Some of the greatest diagnostic dilemmas in cytopathology are in

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1 CANCER CYTOPATHOLOGY 293 Immunocytochemistry in Effusion Cytology A Contemporary Review Patricia A. Fetsch, M.T. (ASCP) Andrea Abati, M.D. Cytopathology Section, National Institutes of Health/National Cancer Institute, Bethesda, Maryland. BACKGROUND. Cytology plays a pivotal role in the diagnosis of pleural effusions. In many cases, immunocytochemistry (ICC) is required to elucidate the etiology of the atypical cells. Effusions are samples that present unique problems for ICC. To date there is no standardization of ICC methods for effusions and cytology in general. METHODS. The authors review the most commonly used cytologic preparations, fixatives, and antibodies used in effusion ICC. RESULTS. Through the utilization of cell block preparations and a panel of antibodies appropriate for the differential diagnosis in question, ICC conditions utilized in surgical pathology can be most closely replicated. CONCLUSIONS. ICC may provide reliable insights into various diagnostic dilemmas in effusion cytology, provided that laboratory standardization practices are followed. Cancer (Cancer Cytopathol) 2001;93: American Cancer Society. KEYWORDS: immunocytochemistry, effusion, adenocarcinoma (ACA), malignant mesothelioma (MM), cytospin, cell block, ThinPrep. Presented in part as an American Society of Clinical Pathologists Teleconference, May 4, 2000 and at the International Academy of Pathology Annual Meeting, Nagoya, Japan, October 19, Address for reprints: Andrea Abati, M.D., Cytopathology Section, National Institutes of Health/National Cancer Institute, Building 10, Room 2A19, Bethesda, MD ; Fax: (301) ; abatia@mail.nih.gov Received March 30, 2001; accepted May 29, Some of the greatest diagnostic dilemmas in cytopathology are in the realm of effusion cytology. Hyperplastic mesothelial cells observed in various benign conditions can undergo cytologic alterations, mimicking malignant cells. 1 4 Extensive morphologic overlap also exists between malignant mesothelial cells and metastatic carcinoma cells. 1 4 In many cases, a definitive diagnosis cannot be reached based on morphology alone; thus, the diagnostic accuracy of effusion cytology is enhanced though the utilization of ancillary techniques. Electron microscopy has long been considered the gold standard for the diagnosis of malignant mesothelioma (MM) through the demonstration of numerous long, complex microvilli. Unfortunately, microvilli can vary in length and number from tumor-to-tumor as well as within a tumor, and the presence of short microvilli does not always exclude a diagnosis of MM. 5 Currently, immunohistochemical stains that are widely utilized as diagnostic discriminators for the aforementioned diagnoses recognize both epithelial-derived and mesothelial cells. The seminal studies outlining the sensitivity and specificity of these antibodies were performed on formalin fixed, paraffin embedded surgical pathology specimens. However, large-scale cytology studies evaluating the same antibodies for use in effusion cytology have been performed on a myriad of preparations (i.e., ethanol-fixed smears and cytospins, ThinPrep [Cytyc Corporation, Boxbourough, MA] preparations, airdried cytospins, and formalin fixed cell blocks), thus accounting for less than reliable and reproducible results. In addition, a wide variety of opinions exist among pathologists with regard to the relative effectiveness of some markers used for this differentiation American Cancer Society

2 294 CANCER (CANCER CYTOPATHOLOGY) October 25, 2001 / Volume 93 / Number 5 In keeping with the contemporary viewpoint that the standardization of immunohistochemical methods is essential for reliable and reproducible results, we present what we have found to be the optimal approach for the utilization of immunohistochemistry in effusion cytology, a view that we believe approximates the current procedures utilized in surgical pathology samples most closely. 6 9 In our experience, participation in the College of American Pathologists laboratory inspection and proficiency testing programs has contributed greatly to improving our immunocytochemistry (ICC) services, moving them toward a higher degree of standardization. 6 9 Specimen Processing To achieve optimal specimen processing for ICC, effusion samples should be received in the cytology laboratory in the fresh state. With appropriate refrigeration (2 8 C), cellular integrity is well preserved for at least 72 hours and up to 1 week. The previous conventional wisdom of fixation with equal volumes of 50% ethanol should be reserved only for those cases that will require long-term storage prior to processing, as ethanol fixation largely precludes the performance of Diff-Quik staining and staining with some ICC markers. Although ICC performed on formalin fixed, paraffin embedded cell blocks of effusion samples is considered ideal because it simulates surgical pathology preparations most closely, ICC can be performed on other cytologic preparations (i.e., cytospins, smears, and ThinPrep preparations) as the situation dictates. 6,10,11 Cytospins Air-dried cytospins can be used for ICC when the sample volume is minimal. Ideally, the cytospin should be a monolayer of cells with a minimum of 100 tumor cells per slide. The sample may be concentrated or diluted with RPMI-1640 (Gibco BRL, Grand Island, NY). For sample optimization, lysis of red cells (ACK lysing buffer; Quality Biological, Inc., Gaithersburg, MD) or ficolling (Accu-Prep Lymphocytes, Westbury, NY) is acceptable if necessary. Cytospins are prepared using charged or silanated microscope slides (Fisher Superfrost Plus; Fisher Scientific, Pittsburgh, PA). Albumin should not be added to the cytofunnel when preparing the cytospins because this may result in unnecessary background on the immunostained slides. After cytocentrifugation (Shandon, Pittsburgh, PA) at 500 revolutions per minute for 5 minutes, the slides are air dried for 30 minutes at room temperature to increase cell adhesion and reduce loss during the subsequent staining procedure. The unfixed slides then are put in a plastic microscope slide storage box, placed secondarily in a sealed plastic bag with half a cup of desiccant (Drierite; W.A. Hammond Drierite Co., Xenia, OH), and stored in a refrigerator until immunostaining for at least 2 weeks without detriment. After removal from the refrigerator prior to immunostaining, the bag is allowed to equilibrate to room temperature for 30 minutes before opening. If the bag is opened while it still is cold, condensation will form on the slides and potentially cause the cells to rupture. Fixation of the cytospins, which is necessary to preserve cellular integrity, is antibody specific and is performed immediately prior to immunostaining. When titering a new antibody, a variety of fixatives (i.e., acetone, alcohol, formalin, or paraformaldehyde) as well as fixation times should be examined to determine optimal immunoreactivity. In most instances, the manufacturer s antibody specification sheet will provide information regarding appropriate fixation methods. Be aware that fixation time is critical since longer exposure to fixatives may decrease sensitivity in detecting certain cellular antigens. In general, cytospin samples requiring lymphoid markers are fixed for 10 minutes at room temperature in acetone, whereas those necessitating epithelial/carcinoma markers are fixed for 5 minutes at room temperature in alcohol (either 95% ethanol or, preferably, a 1:1 mixture of methanol:100% ethanol). 6 The slides are allowed to air dry thoroughly prior to the ICC procedure utilizing either of these methods. Many nuclear antigens may require a minute fixation in either 3.7% buffered formalin or 4% paraformaldehyde. This usually is followed by a short membrane permeabilization step with a dilute solution (0.25%) of Brij or Triton (Sigma Chemical Co., St. Louis, MO) or by further fixation in cold methanol followed by cold acetone. These slides are not allowed to air dry prior to the ICC procedure. 6 Cell blocks Formalin fixed, paraffin embedded thrombin clot cell blocks are ideal for effusion ICC, with results comparable to those of surgical pathology material. 11,12 Avoidance of formalin substitutes and alcohol for the fixation of cell blocks is prudent because the majority of published studies have not been performed using these fixatives. Cell blocks should be cut in 4-5- m sections and mounted on charged slides. Slides must be dried thoroughly and baked for at least 1 hour to melt the paraffin. Baking the slides at temperatures 60 C is not recommended. It is best to use recently prepared slides, as storage of paraffin sections may lead to reduced immunoreactivity for some antigens. Because formalin fixation forms cross-links to preserve protein

3 Immunocytochemistry in Effusions/Fetsch and Abati 295 FIGURE 1. LeuM1 immunostaining in (left) cell block section and (right) corresponding ThinPrep of a reactive pleural effusion. The high background observed in the ThinPrep sample, which was not evident in the cell block, can interfere with interpretation (diaminobenzidene structure, these may mask certain antigens and pretreatment steps are required in some instances. These pretreatment steps include proteolytic digestion and heat-induced epitope retrieval, in which tissue sections are subjected to high temperature heating in a buffer solution using microwave irradiation, autoclaving, pressure cookers, or steaming in an effort to unmask relevant antigens We find it prudent to follow the manufacturer s recommendations for any pretreatment steps as temperature, ph/type of buffer, and heating times can vary. The overuse of microwave pretreatment in particular can cause erroneous results. 13,14 Cytospins/smears/ThinPrep versus cell block Although cytospin preparations often are used successfully for ICC in many types of specimens, they are less than ideal for effusions. Nonspecific/unexpected immunoreactivity can be a major problem in effusion samples due to the nature of the protein-rich fluid the cells are floating in. Large, three-dimensional cell groups commonly encountered in cytospin preparations may entrap immunologic reagents in the center of groups and lead to false-positive results. In addition, crushed, degenerated, or necrotic cells also may contribute to background staining. 10 In a previously published study, we compared the immunoreactivity of nine mesothelioma cell lines in air-dried cytospin preparations and their corresponding cell block sections to determine the optimal type of sample preparation. 17 We reviewed the panel of markers most often utilized in effusion samples at the time of the study and determined that the expected results were observed in the cell block sections, whereas significant nonspecific immunoreactivity was observed in the cytospin samples. Although some laboratories claim excellent results utilizing smears and ThinPrep preparations for ICC, they may perform in a manner similar to that of cytospins; high background staining is often encountered because of three-dimensional clusters of cells (Fig. 1). Cellular shrinkage in ThinPrep samples can make interpretation of staining patterns difficult. 21 Furthermore, because of the methanol fixation of ThinPrep samples, many antigens are not preserved, particularly lymphoid markers, thus limiting antibody panels when using this type of preparation. 19 A recently published technique has been developed that is reported to have excellent results obtained on air-dried smears that were rehydrated and then postfixed in formal saline for 2 14 hours, followed by a 10-minute alcohol fixation. For all the antibodies used, a short heat-induced antigen retrieval pretreatment step was incorporated. 11,22,23 We have no experience with this technique. We find formalin fixed cell block sections to be the most suitable form of sample preparation when performing immunostaining on effusions due to ease of morphologic interpretation, standardized like-like comparison with surgical pathology material, least amount of background stain, and expected immunostaining patterns. In addition, numerous sections obtained from a single sample permit the evaluation of a large number of antigens typically at the lowest cost/ test, and archival material then is made available if needed for future studies.

4 296 CANCER (CANCER CYTOPATHOLOGY) October 25, 2001 / Volume 93 / Number 5 FIGURE 2. Negative specimen control in (a) cytospin and (b) cell block preparations of a pleural fluid specimen of ovarian adenocarcinoma. Note the high background staining in the cytospin preparation, which was not evident in the cell block sample (diaminobenzidene Controls Although every ICC run must include positive controls for each antibody, a negative control for each sample is imperative for assessing nonspecific background staining. 8,9 For the negative control, a duplicate slide from the case under study is tested, and an irrelevant antibody or nonimmune serum from the sample animal (i.e., mouse or rabbit) is applied in place of the primary antibody. 9,10 This slide then is used to assess nonspecific staining of the sample due to cross-reactivity of immunogens other than those the primary antibody is testing for (Fig. 2). True immunoreactivity in the test samples must be of a greater intensity than that observed on the negative control sample. Other qualities of a true positive immunoreaction are crispness of the stain, site of antigen localization within a cell as is specific for the antibody being tested (i.e., membrane, cytoplasm, or nucleus), and, typically, a heterogenous distribution within a group of cells. Internal controls such as vimentin, leukocyte common antigen (LCA) and cytokeratin (CK) may be used to assess the fixation/processing of specimens. Our institution previously published detailed procedure and troubleshooting guidelines for additional reference purposes. 6,7 Variations in Results Variations in results obtained from surgical pathology tissues and cytologic fluids are not uncommon, with immunoreactivity rates fluctuating significantly with

5 Immunocytochemistry in Effusions/Fetsch and Abati 297 FIGURE 3. (a) Diff-Quik and (b) HMB-45 immunostaining in a cytospin preparation of a pleural fluid specimen of malignant melanoma (diaminobenzidene particular antibodies. Differences in antigen expression may be due to sample size and type, fixation, pretreatment method (i.e., heat-induced epitope retrieval, enzyme digestion, etc.), procedural technique, interlaboratory sensitivity, study size (large-scale vs. small-scale studies), and antibody clone. 5,24 As stated previously, the effusion itself, in which cells are floating in the highly proteinaceous cavitary fluid, may contribute to nonspecific/unexpected immunoreactivity. Membrane staining of tumor cells may be difficult to distinguish from nonspecific staining of adjacent inflammatory cells. Finally, interpretation of immunoreactivity between pathologists also can vary; in some studies, staining of a single cell was considered positive whereas in other studies a cutoff level was used, and in still other studies only lateral membrane staining with particular antibodies was considered to be positive. 25 Antibody Panels When dealing with a neoplasm of unknown etiology in an effusion, depending on laboratory philosophy, an initial antibody screen may be performed to determine whether the malignancy is epithelial, lymphoid, melanoma, etc. using antibodies to CK, LCA, S-100, and HMB-45/MART-1. 10,26 28 After this initial screen, other confirmatory antibodies then may be utilized, the choice of which depends on the differential diagnosis. 10 For example, HMB-45 is a melanoma-

6 298 CANCER (CANCER CYTOPATHOLOGY) October 25, 2001 / Volume 93 / Number 5 TABLE 1 Typical Immunoreactivity of Adenocarcinoma and Malignant Mesothelioma in Effusion Samples. Note That Unexpected Reactivity Has Been Reported for Virtually All Antibodies CK EMA CEA LeuM1 B72.3 BerEP4 CA 19-9 Calretinin E-cadherin HBME-1 ACA / / MM / ACA: adenocarcinoma; MM: malignant mesothelioma; CK: cytokeratin; EMA: epithelial membrane antigen; CEA: carcinoembryonic antigen; : positive; : negative. specific antigen and therefore can be utilized as a diagnostic marker in effusions of suspected malignant melanoma (Fig. 3). 26 A minimum of two markers should be selected as the expression of antigens in metastatic malignancies often is heterogenous. Furthermore, there is overlapping immunoreactivity in many tumors so it is best to use a number of antibodies to obtain the appropriate diagnosis. Whenever possible, we use monoclonal antibodies because they provide the greatest specificity and produce the least amount of background staining. Adenocarcinoma versus Mesothelioma Some of the more commonly used antibodies in the differentiation of adenocarcinoma (ACA) from MM are listed in Table 1. 4,5,29 38 Traditionally, the diagnosis of MM has been that of a negative staining pattern with antibodies to BerEP4, LeuM1, B72.3, and carcinoembryonic antigen (CEA). 4,5,25,33,35,36,39,40 However, difficulties have arisen with this panel due to unexpected staining of mesothelial cells (benign or malignant) in a small proportion of samples. 17,37,41,42 Thus, the quest for definitive markers continues. Over the last several years, other epithelial/carcinoma markers (including MOC-31, blood group-related antigens, HMFG-2, and E-cadherin) have been studied, but often with conflicting results. 5,18,29,36, 39 41,43 51 Positive mesothelial markers also have been introduced as part of the panel, including OV632, thrombomodulin, CK 5/6, calretinin, HBME-1, N-cadherin, CD44S, and WT1, also with varying results. 5,18,20,24,25,29,30,35 37,39,40,43,45 49,51 63 Finally, there is speculation that markers such as p53, E-cadherin, and MOC-31 may be useful for the identification of reactive mesothelial cells. 43,46,47,64 In our laboratory, we routinely use an antibody panel comprised of B72.3, BerEP4, CA 19-9, and calretinin for the differentiation of ACA from MM in effusions. Although they are highly specific for ACA, we typically omit both CEA and LeuM1 from our panel since the sensitivity of these antibodies is quite low and the staining of inflammatory cells can interfere with interpretation. In the majority of cases, this panel can differentiate ACA from MM, but cannot reliably distinguish reactive from malignant mesothelial cells or determine the primary tumor site in cases of metastatic ACA. A brief description and immunoreactivity rates of the most commonly used antibodies is shown in Table 2. Cytokeratins CKs are intermediate-sized (10 nanometers) monofilaments found in the cytoplasm of nearly all true epithelial cell types and form part of the cytoskeletal complex in the epidermis and in most other epithelial tissues. 5,24,65,66 The tissue-specific distribution of these intermediate filaments generally is well preserved in neoplasms and forms the basis for the use of antibodies to keratins Antibodies to CK are used to identify normal and malignant cells of epithelial origin, with a cytoplasmic staining pattern (Fig. 4). It is reported that normal surface mesothelial cells and epithelial MM express both low and high molecular weight keratins, whereas ACA generally express low molecular weight keratin; this is of potential use in the identification of these two entities. 35 However, this variation in CK expression is not always a reliable means of distinguishing between the two entities. In one tissue study, both ACAs and MMs were found to express low molecular weight CK. 5 In addition, although epithelial MM has been shown to express both high and low molecular weight CK, spindled MM is reported to express only low molecular weight CK. 35 Thus, because MM and the majority of ACA cases demonstrate intense immunoreactivity with CKs, the addition of this marker to a panel for the differential diagnosis of these two entities appears to have no significant impact for the distinction of these two entities. It does, however, establish immunoreactivity. 5,67 EMA Epithelial membrane antigen (EMA) belongs to a group of proteins known as human milk fat globule membrane proteins. It is present in a wide variety of epithelia of both normal and neoplastic types. 24,31,33,34,40,56,68 Immunoreactivity, indicated by a membranous staining pattern, is evident in mammary epithelium, glandular epithelia, epithelial and squamous cell tumors, and in

7 Immunocytochemistry in Effusions/Fetsch and Abati 299 TABLE 2 Immunoreactivities of Commonly Used Antibodies for the Differentiation of Adenocarcinoma, Mesothelioma, and Reactive Mesothelial Cells Antibody Staining pattern Normal tissue ACA (%) MM (%) RM (%) References CEA C/S Mature PMN leukocytes , 33, 41, 44, 56, 64, and 75 LeuM1 C/S Myeloid cells, monocytes, , 33, 76, and 77 macrophages, epithelial cells B72.3 S Secretory endometrium , 41, 56, 69, and 70 BerEP4 C/S Epithelial cells , 33, 41, 42, and 71 CA 19-9/BG-8 C/S Columnar epithelium of pancreas, stomach, , 29, 41, 44, and 72 gallbladder; bronchial glands of lung E-cadherin S Epithelial cells , 43, 45, and 78 Calretinin C/N Neural, mesothelial, Leydig/ Sertoli, endometrium/ovarian stromal, adrenal cortical cells; eccrine glands, kidney tubules 0 9 (focal) , 60 62, 79, and 80 HBME-1 S Mesothelial cells , 47, and 81 ACA: adenocarcinoma; MM: malignant mesothelioma; RM: reactive mesothelial cells; CEA: carcinoembryonic antigen; C: cytoplasmic; S: surface; PMN: polymorphonuclear; N: nuclear. FIGURE 4. Cytokeratin cocktail (AE1/ AE3) positivity in a cell block section from a pleural fluid specimen of malignant mesothelioma (diaminobenzidene some lymphoid neoplasms (i.e., anaplastic large cell lymphomas). Mesothelial cells also will be stained in serous effusions, but the reaction may be weak. A distinctive EMA pattern in MM shows a characteristic thick cell membrane staining in the periphery of cell clusters that highlights the long microvillus projections (Fig. 5). 35,68 In comparison, the staining pattern of ACA cells is usually strong and diffusely cytoplasmic. 35,68 Effusion studies have shown EMA positivity rates of % for MM, 91% for ACA, and 6% for reactive cases. 31,56 Tissue studies report similar immunoreactivity rates of 89.5% for MM and 100% for ACA. 5 Due to difficulty in the interpretation of staining patterns, EMA usually is not a practical addition to the panel of markers for the differential diagnosis of ACA and MM. 36

8 300 CANCER (CANCER CYTOPATHOLOGY) October 25, 2001 / Volume 93 / Number 5 FIGURE 5. EMA positivity in cell block sections of pleural fluid specimens of malignant mesothelioma. Note the distinct membrane stain, attributed to the thick bushy microvilli on the surface of the cells (diaminobenzidene and hematoxylin, 400). FIGURE 6. B72.3 positivity in pleural effusion. Cell block section of metastatic colon carcinoma. Note the distinct pattern of membrane staining (diaminobenzidene TAG-72 (B72.3) B72.3 is an antibody that recognizes a tumor-associated oncofetal antigen. It is present in a wide variety of ACAs, including those originating from the lung, gastrointestinal (GI) tract, pancreas, breast, endometrium, and ovary. 69 It is not expressed by leukemias, lymphomas, sarcomas, melanomas, or benign tumors. The antigen is present on normal secretory endometrium, but not on other normal tissues. 4,24,31,41,56 Positive immunoreactivity is indicated by a membranous staining pattern (Fig. 6). 69 Tissue studies have reported ACA positivity rates of 44 83%; positivity rates for MM have been reported to be 5%. 5,25,32,36 Effusion studies have reported similar findings; ACA positivity rates range from 44 80%, 31,41,70 whereas MM and reactive effusions typically are negative or rarely positive ( 10%). 31,41,56 Human epithelial antigen (BerEP4) BerEP4 is an antibody prepared by the immunization of mice with cells from the MCF7 breast carcinoma cell line that reacts with two glycoproteins present on the surface and in the cytoplasm of epithelial cells. 24,31,41,71 In contrast to all other antiepithelial antibodies, the antibody does not label mesothelial cells and only rare cases of MM appear to be immunoreactive. The antibody does not react with nerve, glial, muscle, or mesenchymal tissue, including lymphoid

9 Immunocytochemistry in Effusions/Fetsch and Abati 301 FIGURE 7. BerEP4 positivity in pleural effusion. Cell block section of adenocarcinoma. Note the distinct pattern of membrane staining (diaminobenzidene tissue. 71 Positive reactions with BerEP4 may be evidenced by membrane staining, which can be observed in conjunction with cytoplasmic staining (Fig. 7). Reported percentages of BerEP4 positivity vary widely, reportedly ranging from 0 88% in MM cases and from % in ACA cases. 25,31 33,36,41,42 Effusion studies with BerEP4 typically have shown essentially negative immunostaining with MM and reactive cases, and positivity rates for ACA of between 32 96%. 31,33,41,42 One large-scale effusion study demonstrated 83% positivity in the ACA cases tested, which included immunoreactivity rates of 93% in ACAs of the ovary, 88% in ACAs of the GI tract, 81% in ACAs of the lung, and 73% in ACAs of the breast. 42 A recent tissue study reported positivity rates of 26%% in MM, with focal patterns of staining, and of 92% in ACA, with strong and diffuse patterns of staining present in some cases and focal staining noted in other cases. 36 Blood group-related antigens (CA 19-9/BG8) CA 19-9 is a carbohydrate antigenic determinant identified as a sialylated lacto-n-fucopentose II, which is related biochemically to the Lewis (a) blood group substance. 72 BG8 is a monoclonal antibody raised against SK-LU-3 lung carcinoma that recognizes the blood group antigen Lewis (y). 25,73 Antibodies to these blood group antigens have shown positive staining of tumor cells in most of ACAs of the pancreas and stomach, and in a large proportion of colon and gallbladder tumors. 29,35,41,44,55,74 They also are present in approximately half of primary and metastatic ovarian tumors, 35 as well as salivary gland mucoepidermoid carcinomas. 74 In normal tissues, immunoreactivity is observed in columnar epithelium of the pancreas, stomach, liver, and gallbladder and in bronchial glands of the lung. The staining pattern of this antibody has been described as cytoplasmic and luminal (Fig. 8). 5,44,74 Blood group antigens can be used as part of a panel for the differentiation of ACA and MM due to their high specificity, keeping in mind that the sensitivity of these antibodies is somewhat variable. 36,48,55 Unfortunately, both lung carcinoma and MM most often are nonimmunoreactive with anti-ca 19-9, so this marker does not appear to be able to differentiate between these two entities. 29,72 Previous tissue studies have shown positivity rates in ACA of between 39 84%, and only rare positivity in MM cases. 51,55 Effusion studies also have shown a similar pattern of immunoreactivity, with positivity rates in ACA of between 49 86% and only rare positivity in cases of MM. 29,41 Thus, a positive staining reaction with this antibody would make the diagnosis of MM unlikely. Carcinoembryonic antigen CEA first was described as a marker for colon carcinoma. 4,24,31,41,44,56,64 It is highly specific for ACA cells, and typically is negative in benign, reactive, and MM cells. 31,75 There is little or no reactivity with nonmalignant tissues, except for granulocytes. 75 Immunoreactivity with this antibody, indicated by a cytoplasmic staining pattern, is associated with colorectal carcinomas, as well as some lung, breast, and gastric carcinomas. CEA immunoreactivity varies considerably de-

10 302 CANCER (CANCER CYTOPATHOLOGY) October 25, 2001 / Volume 93 / Number 5 FIGURE 8. CA 19-9 positivity in a pleural effusion. Cell block section of metastatic pancreatic carcinoma (diaminobenzidene pending on the antibody clone used and the range of tumor types tested in various studies. 31 Polyclonal antibodies are the most sensitive; however, they have the disadvantage of strong staining of neutrophils and macrophages, and have shown cross-reactivity with substances closely related to CEA that have been identified as nonspecific cross-reacting antigens. 31,36 Cross-reactivity of polyclonal anti-cea may be responsible for the reported reactivity of mesothelial cells, because CEA positivity in MM usually is not encountered with monoclonal CEA. 35,37 In surgical pathology, CEA is considered the most widely accepted marker for the differential diagnosis of ACA and MM. 5 Tissue studies have shown positivity rates of ACA ranging from 85 94%, 5,25,32 with MM reported to be essentially nonimmunoreactive. 5,25,35 ACAs of the GI tract typically are CEA-positive, whereas breast, lung, and mucinous ovarian carcinomas show variable positivity, depending on the antibody clone used. 5,25 Nonmucinous ovarian ACAs are usually CEA negative. 5,25 Reactive and MM effusion samples are also typically negative for CEA. 31,41,56,64 However, positivity rates for ACA effusions range from 21 79%, which is somewhat lower than that reported for tissue studies. This wide range of immunoreactivity most likely is due to the type of antibody used, since monoclonal antibodies to CEA are less sensitive than polyclonals. 31,41,44,64 In our experience, the use of polyclonal CEA in effusion cytology results in a significant background stain that impairs interpretation. Therefore, we routinely omit this marker from our panel. CD15 (LeuM1) CD15 (cluster designation group 15) is a granulocyteassociated antigen that first was described on cells of myelomonocytic lineage. It encompasses a variety of monoclonal antibodies that include, among others, LeuM1. 76 These antibodies have similar immunoreactivity patterns, the majority are of the immunoglobulin (Ig) M isotype, and all react with mature neutrophils. LeuM1 is an antibody that recognizes the human myelomonocytic antigen, lacto-n-fucopentose III, which is present on mature peripheral blood eosinophils, neutrophils, monocytes, Reed-Sternberg cells of Hodgkin disease, and many ACAs. 4,24,31,76,77 It shows both a membranous and cytoplasmic immunostaining pattern. 76 LeuM1 can be a valuable part of a panel in acute leukemias, in the diagnosis and classification of Hodgkin disease, and in the differential diagnosis of ACA versus MM. 76 Tissue studies have shown positivity rates for ACA ranging from 50 78% 5,32,47 and rates for MM ranging from 0 34%. 5, 39 Reactive pleural tissue typically is negative. 47 The sensitivity of LeuM1 staining for ACA in effusions generally is lower than in histologic studies. 31 One report of effusions found a positivity rate of 32% in ACAs with only focal staining, but all reactive and MM samples were nonimmunoreactive. 31,76 This characteristic focal staining pattern in ACA, although strong, may contribute to the negative results observed in small-sized samples. 35 Thus, the main liability of LeuM1 is staining heterogeneity leading to limited utility in samples of

11 Immunocytochemistry in Effusions/Fetsch and Abati 303 FIGURE 9. E-cadherin positivity in pleural effusion. Cell block section of ovarian carcinoma. Note the distinct membranous staining pattern (diaminobenzidene moderate to low cellularity. Although it is a highly specific marker for distinguishing ACAs from MMs, the sensitivity of LeuM1 is quite low and therefore we do not utilize this marker routinely in our panel. 25,36 E-cadherin (HECD-1) Cadherins are a family of transmembrane glycoproteins that are responsible for calcium-dependent intercellular adhesion. E-cadherin (epithelial cadherin or uvomorulin) is a member of this family and is expressed on the cell surfaces of nonneoplastic epithelial cells and their malignant counterparts. 43,78 E- cadherin is potentially useful for distinguishing reactive mesothelial cells from well differentiated carcinoma cells in fluid specimens, and also may be helpful in the differentiation of ACA from MM. 18,36,43,45,55 Tissue studies have shown typically negative or only rare immunoreactivity in MM, but immunoreactivity has been reported in 81 93% of ACA cases. 45,55 Effusion studies have not been as conclusive, and varied study results have been attributed, in part, to the different antibody clones used. 25,55 In a previous effusion study performed at our institution, we found E-cadherin to be present in 97% of ACA cases, 46% of MM cases, and 14% of reactive mesothelial cells. 43 The staining pattern is typically membranous (Fig. 9), and may be in conjunction with cytoplasmic staining as well. 43,78 Conversely, another group examining cell block sections of effusions found that all the MM samples tested were negative, and all the ACA samples tested were positive. 45 Other cytologic sample preparations have produced similarly conflicting results; in a study using Papanicolaou-stained smears, 100% of the MM cases were positive, 87% of the ACA cases were positive, and all reactive effusions were negative. 18 Another study using ThinPrep samples found that in the majority of cases of carcinoma, the tumor cells showed a membranous staining pattern, and only one benign case was found to be positive. 50 Calretinin Calretinin is a neuron-specific calcium-binding protein that is strongly expressed in neural tissues and certain nonneural cell types, including mesothelium. 18,30,39,47,54,59,79,80 Antibodies to this protein are strongly immunoreactive with malignant and benign mesothelial cells, as evidenced by both a cytoplasmic and nuclear type of staining pattern, described by one group as a fried-egg appearance (Fig. 10). 62 In ACAs, this antibody generally is nonimmunoreactive or, rarely weakly, immunoreactive, exhibiting only a cytoplasmic pattern. 30,54,79,80 Differences in sensitivity and specificity exist, as many of the published studies have been performed using polyclonal antibodies from various manufacturers. 25,36,37 In fact, original studies with these original antibodies to calretinin concluded that calretinin was an unreliable marker. 43,47,48 However, the diagnostic reliability of calretinin immunostaining for the positive identification of MM versus ACA in histologic and cytologic preparations has become more and more evident with the advent of purified antibodies. 36,59 Tissue studies have shown rates of immunoreactivity for MM ranging from %, with only rare ACAs exhibiting nuclear immunoreactivity. 30,54 Numerous effusion studies using various types of cyto-

12 304 CANCER (CANCER CYTOPATHOLOGY) October 25, 2001 / Volume 93 / Number 5 FIGURE 10. Calretinin positivity in pleural effusion. Cell block section of mesothelioma. Note the strong cytoplasmic and nuclear staining in the malignant cells (diaminobenzidene and hematoxylin, 400). logic preparations including cell blocks, cytospins, and Papanicolaou-stained smears of effusions also have shown calretinin to be a sensitive and specific marker of reactive and malignant mesothelial cells, with positivity rates of % for MM and % for reactive effusions. 18,60-62, 80 We have found anticalretinin to be invaluable in a panel of markers used in diagnostic effusion cytology, particularly when there is a need to distinguish between single ACA cells and benign reactive mesothelial cells. Human mesothelial cell (HBME-1) HBME-1 (mesothelial cell antibody) was derived from a suspension of human mesothelioma cells from patients with malignant epithelioid mesothelioma, and reacts with an antigen present on the membrane of mesothelial cells. 29,47,57,81 No immunoreactivity has been observed in normal cells other than mesothelial cells; however, HBME-1 has been reported to react with some sarcomas, chordomas, and lymphomas, and is strongly positive in papillary and follicular carcinomas of the thyroid. 25,57 The staining observed in epithelial mesotheliomas most often is characterized by a thick membranous staining pattern, whereas in ACA this pattern is more commonly cytoplasmic, or thin surface staining (Fig. 11). 29,81 Desmoplastic/sarcomatous MM commonly are nonimmunoreactive, which has been attributed to the loss of cell surface microvilli in these variants. 57 Unfortunately, many tissue studies have shown overlapping staining patterns with anti-hbme-1 in MM and ACA. 25,36,48 One study found a sensitivity of 89% and a specificity of 70% for MM, and concluded that HBME-1 was less efficient than other markers for the positive identification of MM. 48 Similarly, another study reported immunoreactivity in 76% of MM cases tested, 73% of ACA cases tested, and 83% of reactive pleura samples tested. 47 A previous effusion study performed in our laboratory demonstrated 89% immunoreactivity with MM and 65% positivity with ACA, with both thick and thin membranous staining patterns present in selected cases of both entities. 29 Thus, in general, the utility of HBME-1 is limited by its relative lack of specificity for cells of mesothelial origin and overlapping staining patterns. Miscellaneous Mesothelium-Associated Antibodies CD44 CD44 is a transmembrane protein that acts as the principal receptor for hyaluronic acid, the presence of which is a major feature of MM. Tissue studies using antibodies to CD44 have been contradictory, with positivity rates of 72 91% reported for MM and positivity rates of 45 48% reported for ACA. 54,55 A recently developed probe that specifically binds to hyaluronate was found be predictive for MM in tissue samples, but was unreliable for use in effusions. 53,58 CK 5/6 The use of CK 5/6 is based on the relatively select expression of CK 5 in mesothelial cells when compared with ACA cells. 25,54 Various immunohistochemical studies on tissues have shown CK 5/6 to be a promising marker for MM. 25,36,54 In one tissue study, 92% of MM cases were immunoreactive, whereas only 14% of ACA cases were positive. 54 Similarly, a second study found that 100% of MM cases were immunore-

13 Immunocytochemistry in Effusions/Fetsch and Abati 305 FIGURE 11. HBME-1 staining in pleural effusion. (Left) Cell block and (right) ThinPrep of mesothelioma. Note the pattern of thick staining of the membrane that is clearly visible in the cell block preparation but is not discernible in the ThinPrep preparation (diaminobenzidene active, all pulmonary ACA cases were nonimmunoreactive, and only weak immunoreactivity was present in a small number of the other types of ACAs tested. 36 Currently, there are no published studies regarding effusions using this antibody. Thrombomodulin Thrombomodulin is a glycoprotein that normally is present in endothelial and mesothelial cells, megakaryocytes, mesangial cells, some squamous cells, epithelial cells, and synovial cells. 35 The pattern of positive immunostaining is reported to be cell membranous; cytoplasmic staining is most likely nonspecific. 25 Tissue studies using antibodies to thrombomodulin have found variable sensitivity rates for MM (range, 43 90%), but relatively high specificity rates (range, 81 90%). 36,48,54 Unfortunately, an effusion study performed in our institution found that 67% of MM samples and 53% of ACA samples tested were immunoreactive, thereby limiting the use of thrombomodulin in cases with this differential diagnosis. 29 WT-1 The Wilms tumor gene is a tumor suppressor gene involved in the development of MM. Antibodies to WT-1 may be potentially useful in the differentiation of MM from pulmonary ACA. 25,36 Tissue studies have shown positivity rates of 72% for MM, 100% for reactive pleural tissue, and 83% for ovarian ACA, whereas only rare immunoreactivity has been reported for all other types of ACA. 47,55 To date, studies have not been performed to verify the value of this marker in effusion samples. Antimesothelial cell antibody This polyclonal antibody directed against a cytoplasmic protein of mesothelial cells was developed by Adalberto Donna, M.D. (City Hospital, Alessandria, Italy), and found to be immunoreactive in all effusions of MM tested and nonimmunoreactive with all ACAs. 56 Unfortunately, to our knowledge, no further published studies with this marker are available. N-cadherin N-cadherin is found in nerve cells, developing skeletal muscle, cardiac muscle, and mesothelial cells. 36 Because it is expressed in normal, reactive, and neoplastic mesothelial cells, it does not discriminate between benign and malignant mesothelial proliferations. 45 One study that examined both tissues and effusions found 92% of MM cases to be positive for N-cadherin and all ACAs were found to be negative. 24 Unfortunately, other studies have reported low sensitivity and specificity rates for MM. 25,33 An effusion study performed in our institution found immunoreactivity rates of 77% in reactive cases, 35% in MM cases, and 48% in ACA cases. 43 Miscellaneous ACA Markers HMFG-2 Human milk fat globule is a cell surface glycoprotein that has been used to distinguish between ACA and MM. In general, a strong cytoplasmic stain is charac-

14 306 CANCER (CANCER CYTOPATHOLOGY) October 25, 2001 / Volume 93 / Number 5 teristic of ACA, whereas the absence of a cytoplasmic stain favors a diagnosis of MM. 35 Conflicting results are believed to be due to the antibody source, as well as disagreement in interpretation among pathologists. 5,24,35 One tissue study found a positivity rate of 91% for ACA and a positivity rate of 47% for MM. 5 MOC-31 MOC-31 is an antihuman epithelial-related antigen raised against a cell surface glycoprotein using the GLS-1 small cell lung carcinoma cell line, and is present on the majority of normal and malignant epithelia. 36 MOC-31 has been shown to react with the majority of ACAs but only rarely with MM and reactive 25,36,46 48 pleural tissues. Thyroid transcription factor-1 Thyroid transcription factor-1 (TTF-1) is a tissue-specific transcription factor expressed in the thyroid and lung. It has been shown to be useful in the differential diagnosis of MM and lung ACA. 55 A recent study reported that 75% of lung ACAs and 100% of thyroid ACAs were immunoreactive with TTF-1, whereas all other ACA tissues (both primary tumors and lung metastases) and MMs tested were negative. 55 Conclusions In our laboratory, we find the use of antibodies to B72.3, BerEP4, CA 19-9, and calretinin to be the optimal panel with which to differentiate ACA from MM in effusions. Cell blocks provide the most suitable form of sample preparation when performing immunostains on effusions due to ease of morphologic interpretations, standardized like-like comparison with surgical pathology material, minimal background staining, and expected immunostaining patterns. In addition, numerous sections obtained from a single cell block sample permit the evaluation of a large number of antigens, typically at the lowest cost/test. Archival material also is then made available if needed for future studies. The number of antibodies that can be used in ICC has increased dramatically over the past few years, and the future of diagnostic cytopathology will continue to expand as more and more cellular markers are validated and used. The advent of heat-induced epitope retrieval techniques has improved staining markedly for a wide range of these antibodies. Automated instrumentation and prepackaged test kits have contributed to improving the reliability and reproducibility of the technical approach. Through participation in external proficiency testing and laboratory inspections, which require appropriate documentation, updated procedures, equipment maintenance, and qualified testing personnel, we will continue to move toward our goal of a standardized immunocytochemistry clinical laboratory section. REFERENCES 1. Koss LG. Effusions. In: Diagnostic cytology and its histopathologic bases. Philadelphia: J.B. Lippincott Co., 1961: Bibbo M. Pleural, peritoneal, and pericardial fluids. 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