Comparison of Keratin Monoclonal Antibodies MAK-6, AE1AE3, and CAM-5.2

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1 Comparison of Keratin Monoclonal Antibodies MAK6, AEAE, and CAM5. MARGARET B. LISTROM, M.D. AND LESLIE W. DALTON, M.D. Two routinely used antikeratin monoclonal antibodies, AELAE (Hybritech Inc., La Jolla, CA) and CAM5. (BectonDickinson, Mountain View, CA), were compared with a new antikeratin monoclonal antibody mixture, MAK6 (Triton Biosciences, Inc., Alameda, CA). Various poorly differentiated epithelial neoplasms, lymphomas, melanomas, and sarcomas were studied with the use of the avidinbiotinperoxidase technic. All three antibodies had similar staining profiles, however, some differences were noted. The MAK6 and CAM5. antibodies illustrated stronger staining for some grade III transitional cell carcinomas, whereas the AE:AE was variably positive to negative in all cases. Three renal cell carcinomas were positive with all three antibodies, two were negative with all three, and one had scattered positive cells with MAK6 only. All lymphomas and plasmacytomas were negative with MAK6 and CAM5., however, AE:AE faintly stained two of three plasmacytomas and two of the seven large cell lymphomas. Two out of three hepatomas evaluated were strongly positive with CAM5. and MAK6 and variably positive with AE:AE. The third case had both positive and negative cells with all three antibodies. In conclusion, MAK6 antikeratin antibody is as useful as AE:AE and CAM5. for identification of poorly differentiated epithelial neoplasms. (Key words: MAK6 ; AE:AE ; CAM5. ; Keratin; Epithelial neoplasms) Am J Clin Pathol 987; 88: 970 KERATIN MONOCLONAL ANTIBODIES have been useful in identification of the epithelial nature of neoplasms. 9I60 These antibodies are perhaps most useful in the differentiation of poorly differentiated epithelial malignancies from those other nonepithelial neoplasms, especially lymphomas and melanomas. Keratins are intermediate filaments with welldescribed properties. 6 " 8 ' 0 Group A (acidic) and Group B (basic) keratins are separated into 9 members by molecular weight, charge specificities, and immunoreactivity with monoclonal antibodies with restricted specificities. 6 ' 8 Most diagnostic surgical pathology laboratories use one or more antikeratin monoclonal antibodies to determine the epithelial nature of poorly differentiated neoplasms. Both Received September, 986; received revised manuscript and accepted for publication January 0, 987. Address reprint requests to Dr. Listrom: Laboratory Service (), Veterans Administration Medical Center, 00 Ridgecrest SE, Albuquerque, New Mexico 87. Department of Pathology, Veterans Administration Medical Center and University of New Mexico Hospital, Albuquerque, New Mexico AE :AE and CAM5. are routinely used in our own laboratory. These two antibodies can identify all of the Group B keratins (AE) and most of the Group A keratins (AE, CAM5.). We recently studied a new antikeratin monoclonal antibody called MAK6 that is mixture of two monoclonal antikeratin antibodies. The purpose of this study was to compare and evaluate the sensitivity and specificity of MAK6 with AELAE and CAM5.. Case Selection Materials and Methods Sixtyseven cases of various human malignancies were selected from the files of the Department of Surgical Pathology at the Veterans Administration Medical Center and the University of New Mexico Hospital. All slides were reviewed and, where appropriate, clinical history obtained. Emphasis was placed on studying cases where the immunohistochemical approach would be most useful, when the differential diagnosis involved a poorly differentiated neoplasm composed of cells not readily classified by light microscopic examination. Other cases were not diagnostic problems but were selected to compare the staining patterns of the three antibodies. In cases that presented with metastatic disease, the metastases were used for this study. One or occasionally two representative blocks were cut from each case. When possible, a cm square of tumor was studied. In cases with small biopsy samples, the entire amount of tissue available was studied. Areas of necrosis and hemorrhage were avoided, because these areas tend to trap antibody, which increased the background staining and made interpretation difficult. A wide spectrum of epithelial and nonepithelial malignancies was selected to define the range and reactivity of all three antibodies. An attempt was made to include 97 Downloaded from on 0 September 08

2 98 LISTROM AND DALTON A.J.C.P. September 987 Tumor Type Breast Lobular Ductal* Ovary* Prostate* Renal cell Transitional* Mesothelioma Small cell carcinomaf Lung* Chondroid chordoma Glioblastoma Synovial sarcoma Melanoma Lymphoma Plasmacytoma Leiomyosarcoma Ewing's sarcoma Table. Staining Pattern No. Cases 7 MAK6 AE:AE CAM5. _ Poorly differentiated tumors. t Primary tumors: lung, stomach, and pancreas. Staining pattern. = trace or nonreactivity: = weak variable staining; = mixture of positive and negative cells; = consistent positive staining. at least four separate cases from each diagnostic category. If fewer than four cases were available for study, more than one block was evaluated. These included epithelial neoplasms, mesotheliomas, 8 lymphomas, plasmacytomas, melanomas, monophasic synovial sarcoma, Ewing's sarcomas, chondroid chordomas, glioblastomas, and leiomyosarcomas. All tissues were paraffin embedded and fixed in 0% neutral buffered formalin. AELAE Antibodies The mouse monoclonal antibody mixture AE:AE was purchased from Hybritech, (La Jolla, CA). It is a :0 mixture of the antibodies AE and AE. This antibody pool is well characterized and recognizes almost all known human epithelial keratins. 8 AE reacts with Group A acidic keratins of four different molecular weights, 0 kd, 50 kd, 5 kd, and 56.5 kd. AE reacts with all eight Group B basic keratins with molecular weights of 5 kd, 5 kd, 56 kd, 58 kd, 59 kd, 6 kd, 65 kd, and 67 kd. 5 A dilution of :0 was used for optimal staining. CAM5. Antibodies The mouse monoclonal antibody CAM5. was purchased from BectonDickinson (Mountain View, CA). This antibody is raised against colon carcinoma cell line HT9. It has been characterized by immunoblotting and has been shown to react with one Group B keratin, molecular weight 50 kd, and two Group A keratins, kd and 9 kd." It was used at a dilution of :5. MAK6 Antibodies The mouse monoclonal antibody MAK6 was supplied to us by Triton Biosciences, Inc. (Alameda, CA). It is a mixture of two monoclonal antibodies: KA, which by gel electrophoresis analysis has been shown to react with Group A keratins with molecular weights of 50 kd and 0 kd and one Group B keratin (56 kd); and UCD/PR0,, which reacts with one Group A keratin (5 kd) and one Group B keratin (5 kd). 5 It was used at a dilution of :5. Immunohistochemical Staining Pilot testing with several tumor blocks was done to find the optimal dilution for each antibody. Optimal dilution for use of an antibody was defined as that dilution at which the greatest contrast was achieved between the desired (specific) staining and unwanted (nonspecific) background. Staining was carried out on 5^m sections of paraffinembedded tissue with the use of the avidinbiotinperoxidase method. 7 Slides were placed in a 7 C oven overnight before staining. After the sections were deparaffinized and rehydrated, endogenous peroxidase activity was blocked using % H 0 in methanol for 0 minutes. Sections were then incubated with porcine trypsin (Sigma Chemical Co., 5 mg/dl in 0. g/dl or 0.0 mol/l CaCl in 0.05 mol/l TRIS buffer, ph 7.8) for one hour at 7 C. Previous reports indicate that prior trypsinization is necessary for optimal detection of keratin proteins in paraffinembedded tissue. 5 Slides were then incubated with diluted horse serum (:00) (Vectastain mouse kit) for 0 minutes at room temperature. Afterward the primary antibodies were applied to the sections and incubated overnight at C. The Vectastain mouse kit provided the linking antibody (biotinylated horse antimouse) and the avidinbiotinperoxidase complex. Both these incubations required one hour at 7 C. Antibody localization was deter Downloaded from on 0 September 08

3 FIG.. Transitional cell carcinoma stained with MAK6 (A, upper, left), AE :AE (B, upper, center), and CAM5. (C. i/pper, right). The MAK6 and CAM5. antibodies have similar staining patterns, whereas the AEl is negative (X0). DF. Different transitional cell carcinoma stained with MAK6 (D, center, left), AE:AE (E, center, center), and CAM5. (F, center, right). The MAK6 and CAM5. antibodies illustrate the paranuclear staining pattern (arrows), and in this case the AE:AE was negative (X00). GI. Hepatoma stained with MAK6 (G. lower, left), AE:AE (H, lower, center), and CAM5. (/, lower, right). Both positive and negative cells are seen with all three antibodies (X0). (All cases counterstained with methylene blue.) Downloaded from on 0 September 08

4 00 LISTROM AND DALTON A.J.C.P. September 987 mined with the use of,diaminobenzidine (5 mg/00 ml of buffer with 0. ml of 0% H 0 ). Slides were counterstained with methylene blue. Positive controls included sections of skin and glandular epithelium, and negative controls were performed by substituting the primary antibody with normal mouse serum. Results Table lists immunoreactivity for AELAE, MAK6, and CAM5. in the various tumors. The number of reactive cells was estimated and the staining scored as follows: trace or nonreactivity (); weak variable staining (); mixture of positive and nonreactive cells (); consistent positive staining (). A wide range of reactivity among the cases of small cell carcinomas was noted with all three antibodies. This variation may be related to the variable amounts of tumor necrosis, technical errors, or, more important, adverse handling of tissues that cannot be easily excluded. In addition, many tumors exhibit heterogeneity in their production of keratin intermediate filaments. The AELAE and CAM5. faintly stained two of four melanomas, and the AE: AE alone exhibited faint cytoplasmic staining in two of seven large cell lymphomas as well as both plasmacytomas. This was interpreted as nonspecific background. Transitional cell carcinomas were more likely to stain with MAK6 and CAM5. than with AELAE, which was either negative or only variably positive, however, one case was negative with all three antibodies. When positive, the malignant transitional cells had two types of staining patterns. One was a diffuse cytoplasmic positivity, which was the typical pattern seen in most epithelial tumors. The other staining pattern appeared as a discrete, round paranuclear cytoplasmic structure resembling a cytoplasmic inclusion body, similar to that seen in the small cell carcinomas. This discrete cytoplasmic staining pattern has been previously described in neuroendocrine tumors of the skin as well as in small cell carcinomas of the lung ' 9 (Fig. ). There was no instance of false positive staining with any of the antibodies. All three antibodies yielded false negative staining of small cell carcinomas more often than in other epithelial tumors. In general, MAK6 illustrated similar staining patterns as the CAM5. and AELAE. Discussion Keratin proteins are intermediate filaments found in epithelial cells and are effective tumor markers that can be detected in paraffin sections. 09 In this study, most neoplasms with epithelial differentiation stained strongly with all three antibodies. False negative staining of small cell carcinomas occurred with equal frequency with all three antibodies and may be related to tumor necrosis, crush artifact, or technical problems. We suggest the use of a battery of complementary antibodies, such as antibodies to leukocyte common antigen, epithelial membrane antigen, carcinoembryonic antigen, and vimentin, to help determine the cell lineage in cases of small cell poorly differentiated neoplasms. The MAK6 and CAM5. antikeratin antibodies were somewhat more sensitive than AE:AE in staining highgrade transitional cell carcinomas. These differences are probably not clinically significant, because most patients with transitional cell carcinoma of the bladder are not diagnostic problems. The MAK6 antibody contains the monoclonal antibody UCD/PR 0., which has been reported to have more intense staining when compared with CAM5. and AE:AE,' and other authors have described negative staining of genitourinary neoplasms with AE antikeratin. ' 6 Combinations of several monoclonal antibodies, as cocktails, can result in broader specificity and greater sensitivity. Small laboratories, however, may not be able to afford the cost of many different monoclonals and therefore must choose one or two that have wide specificjty and high sensitivity. MAK6 fulfills the above criteria as a useful antikeratin antibody. References. Battifora H: Diagnostic uses of antibodies to keratins, Progress in surgical pathology (preprint). Edited by CM FenoglioPreiser, M Wolf, F Rilke. Philadelphia, Field and Wood, Pennsylvania, 987 (In press).. Battifora H, Silva EG: The use of antikeratin antibodies in the irnmunohistochemical distinction between neuroendocrine (Merkel Cell) carcinoma of the skin, lymphoma, and pat cell carcinoma. Cancer 986; 58: Battifora H, Sun TT, Baker RM, Rao S: The use of antikeratin antiserum as a diagnostic tool. Thymoma versus lymphoma. Hum Pathol 980; : Cooper D, Schermer A, Sun TT: Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: Strategies, applications and limitations. Lab Invest 985; 5:5. 5. Edwards BF, Hu R, Chan R, Rosito PL), Min BH, Cardiff R: Distribution of type 8 and 8 cytokeratins in normal and neoplastic human tumors. Am J Pathol (In press). 6. Eichner R, Bonitz P, Sun TT: Classification of epidermal keratins according to their immunoreactivity, isoelectric point, and mode of expression. J Cell Biol 98; 98: Hsu SM, Raine L, Fanger HA: The use of antiavidin antibody and avidinbiotinperoxidase complex in immunoperoxidase technique. Am J Clin Pathol 98; 75: Kahn JH, Huang SN, Hahna WM, Baumal R, Philips MJ: Immu: nohistochemical localization of epidermal and mallory body cytokeratin in undifferentiated epithelial tumors. Am J Clin Pathol 98;8: Knight J, Gusterson B, Jones RR, Landells W, Wilson P: Monoclonal antibodies specific for subsets of epidermal keratins: Bio Downloaded from on 0 September 08

5 Vol. 88 No. KERATIN ANTIBODY COMPARISON 0 chemical and immunocytochemical characterizationapplications in pathology and cell culture. J Pathol 985; 5: Madri J A, Barwick JW: An immunohistochemical study of nasopharyngeal neoplasms using keratin antibodies. Epithelial versus nonepithelial neoplasms. Am J Surg Pathol 98; 6:9.. Makin CA, Bobrow LG, Bodmer WF: Monoclonal antibody to cytokeratin for use in routine histopathology. J Clin Pathol 98;7: Moll R, Franke FF, Schiler DL: The catalog of human cytokeratins: Patterns of expression in normal epithelia tumors and cultured cells. Cell 98; :.. Nagle RB, Bocker W, Davis JR, et al: Characterization of breast carcinomas by two monoclonal antibodies distinguishing myoepithelial from luminal epithelial cells. J Histochem Cytochem 986;: Pinkus GS, Etheridge CL, O'Connor EM: Are keratins proteins a better tumor marker than epithelial membrane antigen? A comparative immunohistochemical study of various paraffinembedded neoplasms using monoclonal and polyclonal antibodies. Am J Clin Pathol 986; 85: Pinkus GS, O'Connor EM, Etheridge CL, Corson JM: Optimal immunoreactivity of keratin proteins in formalinfixed, paraffin embedded tissue requires preliminary trypsinization. An immunoperoxidase study of various tumors using polyclonal and monoclonal antibodies. J Histochem Cytochem 985; : Spagnolo DV, Michie SA, Crabtree GS, Warnke RA, Rouse RV: Monoclonal antikeratin (AE ) reactivity in routinely processed tissue from 66 human neoplasms. Am J Clin Pathol 985; 8: Sun TT, Eichner R, Nelson WG, et al: Keratin classes: Molecular markers for different types of epithelial differentiation. J Invest Dermatol 98; 8:09sl 5s. 8. Tseng SLG, Jarvinen MJ, Nelson WG, Huang JW, Woodcock Mitchel J, Sun TT. Correlation of specific keratins with different types of epithelial differentiation: Monoclonal antibody studies. Cell 98; 0: Van Muijen GNP, Ruiter DJ, Van Leewen C, Prins FA, Reitsema D, Warnaar SO: Cytokeratin and neurofilament in lug carcinomas. Am J Pathol 98; 6: Walts AE, Said JW, Shintaku IP, Sassoon AF, BanksSchlegel S: Keratins of different molecular weight in exfoliated mesothelial and adenocarcinoma cellan aid to cell identification. Am J Clin Pathol 98; :6.. WoodcockMitchell JR, Eichner WG, Nelson TR, Sun TT: Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies. J Cell Biol 98; 95: Downloaded from on 0 September 08