Use of a Panel of Markers in the Differential Diagnosis of Adenocarcinoma and Reactive Mesothelial Cells in Fluid Cytology

Transcription

1 Anatomic Pathology / IMMUNOCYTOCHEMICAL PANEL FOR FLUID SPECIMENS Use of a Panel of Markers in the Differential Diagnosis of Adenocarcinoma and Reactive Mesothelial Cells in Fluid Cytology Ellen C. Ko, MD, Nirag C. Jhala, MD, Jana J. Shultz, MT(ASCP)SH, QIHC, and David C. Chhieng, MD Key Words: Immunocytochemistry; Serous effusion; Cytology; Adenocarcinoma; Mesothelial cells; Calretinin; Carcinoembryonic antigen; B72.3; Mucicarmine Abstract To evaluate the use of a panel of markers to differentiate adenocarcinoma and the reactive/ inflammatory process in fluid cytology, we stained 29 formalin-fixed, paraffin-embedded cell blocks of effusion fluid from patients with metastatic adenocarcinoma and 24 cell blocks from patients with benign effusion with mucicarmine and antibodies to carcinoembryonic antigen (CEA), B72.3, and calretinin. Positive staining with CEA, B72.3, and mucicarmine was seen in 22 (76%), 20 (69%), and 18 (62%) adenocarcinoma cases, respectively. All except 1 adenocarcinoma was negative for calretinin. No benign cases were positive for B72.3 and mucicarmine. In 1 benign case, scattered epithelial cells demonstrated weak positivity for CEA. The majority of combinations were 100% specific for adenocarcinoma. The highest sensitivity (86%) for adenocarcinomas was achieved with the staining combination of negative for calretinin and positive for any adenocarcinoma marker (CEA, B72.3, or mucicarmine). The use of a panel of markers that recognize adenocarcinoma and mesothelial cells is useful in the differential diagnosis between metastatic adenocarcinoma and the reactive/inflammatory process. The profile of positive staining with at least one of the adenocarcinoma markers and negative calretinin staining is highly specific and sensitive for identifying adenocarcinoma in fluid cytology. The cytologic differential diagnosis of mesothelial cells and adenocarcinoma in fluid specimens can be challenging. Difficulties are often due to overlap in the morphologic features of mesothelial cells and carcinomatous cells. Various ancillary studies have been used to increase the diagnostic accuracy of cytology. 1-8 Immunocytochemical analysis is the most commonly used special technique and often involves the use of a panel of antibodies. There is a whole body of literature examining the role of immunocytochemical analysis and the value of individual and panels of markers in fluid cytology However, there are great differences of opinion about which markers should be included in the panel for routine clinical settings. Markers, including carcinoembryonic antigen (CEA), epithelial membrane antigen, B72.3, Ber-Ep4, and Leu-M1, that are used commonly in the panel recognize molecules expressed by adenocarcinomas but not by mesothelial cells Markers that recognize molecules expressed by mesothelial cells are disappointedly scarce and rarely used for diagnostic purposes because of their limited specificity or sensitivity Calretinin, a calcium-binding protein, is expressed normally in neurons of the central and peripheral nervous system. 21 Various investigators have shown that calretinin is a reliable and specific marker for cells of mesothelial origin in both histologic and cytologic preparations The usefulness of a panel with markers of adenocarcinomas and mesothelial cells in fluid cytology has not been studied. The purpose of the present study was to determine the value of a panel that consisted of 3 markers that are used commonly to identify adenocarcinoma (CEA, B72.3, and mucicarmine) and a mesothelial marker (calretinin) for Am J Clin Pathol 2001;116:

