Keratins of Different Molecular Weight in Exfoliated Mesothelial and Adenocarcinoma Cells An Aid to Cell Identification

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1 Keratins of Different Molecular Weight in Exfoliated Mesothelial and Adenocarcinoma Cells An Aid to Cell Identification ANN E. WALTS, M.D., JONATHAN W. SAID, M.B., CH.B., I. PETER SHINTAKU, PH.D., AARON F. SASSOON, B.S. AND SUSAN BANKS-SCHLEGEL, PH.D. Keratin profiles of exfoliated mesothelial and adenocarcinoma cells were determined using antisera to different molecular weight keratins (45, 46, 55, 63 kdaltons) and the immunoperoxidase technic. Most metastatic adenocarcinomas in effusions stained for low (45, 46 kdaltons) and intermediate (55 kdaltons) molecular weight keratins but were negative for 63 kdalton keratin. In contrast, most reactive and malignant mesothelial cells in effusions stained strongly for 63 kdalton keratin and keratins of lower molecular weight. This is the first report of high molecular weight (greater than 60 kdaltons) keratin in exfoliated cells of nonepidermal origin. Differences in staining for 63 kdalton keratin between mesothelial and adenocarcinoma cells may help to distinguish these cells in effusions. (Key words: Keratin; Immunoperoxidase; Mesothelial cells; Adenocarcinoma cells; Mesothelioma; Cytology) Am J Clin Pathol 1984; 81: MESOTHELIAL CELLS may be difficult to distinguish from adenocarcinoma cells in cytologic specimens. 17 Exfoliated adenocarcinoma cells frequently are mucicarmine positive and contain periodic acid-schiff-positive, diastaseresistant, Alcian blue-positive, hyaluronidase-resistant cytoplasmic globules. However, hyaluronic acid may be difficult to demonstrate in small amounts 15 and mucicarmine positivity, though commonly associated with glandular tumors, has been reported in up to 40% of mesotheliomas. 8 Electron microscopy, although contributory, 34,35 is time consuming, suffers from sampling error, and requires special tissue processing. Since the cytology of effusions may determine future diagnostic studies, therapy, or prognosis, additional technics have been sought to improve the accuracy of distinguishing reactive mesothelial cells from adenocarcinoma cells. Studies in paraffin sections suggest that immunoperoxidase staining for whole keratin proteins is an adjunct in the differential diagnosis of pulmonary adenocarcinoma Received July 1, 1983; received revised manuscript and accepted for publication September 8, Address reprint requests to Dr. Walts: Division of Anatomic Pathology, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California Division of Anatomic Pathology, Cedars-Sinai Medical Center and Department of Biology, University of Southern California, Los Angeles, California; and Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (negative to weakly keratin positive) and mesothelioma (strongly positive). 524,25 ' 27 This decreased staining of adenocarcinomas in formalin-fixed paraffin sections presumably represents loss of antigen during tissue fixation and processing. 419 Studies on frozen sections 12,26 as well as our previous report on cytologic material 33 have shown that keratin proteins are present in adenocarcinoma as well as mesothelial cells. Recently human epidermal keratins have been shown to constitute a family of proteins with molecular weights that range from 40 to 67 kdaltons." 128 Characteristic profiles of these keratin proteins have been associated with different tissues. 2,7,9 "' 28 ' 31 ' 37 This immunoperoxidase study utilizing specific antisera to different molecular weight keratin proteins (45-63 kdaltons) was undertaken to characterize the keratin profiles of exfoliated mesothelial and adenocarcinoma cells. The potential of keratin protein profiles to aid in the identification of exfoliated mesothelial and adenocarcinoma cells is discussed. Materials and Methods Fifteen benign and 34 malignant specimens submitted to the cytology laboratory at Cedars-Sinai Medical Center were studied. All of the benign cases were body cavity fluids with large numbers of reactive mesothelial cells. The malignant specimens consisted of 22 adenocarcinomas (16 effusions and 6 fine-needle aspirates) and 12 malignant mesotheliomas (all effusions). All specimens were submitted unfixed. Both Papanicolaou-fixed and Papanicolaou-stained as well as air-dried Wright-Giemsa stained cytospins were prepared in a routine manner, 442

2 Vol. 81 -No. 4 KERATINS OF DIFFERENT MOLECULAR WEIGHT 443 using the Shandon cytocentrifuge. Where material was sufficient, paraffin sections of Bouin's fixed histologic buttons were stained with hematoxylin and eosin. Mesothelioma-containing effusions retrieved from the cytology files included filter and cytospin preparations that were up to five years old. Cytologic diagnoses were based on accepted criteria 17 and all malignant cytologic diagnoses were supported by histologic material. The primary site of each adenocarcinoma was clinically evident and/ or histologically confirmed. Benign cytologic diagnoses were supported by clinical course. Specimens were stored at 4 C for up to two days before preparation for immunoperoxidase staining. Immunoperoxidase Staining Immunoperoxidase staining was performed by a modification of reported methods 18 ' 29,30 as previously described. 32 Papanicolaou-fixed and Papanicolaou-stained preparations were rehydrated and placed at room temperature in methanolic peroxide (five parts methanol and one part 3% hydrogen peroxide) for 10 minutes to consume endogenous peroxidase. The slides were washed in phosphate-buffered saline (PBS) at ph 7.4 and incubated with rabbit antihuman antiserum specific for 45, 46, 55, and 63 kdalton keratin proteins. Details of preparation of the antiserum and specificity have been described previously. 3,36 Optimal dilutions were determined for each antibody, and working dilutions ranged from 1:50 to 1:800, with incubation times varying from 45 minutes to 24 hours. Following incubation with primary antiserum, slides were washed in PBS and incubated sequentially with swine antirabbit IgG (1:20 dilution) and horseradish peroxidase rabbit antihorseradish peroxidase soluble complexes at 1:100 dilution (latter two reagents Dako Corporation, Santa Barbara, CA). Antibody localization was effected by incubating slides for 5-15 minutes with a freshly prepared solution of 6 mg of 3-3' diaminobenzidine tetrahydrochloride (Sigma Chemicals, St. Louis, MO) in 10 ml of PBS, followed by addition of 0.1 ml 3% hydrogen peroxide, giving a brown reaction product. Slides were counterstained with methyl green. Positive controls, sections of human epidermis, were included in all runs. The low molecular weight keratins (45 and 46 kdaltons) were localized to the basal layer of the epidermis as previously described. 3,1 ' Fifty-five kdalton keratin showed predominant localization to the upper layers of the epidermis with focal faint staining of the basal layer. Sixty-three kdalton keratin was localized to the upper keratinized layers of the epidermis with no staining of the basal layers. Negative controls consisted of parallel cytocentrifuge preparations with omission of the primary antiserum and substitution of PBS or preim- Table 1. Staining Patterns of Different Molecular Weight Keratins in Exfoliated Adenocarcinoma Cells Primary Site of Adenocarcinoma kdaltons kdaltons kdaltons kdaltons Breast (N = 7) Ovary (N = 7) Lung (N = 5) Gastrointestinal (N = 2) Fallopian tube (N = 1) Total 22 6(7)* 7(7) 2(2) 21 (22) 6(7) 6(6) 2(2) 20(21) 5(6) 6(7) 1(2) 18(21) 1(7) 1(7) K5)t 0(2) 0(1) 3(22) Number of cases positive given in each column. Total number of cases evaluated in parentheses. t Most tumor cells were negative, with few tumor cells positive. mune rabbit serum. These preparations were run for each case and revealed no staining. Results Only well-preserved cells were evaluated. A positive staining reaction was defined by the presence of an intracytoplasmic brown granular reaction product. Extracellular staining, including that limited to the outer aspect of the cell surface, and staining related to degenerated cells was interpreted as nonspecific. Intensity of staining varied in individual cells within each specimen. Keratin Staining in Adenocarcinoma Cells Staining patterns for keratin proteins in adenocarcinoma cells are summarized in Table 1. Staining was predominantly diffuse, with peripheral cytoplasmic accentuation in some cells of most preparations. While most of the adenocarcinomas (21 of 22) stained for the low molecular weight keratins (45 and 46 kdaltons), only 3 of 22 stained for 63 kdalton keratin (Fig. 1). Eighteen of the adenocarcinomas evaluated stained for 55 kdalton keratin in addition to keratins of lower molecular weight. Three adenocarcinomas (primary in breast, lung, and ovary) stained for 63 kdalton keratin, as well as for the keratins of lower molecular weight. Only one of the adenocarcinomas, a breast primary, did not stain for any of the keratin proteins. Keratin Staining in Reactive and Malignant Mesothelial Cells Staining for different molecular weight keratins in exfoliated mesothelial cells is summarized in Table 2. All reactive preparations contained mesothelial cells that stained for keratins of 45, 46, 55, and 63 kdalton (Fig. 2). Reactive mesothelial cells in the adenocarcinomatous effusions stained similarly. Most of the malignant me-

3 444 WALTS ET AL. A.J.C.P. April c 3a ft! / % * ** **te# frv ib 2b FlG. 1 (upper). Metastatic adenocarcinoma in cytocentrifuge preparations of pleural fluid show (A) strong staining for 45 kdalton (low molecular weight) keratin by the immunoperoxidase technic (black) and (B) negative staining for 63 kdalton (high molecular weight) keratin. Methyl green counterstain (A, X500) (B, X640). sothelial preparations (10 of 12) stained for 63 kdalton keratin (Fig. 3). One of the 63 kdalton keratin-negative mesotheliomatous effusions stained for 45 kdalton keratin; extra smears of the other 63 kdalton negative effusion were not available for low molecular weight keratin staining. All seven of the mesotheliomatous effusions tested for low molecular weight keratin were positive. Staining was predominantly diffuse, with frequent peripheral accentuation and occasional perinuclear concentration of reaction product. Discussion Cytologic specimens (cytospin and filter preparations), recently prepared as well as retrieved from files, are amenable to immunoperoxidase staining. Prior Papanicolaou staining does not interfere with the immunoperoxidase reaction, provides excellent nuclear detail, and enables one to apply established cytologic criteria. Results of this study suggest that the keratin profiles of most mesothelial and adenocarcinoma cells differ and that staining for 63 kdalton keratin is useful in distinguishing both reactive and malignant mesothelial cells from cells of adenocarcinomas that most frequently metastasize to the body cavities. Staining for low molecular weight keratins (45 and 46 kdalton) was strongly positive in both cell types and hence could not be used in a differential manner. Staining for 55 kdalton keratin did distinguish some mesothelial from adenocarcinoma preparations (mesothelial cells were positive, while some adenocarcinomas were negative), but staining for 63 kdalton keratin correlated best with histologically confirmed cytologic diagnoses. All reactive effusions contained mesothelial cells positive for 63 kdalton keratin, while only 3 of 22 adenocarcinomas stained for this antigen. Keratin profiles were similar in most benign and malignant mesothelial cell proliferations, with 10 of 12 malignant mesotheliomatous fluids staining for 63 kdalton keratin. Immunoperoxidase studies have demonstrated that mesothelial proliferations contain whole keratin, 5,23 " 25-33,37 but this is the first report of a high molecular weight keratin (greater than 60 kdaltons) in human exfoliated benign and malignant mesothelial cells. Similar staining Table 2. Staining Patterns of Different Molecular Weight Keratins in Exfoliated Mesothelial Cells FlG. 2 (center). Reactive mesothelial cells in cytocentrifuge preparations of pleural fluid show strong staining for (A) 45 kdalton keratin and (B) 63 kdalton keratin by the immunoperoxidase technic (black). Intensity of staining varies within the cell population. Methyl green counterstain (A, X500) (B, X600). FlG. 3 (lower). Malignant mesothelioma cells in cytocentrifuge preparations of pleural fluid show strong staining for (A) 45 kdalton keratin and (B) 63 kdalton keratin by the immunoperoxidase technic (black). Methyl green counterstain (A, B X600). Reactive mesothelial cells (N = 15) 15(15)* Malignant mesothelial cells (N =12) 7 (7) kdaltons kdaltons kdaltons kdaltons 14(14) 15(15) 15(15) NDf ND 10(12) * Number of cases positive given in each column. Total number of cases evaluated in parentheses, t Insufficient material available to perform test.

4 Vol. 81 No. 4 KERATINS OF DIFFERENT MOLECULAR WEIGHT 445 has been observed in tissue sections of mesotheliomas. 22 Keratins of less than 60 kdaltons have been demonstrated in mesothelial and epidermal cells in tissue culture, but high molecular weight keratins (greater than 60 kdaltons) have not been detected in these systems, possibly because expression of large keratins is suppressed in tissue culture. 7 "' 28,37 Although the mechanisms that regulate keratin production have not been elucidated, it has been suggested that several genes are involved 10 ' 21 and that the process can be influenced by the microenvironment. 7 ' 20 Identical staining for 45 and 46 kdalton keratins observed in all preparations may suggest close linkage of genes regulating production of these low molecular weight proteins. Changes in the keratin profiles of epidermal cells occur during embryonic development of the skin. 1 Low molecular weight keratins are present during the early stages of embryogenesis. High molecular weight keratins appear later following the onset of stratification and with the development of a thickened stratum corneum. These changes parallel with sequential maturation that occurs in the adult epidermis. 3 " Thus, the 63 kdalton keratin represents a differentiation antigen in the skin and is detected only in the suprabasal layers. 3 " The two malignant mesothelial effusions that were negative for 63 kdalton keratin may represent dedifferentiation or lack of differentiation of tumor cells with loss, absence, or suppression of this antigen. Variable staining within morphologically homogeneous cell populations also may be explained by the presence of cell populations heterogeneous with regard to extent of differentiation. 1 ' 3 ' 6 " Kahn 14 has suggested that mesothelial cells can be identified by the distribution of whole keratin staining within the cytoplasm. However, because of overlap in staining patterns, we were unable to distinguish mesothelial from adenocarcinoma cells in this manner. Preliminary studies suggest that cytoplasmic location of intermediate filaments varies with phase of the cell cycle. 16 Further work utilizing synchronized cell populations is necessary to explore whether cell phase, rather than cell type, determines the location of keratin proteins within cells. The demonstration of 63 kdalton keratin in mesothelial cells but not in cells from most of the adenocarcinomas studied suggests that staining for 63 kdalton keratin may be helpful in the differential diagnosis of cytologic material. Acknowledgments. The authors appreciate the excellent word processing skills of Meyer Bekhore and the photographic assistance of Kai Chien. References 1. Banks-Schlegel SP: Keratin alterations during embryonic epidermal differentiation: a presage of adult epidermal maturation. J Cell Biol 1982;93: Banks-Schlegel SP, Harris CC: Tissue-specific expression of keratin proteins in human esophageal and epidermal epithelium and their cultured keratinocytes. Exp Cell Res 1983; 146: Banks-Schlegel SP, Schlegel R, PinkusGS: Keratin protein domains within the human epidermis. Exp Cell Res 1981; 136: Battifora H, Sun TT, Bahu RM, Sambasiva R: The use of antikeratin antiserum as a diagnostic tool: Thymoma versus lymphoma. Hum Pathol 1980; 11: Corson JM, Pinkus GS: Mesothelioma: Profile of keratin proteins and carcinoembryonic antigen an immunoperoxidase study of 20 cases and comparison with pulmonary adenocarcinoma. Am J Pathol 1982; 108: Damjanov I: Antibodies to intermediate filaments and histogenesis (editorial). Lab Invest 1982;47: Doran TI, Vidrich A, Sun TT: Intrinsic and extrinsic regulation of the differentiation of skin, corneal and esophageal epithelial cells. Cell 1980;22: Ernst CS, Atkinson BF: Mucicarmine positivity in malignant mesothelioma. Lab Invest 1980; 42:113 (abstract) 9. Franke WW, Schiller DL, Moll R, et al: Diversity of cytokeratins: Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues. J Mol Biol 1981; 153: Fuchs EV, Coppock SM, Green H, Cleveland DW: Two distinct classes of keratin genes and their evolutionary significance. Cell 1981;27: Fuchs E, Green H: Changes in keratin gene expression during terminal differentiation of the keratinocyte. Cell 1980; 19: Gabbiani G, Kapanci Y, Barazzone P, Franke WW: Immunochemical identification of intermediate-sized filaments in human neoplastic cells a diagnostic aid for the surgical pathologist. Am J Pathol 1981; 104: Hensen DE: Heterogeneity in tumors (editorial). Arch Pathol Lab Med 1982; 106: Kahn HJ, Wedad H, Yeger H, Baumal R: Immunohistochemical localization of prekeratin filaments in benign and malignant cells in effusions comparison with intermediate filament distribution by electron microscopy. Am J Pathol 1982; 109: Kannerstein M, Churg J, Magner D: Histochemistry in the diagnosis of malignant mesothelioma. Ann Clin Lab Sci 1973; 3: Knapp LW, O'Guin WM, Sawyer RH: Drug-induced alterations of cytokeratin organization in cultured epithelial cells. Science 1983; 219: Koss LG: Diagnostic cytology and its histopathologic bases. Philadelphia, JB Lippincott, 1979, pp , Pinkus GS, Said JW: Specific identification of intracellular immunoglobulin in paraffin sections of multiple myeloma and macroglobulinemia using immunoperoxidase technique. Am J Pathol 1977; 87: Ramaekers F, Puts J, Moesker O, Kant A, Jap P, Vooijs P: Demonstration of keratin in human adenocarcinomas. Am J Pathol 1983; 111: Rheinwald JG, Germain E, Beckett MA: Expression of keratins and envelope proteins in normal and malignant human keratinocytes and mesothelial cells, Human carcinogenesis. Edited by CC Haris and HN Autrup. New York, Academic Press, 1983, pp Roop DR, Hawley-Nelson P, Cheng CK, Yuspa SH: Keratin gene expression in mouse epidermis and cultured epidermal cells. Proc Natl Acad Sci 1983; 80: Said JW, Nash G, Banks-Schlegel S, Sassoon AF, Murakami S, Shintaku IP: Keratin in human lung tumors: patterns of localization of different molecular weight keratin proteins. Am J Pathol 1983; 113: Said JW, Nash G, Lee M: Immunoperoxidase localization of keratin proteins, carcinoembryonic antigen, and factor VIII in adenomatoid tumors: Evidence for a mesothelial derivation. Hum Pathol 1982; 13: Said JW, Nash G, Tepper G: Keratin proteins and carcinoembryonic antigen in lung carcinoma an immunoperoxidase study of 54 cases with ultrastructural correlations. Hum Pathol 1983; 14:70-76

5 446 WALTS ET AL. A.J.C.P. -April Schlegel R, Banks-Schlegel S, McLeod JA, Pinkus GS: Immunoperoxidase localization of keratin in human neoplasms. A preliminary survey. Am J Pathol 1980, 101: Sieinski W, Dorsett B, loachim HL: Identification of prekeratin by immunofluorescence staining in the differential diagnosis of tumors. Hum Pathol 1981; 12: Stein RB, Lasecki M, Croker BP Jr, Johnston WW: Immunologic detection of keratin in respiratory cytologic material (abstract). ActaCytol 1982; 26: Sun TT, Green H: Keratin filaments of cultured human epidermal cells. J Biol Chem 1978; 253: Taylor CR, Burns J: The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffinembedded tissues using peroxidase-labeled antibodies. J Clin Pathol 1974;27: Taylor CR, Mason DY: The immunohistochemical detections of intracellular immunoglobulin in formalin-paraffin sections from multiple myeloma and related conditions using the immunoperoxidase technique. Clin Exp Immunol 1974; 18: Tseng SCG, Jarvinen MJ, Nelson WG, Haung J-W, Woodcock- Mitchell J, Sun TT: Correlation of specific keratins with different types of epithelial differentiation-monoclonal antibody studies. Cell 1982; 30: Walts AE, Said JW: Specific tumor markers in diagnostic cytology immunoperoxidase studies of CEA, lysozyme and other tissue antigens in effusions, washes and aspirates. Acta Cytol 1983; 27: Walts AE, Said JW, Banks-Schlegel S: Keratin and carcinoembryonic antigen in exfoliated mesothelial and malignant cells: an immunoperoxidase study. Am J Clin Pathol 1983; 80: Wang NS: Electron microscopy in the diagnosis of pleural mesotheliomas. Cancer 1973; 31: Warhol MJ, Hickey WF, Corson JM: Malignant mesotheliomaultrastructural distinction from adenocarcinoma. Am J Surg Pathol 1982;6: Warhol MJ, Pinkus GS, Banks-Schlegel SP: Localization of keratin proteins in the human epidermis by a postembedding immunoperoxidase technique. J Histochem Cytochem 1983; Wu YJ, Parker LM, Binder NE, et al: The mesothelial keratins: A new family of cytoskeletal proteins identified in cultured mesothelial cells and non-keratinizing epithelia. Cell 1982; 31: