Distribution of Myoepithelial Cells and Basement Membrane Proteins in the Normal Breast and in Benign and Malignant Breast Diseases

[CANCER RESEARCH 42, 4763-4770, November 1982] 0008-5472/82/0042-0000$02.00 Distribution of Myoepithelial Cells and Basement Membrane Proteins in the Normal Breast and in Benign and Malignant Breast Diseases

Barry A. Gusterson,1 Michael J. Warburton, Diana Mitchell, Morag Ellison, A. Munro Neville, and Philip S. Rudland

Ludwig Institute for Cancer Research (London Branch) [B. A. G., M. J. W., M. E., A, M. N., P. S. R.I and Department of Histopathology Â¡D.M.], Royal Marsden Hospital, Sutton, Surrey, SM2 5PX United Kingdom

ABSTRACT tins occur in epithelial cells, myosin myofilaments occur in smooth muscle cells, fibronectin is produced by stromal cells, An immunocytochemical method for fixed and paraffin- and laminin and type IV collagen are produced by endothelial embedded human breast biopsies is reported for the detection cells. This paper describes the demonstration of keratin, of myoepithelial and epithelial cells using antibodies to myosin myosin, laminin, and type IV collagen using immunocytochem and keratin, respectively, and of basement membranes using ical localization techniques with specific antibodies in fixed and antibodies to laminin and type IV collagen. Using these paraffin-embedded specimens from a range of normal, benign, markers, myoepithelial cells can be clearly distinguished in the and malignant breast biopsies. This technique yields a superior normal breast and in the benign breast diseases sclerosing cytology to that obtained previously using immunofluorescent adenosis, epitheliosis, and fibroadenoma. In sclerosing aden- localization of single antibodies on frozen breast sections and osis, myoepithelial cells form a major cellular component. A delineates the myoepithelial cell in benign breast diseases from stromally derived spindle cell is identified which stains with other reactive stromal cells, e.g., myofibroblasts. These results myosin but not with keratin antibodies (myofibroblast). These are discussed in relation to the normal developmental program cells are seen in one-fifth of the fibroadenomas and in one-half of the myoepithelial cell in the breast. (9 of 18) of the infiltrating carcinomas. Although cells staining with myosin antibodies are seen in the infiltrating component MATERIALS AND METHODS of all 18 carcinomas examined, elongated cells staining with both myosin and keratin antibodies (myoepithelial-like) are Tissue Processing. Biopsy specimens were used from 35 unse- seen in only one infiltrating carcinoma where they are inter lected cases of human breast disease including 18 infiltrating ductal posed at the stromal-epithelial junction of the infiltrating tumor carcinomas. At least 2 pieces of tissue from each specimen were cut into slices approximately 3-mm thick and fixed in 60% methanol:30% cells. In contrast to the situation in benign breast diseases, chloroform:10% acetic acid overnight at 4Â°.Tissues were processed mature myoepithelial cells form a very minor component of the through 5 changes of absolute alcohol, one change of absolute alcohol majority of infiltrating ductal carcinomas. Basement membrane and 1.1.1. trichlorethane (1:1 v/v), 2 changes of 1.1.1. trichlorethane, proteins, laminin, and type IV collagen are present in normal and 2 changes of wax. After a further hr in wax under vacuum, the breast, benign breast disease, and grade I infiltrating ductal slices were finally embedded in fresh wax, the wax blocks cooled at carcinomas but are absent in carcinomas of grades II and III. 4Â°,and 3-fim-thick sections were cut on a sledge microtome fitted with disposable blades (Sukura Fine Technical Co., Japan). Serial sections were mounted on albumin-coated slides and dried overnight at 45Â°. INTRODUCTION Sections were dewaxed by immersion in fresh xylene for three 5-min The relative contribution made by myoepithelial cells to both periods and then by immersion in fresh absolute alcohol for a further three 5-min periods. The sections were rehydrated firstly in water and benign and malignant breast disease is still debated (1, 3, 8, then finally in Ca2+-, Mg2+-free PBS.2 17, 23, 27, 31). Numerous histological methods have been Antibodies. Keratins were purified from human callus and analyzed used to identify myoepithelial cells, but the techniques used on sodium dodecylsulfate:polyacrylamide gels as described by Sun have relied mainly upon poorly characterized enzymatic histo- and Green (41). The molecular weights of the keratins were 43,000, chemical or nonspecific staining methods (25, 32, 33, 35). 45,000, 47,000, 47,000 and 69,000. Antisera were raised by dissolv Characteristic properties of myoepithelial cells, at least in ro ing 1 mg of keratins in 0.5 ml of 1 M urea and 0.14 M/8-mercaptoethanol dents, are the presence of actin and myosin-containing myo- in PBS; this solution was emulsified with 1 ml of Freund's complete filaments and keratin-containing intermediate filaments and the adjuvant, and the resultant mixture was injected s.c. into the nuchal production of basement membrane proteins, fibronectin, lami region of rabbits at monthly intervals. The resultant antisera gave a nin, and type IV collagen (45). Immunofluorescence studies single immunoprecipitin band in Ouchterlony diffusion tests with the using antibodies to actin (12-14, 17, 28) and myosin (17, 26) purified keratins. These antisera are henceforth called anti-keratin. Rabbit antisera to pig uterine smooth muscle myosin (30, 43) and the have demonstrated that these proteins occur in certain cells in mouse type IV collagen (24) and laminin (42) have been described frozen sections of both benign (26, 28) and malignant (12-14, previously. The last 2 antisera specifically precipitated collagen IV and 16, 35) human breast diseases, and keratin immunoreactive laminin, respectively, as analyzed on sodium dodecyl sulfate:poly- materials have been found in both the normal and malignant acrylamide gels (45). IgG fractions of all antisera were obtained by human (5, 18, 37, 40) and rodent breasts (6, 44). None of precipitation of the original sera with 25% ammonium sulfate. The these proteins is unique to myoepithelial cells. Thus, the kera precipitates were collected by centrifugation, dissolved in 0.025 M NaCI:0.05 M Tris-HCI (pH 7.5), and then chromatographed on DEAE-

' To whom requests for reprints should be addressed. Received February 10, 1982; accepted July 20, 1982. ! The abbreviation used is: PBS, phosphate-buffered saline.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1982 American Association for Cancer Research. B. A. Gusterson et al. cellulose columns. All fractionated antisera were diluted between 1/ strongly stained but the secretory cells were only very weakly 100 and 1/500 with 0.5% bovine serum albumin (w/v) in PBS before stained. use. Second antibodies were obtained by conjugating affinity-purified Antibodies to laminin and type IV collagen delineated the sheep anti-rabbit -^globulin to alkaline phosphatase with glutaralde- basement membrane (Fig. 4) as a single line around both the hyde (7). lobules and ducts. No individual cells were stained with these Immunocytochemical Staining. The binding of specific rabbit anti- antibodies. No breaches were seen in the basement membrane sera to histological sections was visualized by an indirect procedure using alkaline phosphatase-conjugated anti-rabbit second antibodies. in either the lactating or resting gland. When sections were partially digested with proteases before incubation Benign Breast Disease. Because of the confusing nomen with antisera, to enhance the resultant staining (laminin, type IV colla clature in benign breast disease, it is necessary to define some gen, and keratin) they were incubated first with PBS and then with of the conditions described in the text. These definitions are trypsin (0.05 mg/ml; type III; Sigma Chemical Co.) in PBS, both at 37Â° taken from Azzopardi (8). He also provides an excellent ac for 15 min, and rinsed in running water. Staining with antisera was count of the histological appearances of the subtypes of ad- carried out on sections without prior protease treatment unless speci enosis. fied to the contrary in the text. All sections were then treated with 15% Adenosis. This is a nonneoplastic glandular hyperplasia. acetic acid (v/v) for 5 min at room temperature to inhibit endogenous Distinct varieties can be recognized, namely blunt duct aden- alkaline phosphatase, subsequently washed in running tap water, and osis, nodular adenosis, and sclerosing adenosis. then rinsed with PBS. Sections were incubated with 100 /jml of the first antiserum at room temperature for 1.5 hr and then washed with several Epitheliosis. This is the name for the solid and quasisolid changes of PBS containing 0.01 % Tween 80 followed by several rinses benign epithelial proliferation which is found predominantly in in PBS. They were incubated with 1/100 alkaline phosphatase-conju small ducts, ductules and lobules. gated sheep anti-rabbit IgG at room temperature for 1.5 hr. The In the 4 cases of adenosis, the myoepithelial cells were sections were again washed with 0.01 % Tween 80 in PBS, followed by stained with the antimyosin serum, and the epithelial cells were a rinse in PBS. The sections were then washed in running tap water, identified by the strong staining with the anti-keratin serum. In rinsed in distilled water followed by incubation with 0.5 mg naphthol blunt duct adenosis, the myoepithelial cells of the terminal AS:B1 phosphoric acid (sodium salt) per ml and 0.5 mg Fast Red TR ducts were very prominent with a ragged appearance to their per ml in veronal acetate buffer, pH 9.2, at room temperature for 1 hr. outer surface. There was a spectrum of changes from blunt The sections were again washed in running water, counterstained with Mayer's hemalum, and mounted in buffered glycerin jelly, pH 7.4. The through nodular to sclerosing adenosis with a progressive loss specificity of staining was confirmed by absorption of each of the of epithelial cells. The reduction in number and distortion of antisera with 1 mg of the appropriate antigen per ml at room tempera the acini is demonstrated by the keratin staining (Fig. 5). At the ture for 1.5 hr. Incubation of histological sections with these absorbed same time, there was a proliferation of myoepithelial cells which antisera resulted in a complete abolition of the alkaline phosphatase took on a spindle cell morphology (Fig. 6) and which appeared stain in both protease-treated and untreated material. to have migrated into the intralobular stroma. In these regions, there was a diffuse staining with antibodies to laminin and type RESULTS IV collagen Â¡nthe intralobular stroma around the myoepithelial cells (Fig. 7). The myoepithelial cells in regions of sclerosing Previous experiments with different fixatives indicated that adenosis and nodular adenosis were weakly positive with the the staining of rodent tissues with all the antisera used here anti-keratin serum before trypsinization and strongly positive was best accomplished by fixation in 60% methanol:30% chlo after trypsinization of the histological sections. In the normal roform: 10% acetic acid in preference to the more conventional glands and benign breast diseases, neither the epithelial nor fixatives (44). Immunostaining was enhanced by pretreatment the myoepithelial cells showed cytoplasmic staining with anti of the histological sections with proteases (44). Similar results bodies to laminin or type IV collagen. were found with human breast tissues. Unlike the rodent sys In the 4 cases of epitheliosis examined, the majority of the tem, prior trypsinization was not required with the keratin intraluminal cells were strongly keratin positive, and a minor antiserum but prior trypsinization of the sections was required population was myosin positive. In one case, cells similar to to give adequate staining with antisera to type IV collagen and those identified morphologically and by their keratin content as laminin. With the exception of the breast, the binding of the epithelial-like, contained myosin-immunoreactive materials. keratin antiserum to different tissues fixed in 60% The basement membrane and normal external myoepithelial methanol:30% chloroform: 10% acetic acid was similar to that cell layer were still intact in areas of epitheliosis. Areas of reported for the same tissues fixed in formol 0.9% NaCI solution apocrine metaplasia showed an accentuation of the myoepithe (37). However, the degree of staining was much stronger using lial cells and weak keratin positively of the epithelial compo the former method of fixation. nent. Normal Human Breast. Antibodies to myosin clearly delin In fibroadenomas, the staining patterns with antibodies to eated the myoepithelial cells in the resting (Fig. 1) and the myosin, keratin, and the basement membrane proteins were lactating mammary gland. Pericytes, smooth muscle cells of similar to those found in the normal breast. In the stroma of the nipple, and blood vessels were stained, but all other stromal one fibroadenoma, spindle-shaped cells stained very strongly elements were consistently negative. with myosin antibodies. Morphologically similar cells in an Without prior trypsinization, keratin antibodies stained the adjacent section failed to stain with keratin antisera even after epithelial cells strongly (Fig. 2) and the myoepithelial cells trypsinization, a technique which enhanced the myoepithelial weakly. This staining pattern was altered by prior trypsinization cell staining in the adjacent ducts. of the sections whereupon the keratin antibodies stained both Intraduct and Infiltrating Carcinoma. Tumors were graded the epithelial and myoepithelial cells (Fig. 3). In the lactating in accordance with Bloom and Richardson (11 ). In the majority breast, the epithelial cells in the nonsecretory areas were of the intraduct lesions, the myoepithelial cells were prominent