2 Ko et al / IMMUNOCYTOCHEMICAL PANEL FOR FLUID SPECIMENS Table 1 Antibody Panel Antibody Source Clone Dilution Pretreatment Carcinoembryonic antigen Ventana, Tucson AZ TF3H8-1 Prediluted Heat-induced B72.3 Ventana B72.3 Prediluted Heat-induced Calretinin Zymed, San Francisco, CA Polyclonal Prediluted Heat-induced distinguishing between reactive mesothelial cells and metastatic adenocarcinoma in effusion fluids. The immunostaining results of individual markers and various combinations were analyzed to identify the immunoprofile that would be most specific and sensitive in the differential diagnosis of these two entities in fluids. Materials and Methods Formalin-fixed, paraffin-embedded cell blocks from 53 cytologic specimens of pleural, pericardial, and peritoneal fluids were retrieved from the files of the Department of Pathology, University of Alabama at Birmingham. These cytologic specimens consisted of 29 metastatic adenocarcinomas from 28 patients and 24 benign specimens from 24 patients without a history of malignancy. For 1 patient with breast carcinoma, 2 specimens were available; the specimens were obtained at different times. The primary sites of origin of the metastatic adenocarcinomas were as follows: breast, 3; gastrointestinal tract, 3; lung, 11; kidney, 1; and ovary, 10. In 34 fluid specimens (20 adenocarcinomas and 14 benign cases), a corresponding pleural, pericardial, or peritoneum (omentum) biopsy specimen was available. Previous surgical pathologic materials were available for review and comparison in the remaining 9 malignant fluid specimens. The remaining 10 benign fluid specimens were from patients with no known malignant neoplasms. H&E-stained sections of each cell block were evaluated for the presence of tumor. Five-micrometer sections were cut and deparaffinized. Immunostaining was performed using a modified avidinbiotin peroxidase technique. Heat-induced epitope retrieval was accomplished by heating in a steamer for 10 minutes in a 0.01-mol/L concentration of citrate buffer, ph 6. The antibodies used are summarized in Table 1. Immunostaining was accomplished with a semiautomated staining machine (Ventana Medical Systems, Tucson, AZ). Appropriate positive and negative controls were included. Cytochemical staining for mucicarmine was performed using the Mayer method. Briefly, 5-µm sections were deparaffinized, rehydrated, and incubated with Weigert hematoxylin and working mucicarmine solution for 7 and 30 minutes, respectively. The stained sections were evaluated independently by 2 observers (E.C.K., D.C.C.) who were unaware of the original cytologic diagnosis and follow-up. The results of staining with CEA, B72.3, and mucicarmine were based on cytoplasmic staining, whereas calretinin results were based on nuclear staining of the atypical cells. The distribution of staining was scored as negative or positive. Positive staining was defined as staining in more than 1% of the atypical cells regardless of the intensity of the staining. Results Table 2 summarizes the results of the staining for each marker. None of the benign cases were positive for B72.3 or for mucicarmine. In 1 benign case, scattered (<10%) epithelial cells demonstrated weak positivity for CEA. The reactive mesothelial cells showed strong immunoreactivity for calretinin in all benign cases Image 1. Positive immunostaining with CEA, B72.3, and mucicarmine was seen in 22 (76%), 20 (69%), and 18 (62%) malignant cases, respectively Image 2. All except 1 malignant case demonstrated negative staining for calretinin. The adenocarcinoma that showed positive staining with calretinin was a pelvic washing specimen obtained from a patient with FIGO (International Federation of Gynecology and Obstetrics) grade III endometrioid ovarian carcinoma. The patient also had histologic evidence of omental involvement. The cytologic specimen consisted of numerous papillary groups of atypical cells that were positive for calretinin and negative for B72.3, CEA, and mucicarmine. Compared with the primary ovarian tumor, the atypical cells present in the cell block preparation had a lower nuclear grade and appeared less pleomorphic. We concluded that the atypical cells in the cytologic specimen most likely were of mesothelial origin and that this false-negative case was due to a sampling error. Four cases of metastatic adenocarcinomas, including 1 renal cell carcinoma, 1 breast carcinoma, and 2 adenocarcinomas of lung, were negative for CEA, B72.3, and mucicarmine. The sensitivity and specificity for individual and combinations of markers for the differential diagnosis 710 Am J Clin Pathol 2001;116:

3 Anatomic Pathology / ORIGINAL ARTICLE Table 2 Results of Staining for Individual Markers and Combinations No. (%) Positive No. (%) Positive Adenocarcinoma Cases Benign Cases Sensitivity for Specificity for Marker (n = 29) (n = 24) ACA (%) ACA (%) Likelihood Ratio One marker Mucicarmine+ 18 (62) 0 (0) B (69) 0 (0) CEA+ 22 (76) 1 (4) Calretinin+ 1 (3) * 24 (100) Two markers Mucicarmine+/calretinin 18 (62) 0 (0) B72.3+/calretinin 20 (69) 0 (0) CEA+/calretinin 22 (76) 0 (0) CEA+/B (59) 0 (0) Other combinations Calretinin /CEA+ or B (79) 0 (0) Positive staining in 1 or more 24 (83) 1 (4) (CEA, B72.3, mucicarmine) Positive staining in 2 or more 18 (62) 0 (0) (CEA, B72.3, mucicarmine) Calretinin and positive staining in 25 (86) 0 (0) or more (CEA, B72.3, mucicarmine) Calretinin and positive staining in 18 (62) 0 (0) or more (CEA, B72.3, mucicarmine) ACA, adenocarcinoma; CEA, carcinoembryonic antigen; +, positive;, negative. * Including the false-negative case. For reactive mesothelial cells. between metastatic adenocarcinoma and a benign process in fluid cytology are listed in Table 2. B72.3 and mucicarmine were both specific markers (100%) for adenocarcinoma, but their sensitivity was 69% and 62%, respectively. CEA showed a specificity of 96% for adenocarcinoma and a sensitivity of 76%. Calretinin was a specific and sensitive marker for mesothelial cells with a sensitivity and specificity of 96% and 97%, respectively. A The best 2-marker combination was CEA and calretinin. The staining combination of positive for CEA and negative for calretinin was 100% specific and 76% sensitive for metastatic adenocarcinoma. The positive and negative predictive values of this combination were 100% and 77%, respectively. Many combinations of 3 or 4 markers were 100% specific for adenocarcinoma. The highest sensitivity of 86% for adenocarcinoma was achieved with the B Image 1 A, A small group of reactive mesothelial cells showing nuclear atypia and cytoplasmic vacuolization (H&E, 1,000). B, Reactive mesothelial cells showing strong positive nuclear staining and membranous accentuation, resulting in a fried egg appearance (calretinin, 1,000). Am J Clin Pathol 2001;116:

4 Ko et al / IMMUNOCYTOCHEMICAL PANEL FOR FLUID SPECIMENS A B C Image 2 A, Metastatic lung adenocarcinoma showing intracytoplasmic mucin production (mucicarmine, 1,000). B, Same tumor exhibiting positive cytoplasmic staining with anti carcinoembryonic antigen (carcinoembryonic antigen, 1,000). C, Same tumor demonstrating positive cytoplasmic staining with anti-b72.3 monoclonal antibody (B72.3, 1,000). D, Metastatic ovarian adenocarcinoma showing negative staining with anticalretinin polyclonal antibody (calretinin, 400). D immunoprofile of negative for calretinin and positive for any adenocarcinoma marker (CEA, B72.3, or mucicarmine). The positive and negative predictive values of this combination were 100% and 86%, respectively, and the likelihood ratio was 50. Discussion Based on morphologic features alone, the cytologic differentiation of reactive mesothelial cells from adenocarcinoma can be difficult. Various cytologic features are characteristic of, but not specific for, mesothelial cells. For example, intercellular spaces (windows), commonly seen in cellular aggregates of mesothelial cells, also can be identified in 13% of cases of metastatic adenocarcinoma. 28 Therefore, ancillary studies often are performed to assist in the differential diagnosis. Electron microscopy has been used for differentiating mesothelial cells from adenocarcinoma by demonstrating long, thin, bushy microvilli in the mesothelial cells. 29,30 Availability of adequate material, sizable costs, and long turnaround times limit the universal implementation of this technique. Furthermore, not all mesothelial cells display the aforementioned characteristic ultrastructural features. 31 The use of cytochemical stains such as mucicarmine also has been studied. In our experience as well as in that of others, 712 Am J Clin Pathol 2001;116:

5 Anatomic Pathology / ORIGINAL ARTICLE demonstration of intracytoplasmic neutral mucin production strongly favors adenocarcinoma over mesothelial cells. 32,33 However, the usefulness of cytochemical stains including mucicarmine is limited because they are generally insensitive. 34,35 Fewer than two thirds of the adenocarcinomas in our study demonstrated neutral mucin production. In recent years, immunocytochemical analysis has contributed greatly to the differentiation between adenocarcinoma and mesothelial cells Most of the available markers, such as CEA, CD15 (Leu M1), BerEP4, and B72.3, recognize molecules commonly expressed by adenocarcinomas but not by mesothelial cells. A diagnosis of metastatic adenocarcinoma is favored if the neoplastic cells demonstrate positive immunoreactivity for CEA, CD15, B72.3, or BerEP4, which are expected to be absent in mesothelial cells. However, these markers are not distributed uniformly among different types of adenocarcinomas. 36 Positive staining with at least 2 of the adenocarcinoma markers would favor a diagnosis of adenocarcinoma. Therefore, a panel of 2 or more antibodies often is used to confirm (or rule out) the possible diagnosis of metastatic adenocarcinoma but cannot support a positive identification of mesothelial cells. 37,38 CEA is the most extensively studied and widely accepted marker for differentiating adenocarcinoma and mesothelial cells. It is an oncofetal glycoprotein, originally extracted from colonic carcinomas. Most studies reported a high detection rate for adenocarcinoma with CEA. 9,13,39,40 In our study, CEA was positive in 76% of adenocarcinomas. On the other hand, nearly all cases of reactive mesothelial cells reported in the literature lacked CEA staining. We noticed focal and weak CEA immunostaining in 1 benign case. Several investigators have reported that CEA stained macrophages and other inflammatory cells owing to nonspecific cross-reactivity and that it has been associated with a 5% to 15% false-positive rate. 12,41 The monoclonal antibody B72.3 is generated with a membrane-enriched fraction of human mammary carcinoma and recognizes the glycoprotein TAG-72 that is expressed in a wide variety of adenocarcinomas. 42 In our experience, B72.3 was expressed in 69% of adenocarcinomas, and it lacked affinity for reactive mesothelial cells. These figures are similar to those reported by other investigators. 40,41,43,44 It has been reported that weak and focal immunoreactivity can be seen in rare cases of malignant mesothelioma with both CEA and B ,14,45,46 Specific markers for mesotheliomas are scarce. Furthermore, their specificity and sensitivity are limited Among the suggested specific mesothelial markers, the ME1 monoclonal antibody, which recognizes a molecule present on the surface of mesothelial cells, reacts with up to 40% of carcinomas. 19 The K1 monoclonal antibody, generated by immunizing mice with the OV-CAR3 ovarian cell line, reacts with epithelial and biphasic mesotheliomas as well as 70% of nonmucinous ovarian carcinomas. 15,16 Thrombomodulin, a transmembrane glycoprotein normally expressed by endothelial and mesothelial cells, stained two thirds of the mesotheliomas and more than half of the adenocarcinomas in effusion cytology. 18 HBME-1, an antigen present on the microvillus surface of the mesothelial cell, was expressed by more than half of the adenocarcinomas. 18 N-cadherin, a member of the cadherin family, was reported to be immunoreactive with 77% of reactive mesothelial cells, 35% of malignant mesotheliomas, and 48% of adenocarcinomas. 47 Calretinin, a 29-kd calcium-binding protein, belongs to a large family of EF-hand proteins. 21 In its structure, it possesses a characteristic helix-loop-helix that acts as the calcium-binding site. It is expressed in central and peripheral neural tissues and is thought to have a key role in somatosensory transduction. 48,49 Several investigators have demonstrated that calretinin is a sensitive and specific marker for both benign and malignant mesothelial cells The biologic function of calretinin in mesothelial cells is unknown. In the present study, the reactive mesothelial cells of all benign cases demonstrated strong nuclear staining with calretinin, whereas all metastatic adenocarcinomas lacked positive nuclear staining to calretinin. However, several authors have reported scattered positive staining for calretinin in 5% to 10% of adenocarcinomas, including colonic and ovarian adenocarcinomas. 22,25 Our experience, as well as that of others, has shown that a panel of antibodies is most helpful for differentiating between adenocarcinoma and mesothelial cells in fluid specimens. 9,13,39,40 The advantage of using a panel of markers is to improve sensitivity and specificity. We included calretinin, a marker that recognizes mesothelial cells, in our panel so that we did not need to rely on a conclusion reached after a series of negative stainings to rule out metastatic adenocarcinoma. The majority of the combinations of markers had high specificity for diagnosing metastatic adenocarcinoma in fluids. The 2-marker combination with the highest sensitivity (76%) for recognizing adenocarcinoma was positive staining for CEA and negative for calretinin. The best combination with the highest sensitivity (86%) and specificity (100%) for adenocarcinoma was positive staining with any one of the adenocarcinoma markers (CEA, B72.3, or mucicarmine) and negative staining with calretinin. The opposite reaction, positive for calretinin and negative for all adenocarcinoma markers, was also highly specific (97%) and sensitive (100%) for reactive mesothelial cells and had a positive predictive value of 96% in the present study. The use of a panel, which included markers that recognized adenocarcinoma as well as cells of mesothelial origin, Am J Clin Pathol 2001;116:

6 Ko et al / IMMUNOCYTOCHEMICAL PANEL FOR FLUID SPECIMENS aided the differentiation between metastatic adenocarcinoma and reactive mesothelial cells. The profile of positive staining with any adenocarcinoma markers and negative staining with calretinin was specific and sensitive for recognizing adenocarcinoma in fluid cytology. From the Department of Pathology, University of Alabama at Birmingham. Presented in part at the 90th Annual Meeting of the United States and Canadian Academy of Pathology, Atlanta, GA, March 3-9, Address reprint requests to Dr Chhieng: Dept of Pathology, th St S, KB 526, Birmingham AL References 1. Bauer Z, Milic N, Handl S, et al. The results of some cytochemical reactions in metastatic malignant tumor cells in pleural and peritoneal effusions. Acta Cytol. 1977;21: Domagala W, Woyke S. Transmission and scanning electron microscopic studies of cells in effusions. Acta Cytol. 1975;19: Ghosh AK, Spriggs AI, Taylor-Papadimitriou J, et al. Immunocytochemical staining of cells in pleural and peritoneal effusions with a panel of monoclonal antibodies. J Clin Pathol. 1983;36: Halter SA, Hunt LS, Roche B, et al. Backscatter electron imaging of cells from effusions. Scan Electron Microsc. 1984(pt 4): Bousfield LR, Greenberg ML, Pacey F. Cytogenetic diagnosis of cancer from body fluids. Acta Cytol. 1985;29: Walts AE, Said JW, Shintaku IP, et al. Keratins of different molecular weight in exfoliated mesothelial and adenocarcinoma cells: an aid to cell identification. Am J Clin Pathol. 1984;81: Unger KM, Raber M, Bedrossian CW, et al. Analysis of pleural effusions using automated flow cytometry. Cancer. 1983;52: Koss LG, Domagala W. Configuration of surfaces of human cancer cells in effusions: a review. Scan Electron Microsc. 1980: Esteban JM, Yokota S, Husain S, et al. Immunocytochemical profile of benign and carcinomatous effusions: a practical approach to difficult diagnosis. Am J Clin Pathol. 1990;94: Cibas ES, Corson JM, Pinkus GS. The distinction of adenocarcinoma from malignant mesothelioma in cell blocks of effusions: the role of routine mucin histochemistry and immunohistochemical assessment of carcinoembryonic antigen, keratin proteins, epithelial membrane antigen, and milk fat globule derived antigen. Hum Pathol. 1987;18: Mason MR, Bedrossian CW, Fahey CA. Value of immunocytochemistry in the study of malignant effusions. Diagn Cytopathol. 1987;3: Silverman JF, Nance K, Phillips B, et al. The use of immunoperoxidase panels for the cytologic diagnosis of malignancy in serous effusions. Diagn Cytopathol. 1987;3: Nance KV, Silverman JF. Immunocytochemical panel for the identification of malignant cells in serous effusions. Am J Clin Pathol. 1991;95: Delahaye M, Hoogsteden HC, Van der Kwast TH. Immunocytochemistry of malignant mesothelioma: OV632 as a marker of malignant mesothelioma. J Pathol. 1991;165: Chang K, Pastan I, Willingham MC. Frequent expression of the tumor antigen CAK1 in squamous-cell carcinomas. Int J Cancer. 1992;51: Chang K, Pai LH, Pass H, et al. Monoclonal antibody K1 reacts with epithelial mesothelioma but not with lung adenocarcinoma. Am J Surg Pathol. 1992;16: Donna A, Betta PG, Bellingeri D, et al. Cytologic diagnosis of malignant mesothelioma in serous effusions using an antimesothelial-cell antibody [published correction appears in Diagn Cytopathol. 1992;8:661]. Diagn Cytopathol. 1992;8: Fetsch PA, Abati A, Hijazi YM. Utility of the antibodies CA 19-9, HBME-1, and thrombomodulin in the diagnosis of malignant mesothelioma and adenocarcinoma in cytology. Cancer. 1998;84: Skov BG, Stahel RA, Hirsch F. ME1-antibody labelling of primary bronchogenic tumours and extrapulmonary malignancies. Lung Cancer. 1994;11: Clark SP, Chou ST, Martin TJ, et al. Parathyroid hormone related protein antigen localization distinguishes between mesothelioma and adenocarcinoma of the lung. J Pathol. 1995;176: Rogers JH. Calretinin: a gene for a novel calcium-binding protein expressed principally in neurons [published correction appears in J Cell Biol. 1990;110:1845]. J Cell Biol. 1987;105: Doglioni C, Tos AP, Laurino L, et al. Calretinin: a novel immunocytochemical marker for mesothelioma. Am J Surg Pathol. 1996;20: Gotzos V, Vogt P, Celio MR. The calcium binding protein calretinin is a selective marker for malignant pleural mesotheliomas of the epithelial type [published correction appears in Pathol Res Pract. 1996;192:646]. Pathol Res Pract. 1996;192: Riera JR, Astengo-Osuna C, Longmate JA, et al. The immunohistochemical diagnostic panel for epithelial mesothelioma: a reevaluation after heat-induced epitope retrieval. Am J Surg Pathol. 1997;21: Leers MP, Aarts MM, Theunissen PH. E-cadherin and calretinin: a useful combination of immunochemical markers for differentiation between mesothelioma and metastatic adenocarcinoma. Histopathology. 1998;32: Barberis MC, Faleri M, Veronese S, et al. Calretinin: a selective marker of normal and neoplastic mesothelial cells in serous effusions. Acta Cytol. 1997;41: Chhieng DC, Yee H, Schaefer D, et al. Calretinin staining pattern aids in the differentiation of mesothelioma from adenocarcinoma in serous effusions. Cancer. 2000;90: Yu GH, Sack MJ, Baloch ZW, et al. Occurrence of intercellular spaces (windows) in metastatic adenocarcinoma in serous fluids: a cytomorphologic, histochemical, and ultrastructural study. Diagn Cytopathol. 1999;20: Wang NS. Electron microscopy in the diagnosis of pleural mesotheliomas. Cancer. 1973;31: Warhol MJ, Hickey WF, Corson JM. Malignant mesothelioma: ultrastructural distinction from adenocarcinoma. Am J Surg Pathol. 1982;6: Am J Clin Pathol 2001;116:

7 Anatomic Pathology / ORIGINAL ARTICLE 31. Dardick I, Jabi M, McCaughey WT, et al. Diffuse epithelial mesothelioma: a review of the ultrastructural spectrum. Ultrastruct Pathol. 1987;11: Benjamin CJ, Ritchie AC. Histological staining for the diagnosis of mesothelioma. Am J Med Technol. 1982;48: Kannerstein M, Churg J, Magner D. Histochemistry in the diagnosis of malignant mesothelioma. Ann Clin Lab Sci. 1973;3: Hammar SP, Bockus DE, Remington FL, et al. Mucin-positive epithelial mesotheliomas: a histochemical, immunohistochemical, and ultrastructural comparison with mucinproducing pulmonary adenocarcinomas. Ultrastruct Pathol. 1996;20: Roggli VL, Kolbeck J, Sanfilippo F, et al. Pathology of human mesothelioma: etiologic and diagnostic considerations. Pathol Annu. 1987;22(pt 2): Battifora H. The pleura. In: Sternberg SS, ed. Diagnostic Surgical Pathology. Vol I. 3rd ed. New York, NY: Raven Press; 1998: Motoyama T, Watanabe T, Okazaki E, et al. Immunohistochemical properties of malignant mesothelioma cells in histologic and cytologic specimens. Acta Cytol. 1995;39: Bedrossian CW, Bonsib S, Moran C. Differential diagnosis between mesothelioma and adenocarcinoma: a multimodal approach based on ultrastructure and immunocytochemistry. Semin Diagn Pathol. 1992;9: Frisman DM, McCarthy WF, Schleiff P, et al. Immunocytochemistry in the differential diagnosis of effusions: use of logistic regression to select a panel of antibodies to distinguish adenocarcinomas from mesothelial proliferations. Mod Pathol. 1993;6: Shield PW, Callan JJ, Devine PL. Markers for metastatic adenocarcinoma in serous effusion specimens. Diagn Cytopathol. 1994;11: Tickman RJ, Cohen C, Varma VA, et al. Distinction between carcinoma cells and mesothelial cells in serous effusions: usefulness of immunohistochemistry. Acta Cytol. 1990;34: Johnston WW, Szpak CA, Lottich SC, et al. Use of a monoclonal antibody (B72.3) as a novel immunohistochemical adjunct for the diagnosis of carcinomas in fine needle aspiration biopsy specimens. Hum Pathol. 1986;17: Martin SE, Moshiri S, Thor A, et al. Identification of adenocarcinoma in Cytospin preparations of effusions using monoclonal antibody B72.3. Am J Clin Pathol. 1986;86: Kuhlmann L, Berghauser KH, Schaffer R. Distinction of mesothelioma from carcinoma in pleural effusions: an immunocytochemical study on routinely processed cytoblock preparations. Pathol Res Pract. 1991;187: Guzman J, Bross KJ, Wurtemberger G, et al. Tuberculous pleural effusions: lymphocyte phenotypes in comparison with other lymphocyte-rich effusions. Diagn Cytopathol. 1989;5: Ordonez NG. The immunohistochemical diagnosis of mesothelioma: differentiation of mesothelioma and lung adenocarcinoma. Am J Surg Pathol. 1989;13: Simsir A, Fetsch P, Mehta D, et al. E-cadherin, N-cadherin, and calretinin in pleural effusions: the good, the bad, the worthless. Diagn Cytopathol. 1999;20: Andressen C, Blumcke I, Celio MR. Calcium-binding proteins: selective markers of nerve cells. Cell Tissue Res. 1993;271: Carafoli E. Intracellular calcium homeostasis. Annu Rev Biochem. 1987;56: Am J Clin Pathol 2001;116: