma, 0.25 ml of thrombin is added, and the suspension is mixed. After 5 minutes, the clot is formed, and it is removed from the test tube and fixed in formalin for light microscopic studies. 6 A smaller portion is fixed in a vial of 2% glutaraldehyde for electron microscopic (EM) examination, if indicated. Ethanol is not an acceptable fixative for EM study, as it destroys the ultrastructure of the fixed cells. Immunocytochemical (IM) staining with commercial antibodies can be done on Papanicolaou-stained smears without previous destaining and on cell block sections, using the routine avidin-biotin-complex technique. A blood-stained effusion requires special preparation to obtain satisfactory smears for cytologic evaluation. Minimal blood contamination may be overcome by fixing smears in Carnoy solution to lyse the RBCs. However, if the fluid sample is contaminated heavily with blood, it should be mixed with an equal volume of a density-gradient solution, such as Ficoll-Hypaque. The mixture is centrifuged subsequently to separate nucleated cells from RBCs, and the layer of nucleated cells is removed with a pipette for preparation of smears and a cell block. 6 Fine-Needle Aspirate Cells from a pleural or lung mass lesion can be sampled by transthoracic FNAB under fluoroscopic or computed tomographic guidance. Usually, disposable 22-gauge and 15- cm-long spinal-type needles are used. Several direct smears are prepared from the needle aspirate. They can be fixed in 95% ethanol for staining with the Papanicolaou method or with H&E, or they can be air dried for staining by the routine rapid Romanowsky technique. Minute tissue fragments retrieved from the needle aspirate should be fixed in 2% glutaraldehyde for EM study. The needle and syringe can be rinsed in a vial of a salt-balanced solution to be used for cell block preparation for IM or EM studies. Cytologic Manifestations of Mesotheliomas Since the first detailed microscopic description of 5 pleural mesotheliomas by Klemperer and Rabin in 1931, 7 numerous published studies have established different histologic variants and IM and EM characteristic features. 1-3,8 The tumor may be classified into 4 main histologic types: epithelial, sarcomatous, biphasic or mixed, and poorly differentiated or undifferentiated. 1,3,8 There are, in addition, a few rare histologic subtypes, such as desmoplastic, small cell, lymphohistiocytoid, and deciduoid mesotheliomas. 1,8 About 50% are epithelial, and they commonly show a tubulopapillary, microcystic, or solid pattern. Usually, 2 or 3 histologic patterns coexist in almost all epithelial and mixed types. 1-3,8 Sarcomatous types and mixed types account for approximately 15% to 20% and 25% to 30% of cases, respectively. 1-3,8 Histologically, a sarcomatous mesothelioma is characterized by spindle-shaped malignant cells arranged in a nonspecific pattern. A mixed mesothelioma is composed of epithelial and sarcomatous elements, and areas with transitions between these 2 cellular elements may be seen. 1 Epithelial and mixed mesotheliomas commonly are associated with pleural effusions containing exfoliated epithelial tumor cells. In contrast, sarcomatous tumors rarely are associated with pleural effusions, and when they are, they seldom exfoliate their cells in the fluids. 6,9-12 Effusion Cytology The first detailed description of the cytologic manifestations of mesotheliomas was reported by Klempman in 1962. 13 More than 20 reports on this topic subsequently have appeared in the literature. 6 Epithelial and Mixed Mesotheliomas Cytology. Pleural effusions associated with epithelial or mixed types usually are hypercellular and contain numerous epithelial tumor cells. Sarcomatous cells from a mixed mesothelioma are seen rarely in the effusion. 6,9-12 About 10% of effusions are acellular or have extremely scant cellularity. 6 The classic cytologic manifestations of epithelial and mixed mesotheliomas have been described and illustrated in several articles 12-19 and in standard cytopathology books. 9-11,20 They may be summarized as follows: more and bigger cells American Society of Clinical Pathologists Am J Clin Pathol 2000;114(Suppl 1):S68-S81 S69

Nguyen / CYTOPATHOLOGY OF PLEURAL MESOTHELIOMAS Image 1 Pleural effusion in an epithelial mesothelioma showing numerous tumor cells present singly, in small clusters, in large ball-like clusters with smooth or lobulated contours, and in papillary clusters (Papanicolaou, low power). Image 2 Cells from an epithelial mesothelioma are seen in pairs and clusters showing abundant, granular, 2-tone cytoplasmic staining with eosinophilic endoplasm and basophilic ectoplasm. A clear space or window is noted in the small cell cluster. A tight cell-embracing-cell arrangement is evident in the large cell cluster (Papanicolaou, high power). Image 3 Long, slender microvilli are present on the free membrane of the larger tumor cell (Papanicolaou, high power). in more and bigger clusters, and morphologic kinship with mesothelial cells. 11 The large ball-like clusters of tumor cells may show knobby, scalloped, or smooth borders and consist of a dozen or a few hundred cells. Papillary tumor tissue fragments may be seen. Admixed with these large cell clusters are single and clustered tumor cells Image 1. In about 50% of the cases, tumor cell clusters and single neoplastic cells are roughly equal, and in the remaining cases either clusters or single cells predominate. 19 The tumor cells that are present singly and in small clusters have enlarged, hyperchromatic, single or double nuclei with chromatin clumping and single or multiple prominent nucleoli. Their cytoplasm may show features of mesothelial cells with ectoplasmic-endoplasmic demarcation and a fuzzy border around the entire perimeter, and a 2-tone cytoplasmic staining with pink-orange endoplasm and blue-green ectoplasm may be observed in tumor cells stained with the Papanicolaou method. 10,11 Long, slender microvilli may be seen on the free surfaces of tumor cells exfoliated from a well-differentiated epithelial mesothelioma. Occasionally, multinucleate giant tumor cells are present. Intercellular clear spaces, or windows, and cell arrangements with configurations, such as cell-embracing-cell, pincer-like grip, and cell-in-cell or cell engulfment commonly are found in small tumor cell clusters 10,11 Image 2, Image 3, Image 4, and Image 5. Intracytoplasmic vacuoles or blebs located at the periphery of the tumor cell cytoplasm or in the paranuclear area are best visualized in airdried smears stained with the rapid Romanowsky or May- Grünwald-Giemsa technique Image 6. Occasional tumor cells with signet-ring configuration are observed commonly. Numerous mesothelial cells with normal and atypical nuclei almost always are present in the cell sample. In a few patients, the cells occur in large ball-like or small clusters, and they have 1 or 2 large intracytoplasmic vacuoles. Large, individual, malignant cells with thick, granular, nonvacuolated cytoplasm commonly are scattered in the smear background Image 7 and Image 8. S70 Am J Clin Pathol 2000;114(Suppl 1):S68-S81 American Society of Clinical Pathologists

Image 4 Cells from an epithelial mesothelioma are present in a large ball-like cluster. A multinucleate giant tumor cell is noted (Papanicolaou, medium power). Image 5 Cells from an epithelial mesothelioma are present singly and in pairs with a cell-embracing-cell arrangement (Papanicolaou, high power). A B Image 6 A, Cells from an epithelial mesothelioma showing dense or vacuolated cytoplasm (Papanicolaou, high power). B, A multinucleate tumor cell displays multiple cytoplasmic vacuoles located in its ectoplasm (rapid Romanowsky, high power). In some cases, the fluid has single and clustered malignant cells with thick and hard cytoplasm mimicking cells of a metastatic keratinizing squamous cell carcinoma Image 9. Rarely, an epithelial or mixed mesothelioma exfoliates numerous single and clustered tumor cells with subtle cellular changes mimicking reactive mesothelial cells Image 10. Cell Block. Sections may have epithelial cells in solid, acinar, and papillary patterns with fibrovascular or hyalinized fibrous cores reflecting papillary tumor fragments broken from the main tumor mass Image 11. The acinar clusters of tumor cells with central hyalinized fibrous cores are of diagnostic value for epithelial mesothelioma, according to Whitaker et al. 6 Cytochemistry and Immunocytochemistry. Cells of an epithelial or mixed mesothelioma are rich in glycogen, and they stain positively with periodic acid Schiff 11 Image 12. They usually express high- and low-molecular-weight keratins and rarely express vimentin. The cells stain negatively with monoclonal antibodies against carcinoembryonic antigen (CEA), Ber-Ep4, B72.3, and Leu-M1 1,6,10,11,21-24 Image 13. Staining with monoclonal antibodies against epithelial membrane antigen (EMA), HMFG-2, 22,24 HBME-1, 23 and thrombomodulin 23-26 often has a positive membranous pattern. The tumor cells stained with these 4 antibodies commonly have a thick and bushy immunopositive membranous pattern American Society of Clinical Pathologists Am J Clin Pathol 2000;114(Suppl 1):S68-S81 S71

Nguyen / CYTOPATHOLOGY OF PLEURAL MESOTHELIOMAS Image 7 Cells from an epithelial mesothelioma in large ball-like clusters showing large, single, or multiple intracytoplasmic vacuoles (Papanicolaou, medium power). Image 8 Single and clustered cells from an epithelial mesothelioma showing large, single, intracytoplasmic vacuoles. A few single malignant cells with scant and nonvacuolated cytoplasm also are noted (Papanicolaou, high power). A B Image 9 Single (A) and clustered (B) cells from an epithelial mesothelioma showing squamoid cytoplasm (Papanicolaou, high power). reflecting long and slender microvilli present on the free cell surfaces 22-26 Image 14. A weak immunopositive cytoplasmic reaction may be noted in tumor cells that have positive membranous staining with the aforementioned antibodies. 22-25 Polyclonal calretinin antibody shows a cytoplasmic immunopositive reaction not only with epithelial tumor cells 27,28 but also with benign and reactive mesothelial cells. 27 Positive nuclear staining may be seen in tumor cells having a positive cytoplasmic reaction with calretinin antibody. 27,28 The epithelial Image 10 Numerous single and clustered tumor cells with features in keeping with atypical and benign-appearing mesothelial cells present in a serous effusion associated with a well-differentiated epithelial mesothelioma (Papanicolaou, high power). mesothelioma cells also express cytokeratins 5 and 6. 29 Ultrastructural Features. Ultrathin sections stained with uranyl acetate and lead citrate commonly reveal polygonal, non mucus secreting epithelial cells with well-formed cell junctions, abundant intracytoplasmic glycogen granules, perinuclear bundles of intermediate filaments, and long, slender microvilli without dense-core rootlets on the free cell surfaces 6,10,11,30,31 Image 15. The presence of long, slender microvilli with a length/diameter ratio greater than 15 1,8 or 12 31 S72 Am J Clin Pathol 2000;114(Suppl 1):S68-S81 American Society of Clinical Pathologists

Image 11 Cell block section showing mesothelioma cells in an acinar arrangement. Central hyalinized cores are noted in 2 tumor cell acini (H&E, medium power). Image 12 A cluster of epithelial mesothelioma cells showing a cell-embracing-cell arrangement and cytoplasm positive for periodic acid-schiff (periodic acid Schiff, high power). A B Image 13 A, Epithelial mesothelioma cells showing immunonegative reaction with carcinoembryonic antigen antibody (avidin-biotin-complex, high power). B, Tumor cells with diffuse or perinuclear immunopositive reaction with keratin antibody (avidin-biotin-complex, high power). Image 14 A cluster of tumor cells in a cell block section stained with epithelial membrane antigen antibody showing an immunopositive, irregular, thick and bushy membranous pattern (avidin-biotin-complex, high power). is the most reliable characteristic feature of epithelial cells from mesotheliomas. Sarcomatous Mesothelioma This type of tumor rarely is associated with a pleural effusion, and it seldom exfoliates its cells in fluid if there is an effusion. 6,9-12 The exfoliated tumor cells may be spindle-shaped and occur singly or in small clusters 6,9,10 Image 16. They also may appear as isolated or loosely clustered pleomorphic malignant cells with no mesothelial features Image 17. The tumor cells usually express keratin and vimentin, and they stain negatively with CEA and other epithelial antibodies. 1-3 Poorly Differentiated Mesothelioma Cells exfoliated from this type of mesothelioma have no distinctive features. They are present singly or in loose American Society of Clinical Pathologists Am J Clin Pathol 2000;114(Suppl 1):S68-S81 S73

Nguyen / CYTOPATHOLOGY OF PLEURAL MESOTHELIOMAS Image 15 Ultrastructure of a cluster of epithelial mesothelioma cells showing well-formed cell junctions, glycogen lakes, and long, slender microvilli on the free cell surfaces (uranyl acetate and lead citrate, original magnification 9,000). Image 16 A spindle-shaped malignant cell from a sarcomatous mesothelioma seen admixed with inflammatory cells (Papanicolaou, high power). Rare Histologic Variants of Mesothelioma Desmoplastic, small cell, lymphohistiocytoid, and deciduoid mesotheliomas are rare histologic subtypes. 1,3,8 Extensive tissue sampling of desmoplastic and small cell neoplasms may reveal foci of an epithelial or mixed mesothelioma. 1,8 Effusions in desmoplastic tumors are commonly negative for malignant cells. The small cell mesothelioma may exfoliate single or clustered tumor cells with scant cytoplasm and inconspicuous nucleoli 18 Image 19. They may stain positively with neuron-specific enolase and Leu-7 antibodies. 1,8 In an effusion, a lymphohistiocytoid mesothelioma may show single and clustered histiocyte-like tumor cells with beanshaped nuclei and vacuolated cytoplasm admixed with numerous lymphocytes. 32 Fine-Needle Aspiration Cytology Image 17 Pleomorphic malignant cells without mesothelial features are noted in a pleural effusion associated with a sarcomatous mesothelioma (Papanicolaou, high power). aggregates and have abundant, granular, or vacuolated cytoplasm. The nuclei are single or multiple with prominent nucleoli. Intracytoplasmic fat droplets or vacuoles are visualized easily in air-dried smears stained with the rapid Romanowsky technique Image 18. In cell block sections, the tumor cells stain negatively with CEA, Leu-M1, Ber-Ep4, and EMA monoclonal antibodies, and they may express keratin and vimentin. 1-3 Ultrastructural features of mesothelial cells usually are not found in ultrathin sections. 1-3 Cytology Only a few mesotheliomas with cytologic evaluation by transthoracic FNAB have been reported. 33-36 An epithelial mesothelioma usually yields tumor cells singly, in sheets, and in ball-like or papillary clusters 33,34,36 Image 20. The tumor cells display well-defined, optically dense cytoplasm, oval nuclei, and prominent nucleoli. Occasional tumor cells show vacuolated cytoplasm Image 21. Needle aspirates from a sarcomatous tumor may reveal spindle-shaped malignant cells with elongated nuclei and scant, granular, or clear cytoplasm, present singly and in clusters or sheets 34-36 Image 22 and Image 23. A mixed mesothelioma is characterized by an admixture of single and clustered malignant S74 Am J Clin Pathol 2000;114(Suppl 1):S68-S81 American Society of Clinical Pathologists

A B Image 18 A, Poorly differentiated mesothelioma showing pleomorphic, single, malignant cells with single or multiple nuclei and prominent nucleoli (Papanicolaou, high power). B, Intracytoplasmic vacuoles aggregated in perinuclear area are seen (rapid Romanowsky, high power). vimentin, EMA, HBME-1, calretinin, thrombomodulin, and cytokeratins 5 and 6 may reveal characteristic staining features of tumor cells as seen in effusions. By EM studies, the epithelial tumor cells show well-formed cell junctions and desmosomes and long, slender microvilli 1,8,31,36 Image 25. Long, slender microvilli in direct contact with collagen fiber bundles in the tumor matrix commonly are seen in minute tumor tissue fragments, a finding that constitutes strong evidence of an invasive epithelial mesothelioma 31,36,37 Image 26. Diagnostic Accuracy Image 19 Small malignant cells in clusters with knobby contours from a small cell mesothelioma. Inconspicuous nucleoli are present in some tumor cells (Papanicolaou, high power). spindle-shaped cells, as well as malignant epithelial cells displaying mesothelial cell features. 34,36 A poorly differentiated mesothelioma yields large malignant cells with illdefined or well-defined granular cytoplasm and prominent nucleoli singly and in aggregates, similar to those of a large cell carcinoma 36 Image 24. Immunocytochemistry and Electron Microscopy Immunostaining of the tumor cells in smears or cell block sections with antibodies against CEA, keratin, The cytologic diagnosis of mesotheliomas is challenging. In a review of the literature, the reported diagnostic accuracy rates of cytology varied tremendously, ranging from 0% to 100%. 6 This may reflect the lack of diagnostic experience of pathologists with this rare neoplasm. 6 A clinical history of occupational asbestos exposure and the presence of pleural-based tumors detected by imaging techniques should alert the cytotechnologist and pathologist to the high probability of a mesothelioma. 6 The cytologic diagnosis of mesothelioma in fluid or in FNAB consists of 2 stages. The first stage is the identification of malignant cells, and the second stage is the identification of the mesothelial features of the neoplastic cells. 1,6,38 According to Whitaker et al 6 and Whitaker and Shilkin, 38 a correct cytologic diagnosis of an epithelial mesothelioma can be made in the presence of many large epithelial clusters or aggregates together with abundant epithelial cells seen singly American Society of Clinical Pathologists Am J Clin Pathol 2000;114(Suppl 1):S68-S81 S75

Nguyen / CYTOPATHOLOGY OF PLEURAL MESOTHELIOMAS A B Image 20 Fine-needle aspirates from an epithelial mesothelioma. A, A large and thick papillary tumor tissue fragment (H&E, medium power). B, Lobulated large and small tumor cell clusters (rapid Romanowsky, medium power). A B Image 21 A, Cells from an epithelial mesothelioma are present in small clusters, and a large intracytoplasmic vacuole is noted in 1 tumor cell (H&E, high power). B, Similar tumor cells present singly and in a ball-like cluster (H&E, high power). and in small groups. Large cellular aggregates are the most valuable for facilitating the diagnosis of a malignant neoplasm, and single cells or clusters of 2 to 6 cells are the most valuable for identifying the mesothelial characteristics of tumor cells. 6 When only large cellular aggregates are found, IM and EM studies of the effusion cell block are necessary for distinguishing an epithelial mesothelioma from an adenocarcinoma. 6 In experienced hands, a correct diagnosis of mesothelioma by effusion cytology may be made in about 50% of the cases by using cytologic criteria alone without IM or EM studies of cell blocks. 38 With effusion cytology and IM studies with or without EM studies of the cell block, a correct diagnosis of epithelial mesothelioma may be made in 60% to 80% of the cases. 19 An 80% accuracy of diagnosis of mesothelioma has been reached by a combination of effusion cytology, IM, and/or EM studies of cell blocks and histologic examination of minute tumor tissue fragments obtained by closed pleural biopsy, 6,38 and pleural biopsy alone can diagnose only 40% to 60% of mesotheliomas. 38 If the cytologic and IM and/or EM studies of the effusions fail to establish a diagnosis of mesothelioma, a pleuroscopy or a thoracotomy should be done to obtain tumor tissue for histologic and EM diagnoses. Since there is no specific mesothelial cell antibody, a cytologic diagnosis of epithelial or mixed mesothelioma is based on negative IM reactions of the tumor cells with CEA, S76 Am J Clin Pathol 2000;114(Suppl 1):S68-S81 American Society of Clinical Pathologists

Image 22 Single spindle-shaped tumor cells from a sarcomatous mesothelioma (rapid Romanowsky, medium power). Image 23 Spindle-shaped malignant cells with clear or granular cytoplasm in a cohesive sheet aspirated from a sarcomatous mesothelioma with extensive clear cell changes (Papanicolaou, high power). Image 24 Clustered pleomorphic malignant cells with illdefined granular cytoplasm and prominent nucleoli aspirated from a poorly differentiated mesothelioma (Papanicolaou, high power). Image 25 Ultrathin section from a cluster of tumor cells aspirated from an epithelial mesothelioma showing intracytoplasmic vacuoles or invagination and long, slender microvilli on the cell surfaces and on the inner aspect of the intracytoplasmic invagination (uranyl acetate and lead citrate, original magnification 9,000). Leu-M1, Ber-Ep4, and B72.3 monoclonal antibodies. 21-25 In recent years, 3 mesothelioma-related antibodies against HBME-1, thrombomodulin, and calretinin have been studied for their potential diagnostic value for epithelial mesotheliomas. 23-28 The results from a few published studies have shown that cells derived from a mesothelioma more commonly react with HBME-1, thrombomodulin, and calretinin antibodies than do those derived from an adenocarcinoma. 23-28 The frequency of expressions of HBME-1, thrombomodulin, and calretinin of mesotheliomas vs adenocarcinomas has been about 79% to 89% vs 40% to 65%, 23-25 49% to 100% vs 6% to 53%, 23-26 and 42% to 100% vs 4% to 20%, 24,27,28 respectively. Recently, cytokeratins 5 and 6 and N-cadherin have been found to be more sensitive American Society of Clinical Pathologists Am J Clin Pathol 2000;114(Suppl 1):S68-S81 S77

Nguyen / CYTOPATHOLOGY OF PLEURAL MESOTHELIOMAS Image 26 Ultrathin section from a tumor tissue fragment aspirated from an epithelial mesothelioma showing 2 tumor cells with long, slender microvilli in direct contact with collagen bundles in the matrix (arrowhead) (uranyl acetate and lead citrate, original magnification 7,500). markers for mesothelioma cells than for adenocarcinoma cells; cytokeratins 5 and 6 appear to be some of the most promising positive markers for mesothelioma. 29 In Ordóñez experience, all 36 mesotheliomas (100%) expressed cytokeratins 5 and 6, and none of the 30 primary adenocarcinomas of the lung (0%) showed reactivity to this antibody. 29 N-cadherin is not entirely sensitive or absolutely specific for mesothelioma, but it is more commonly expressed by mesothelioma than by pulmonary adenocarcinoma, and it may, therefore, assist in the diagnosis of mesothelioma. 29 Thus, adding HBME-1, thrombomodulin, calretinin, and cytokeratins 5 and 6 antibodies to the usual IM mesothelioma diagnostic panel that consists of commonly used epithelial antibodies will be diagnostically helpful. EM study of cells in cell block or obtained by FNAB is probably the most reliable test available to identify epithelial mesothelioma cells. 10,30 Differential Diagnosis The cytologic differential diagnosis between epithelial mesothelioma cells, malignant glandular cells, and reactive mesothelial cells may be challenging. 5,6,7-11,39 Malignant Glandular Cells In effusions, a metastatic adenocarcinoma usually shows 2 distinct cell populations consisting of benign mesothelial cells and malignant glandular cells, 8-11 and cells from an epithelial mesothelioma commonly show a spectrum of nuclear changes ranging from those of normal to atypical and malignant nuclei. 1,6,11,38 However, these 2 classic cellular patterns may be absent or difficult to appreciate on several occasions. 11,38 Therefore, IM studies of smears, cell blocks, or both with antibodies against CEA, Ber-Ep4, Leu-M1, B72.3, EMA, HBME-1, thrombomodulin, calretinin, and cytokeratins 5 and 6 are needed for a more definitive diagnosis Image 27. A cell block from an effusion containing adenocarcinoma cells stained with EMA antibody usually shows a smooth and thin membranous pattern 22 that is different from the characteristic thick and bushy membranous pattern of epithelial mesothelioma cells 22 Image 28. EM study of the cell block or minute tissue fragments retrieved from FNAB of a pleural tumor also is helpful for identifying epithelial mesothelioma cells and adenocarcinoma cells, as glandular cells and mesothelial cells usually show distinctive ultrastructural features. 1,3,8,30,31,36 Reactive Mesothelial Cells The cytologic differential diagnosis between atypical reactive mesothelial cells and epithelial mesothelioma cells can be problematic. Staining of the tumor cells with p53 antibody may show a positive nuclear stain, and reactive mesothelial cells show no immunopositive reaction with this antibody. 1,8,40,41 Ploidy determination by flow cytometry or cell image analysis does not help solve this diagnostic dilemma, as 50% to 85% of epithelial mesotheliomas are diploid. 1,8,42 EM study of the cell block is not helpful for the differentiation between reactive and malignant mesothelial cells, as these 2 cell types may be similar. 31 Some investigators claimed that reactive mesothelial cells do not express EMA and that the tumor cells derived from an epithelial mesothelioma do. 6,22 However, this finding is not supported by the work of Friedman et al. 42 In the diagnosis of epithelial mesotheliomas by FNAB, EM study of aspirated minute tumor tissue fragments may reveal a direct contact between long slender microvilli and collagen bundles. This interesting finding was observed in tissue from an invasive epithelial mesothelioma, 31,36,37 and it is not seen in reactive mesothelial tissue, in the author s limited experience. Finally, all ultrastructural diagnostic criteria for an epithelial mesothelioma are expressed to varying degrees in individual cases, and the absence of long, slender microvilli on the free cell surface does not exclude a diagnosis of the tumor. 43 Spindle-Cell Sarcomas of the Lung and Chest Wall In FNAB specimens, malignant spindle cells from a sarcomatous mesothelioma should be differentiated from those of a benign solitary fibrous tumor of the pleura 1,44,45 and from cells of a primary or metastatic fibrosarcoma, leiomyosarcoma, malignant schwannoma, and malignant S78 Am J Clin Pathol 2000;114(Suppl 1):S68-S81 American Society of Clinical Pathologists

A B Image 27 A, Metastatic malignant glandular cells are present singly and in tridimensional clusters admixed with benign mesothelial cells (Papanicolaou, high power). B, Malignant glandular cells showing immunopositive reaction with carcinoembryonic antigen antibody (avidin-biotin-complex, high power). neoplasms do not show these characteristic ultrastructural features. 31,48 Image 28 Clusters of malignant glandular cells in a cell block section showing an immunopositive, linear, thin, membranous pattern reaction with epithelial membrane antigen antibody (avidin-biotin-complex, high power). fibrous histiocytoma of the lung 44,46 and pleura. IM staining of the needle aspirate with keratin antibody is helpful in this situation, as sarcomatous mesothelioma cells stain positively with keratin antibody, 44 while those of the 5 other aforementioned tumors usually do not. 47,48 Moreover, cells from a benign solitary fibrous tumor of the pleura commonly express CD34. 1,45 Poorly formed cell junctions, intracytoplasmic vacuoles containing proteoglycan, and cell processes may be identified by EM study of aspirated sarcomatous mesothelioma cells. 31 The 5 other aforementioned Carcinosarcoma and Synovial Sarcoma Carcinosarcoma and pulmonary blastoma of the lung and primary or metastatic synovial sarcoma of the lung and pleura should be considered in the differential diagnosis with mixed mesothelioma in cytologic material obtained by FNAB. 44,46 Epithelial and sarcomatous cells aspirated from a mixed mesothelioma are keratin-positive and CEA-negative. 1-3 Epithelial and sarcomatous cells from a carcinosarcoma of the lung may express CEA. 47,48 Cells from a pulmonary blastoma may express neuroendocrine markers, CEA, EMA, keratin, Clara cell antigen, surfactant apoprotein, and, rarely, alpha-fetoprotein. 44,46 Tumor cells from a biphasic synovial sarcoma stain positively with keratin and vimentin antibodies, and the epithelial component of the tumor may express, in addition, CEA. 47,48 EM study of synovial sarcoma cells is not helpful, as its neoplastic epithelial cells may show long and slender microvilli similar to those seen in the epithelial component of a mixed mesothelioma. 31 Cytogenetic study of cells from synovial cell sarcomas reveals a characteristic chromosomal translocation t(x;18)(p11;q11) in more than 90% of cases, 49 while cells from a mesothelioma show no consistent karyotypic abnormalities or specific chromosomal aberrations. 1,8 From the Division of Anatomical Pathology, University of Alberta Hospitals, Edmonton, Alberta. Address reprint requests to Dr Nguyen: Division of Anatomical Pathology, University of Alberta Hospitals, Edmonton, AB, Canada T6G 2B7. American Society of Clinical Pathologists Am J Clin Pathol 2000;114(Suppl 1):S68-S81 S79

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