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Table 1 examined there are very few identifiable myoepithelial cells. Occurrence of myofibroblasts and basement membranes in breast diseases These results are in agreement with earlier morphological The myofibroblasts were defined as anti-myosin-staining spindle cells which studies (1, 8, 23). However, morphological criteria are no failed to stain with antibodies to keratins even after trypsinization of sections. The basement membrane was delineated with antibodies to laminin. The infiltrat longer entirely satisfactory for the recognition of myoepithelial ing carcinomas were graded according to Bloom and Richardson (11), and each cells since a similar spindle cell, the myofibroblast (20, 29), tumor biopsy was from a different patient. has recently been described in certain human breast diseases with with (2, 3, 8, 36) including infiltrating carcinomas (1, 29, 38). In our myofibro basement diseaseFibroadenomasAdenosisEpitheliosisApocrineBreast blasts1000243No.membranes5443300studies, myofibroblasts are observed in one fibroadenoma and one-half of the infiltrating carcinomas. These cells stain strongly with antibodies to myosin but not with antibodies to keratins even under conditions shown to enhance the staining metaplasiaInfiltrating carcinomasGrade of myoepithelial cells. This strongly supports the view that IGrade myofibroblasts originate from the stromal rather than the epi IIGrade thelial component of the breast (29, 38). Positive stromal IIITotal5443486No. markers have not been used to identify these cells, and in these studies it is not possible to exclude the possibility that and clearly identified at the periphery of the ducts (Fig. 8). In trace amounts of keratins are present but that the concentra some areas, however, there was a discontinuity in the myoep- tions are too small to be detected Â¡mmunocytochemically or ithelial cell layer which was too large to be explained by that the epitope recognized by the antibody is masked. The attenuation of the myoepithelial cells (Fig. 9). There was also prsence of large amounts of polymerized myosin in myofibro loss of definition of the basement membrane proteins around blasts does not necessarily suggest that they represent a new some areas of intraduct carcinoma. cell type since similar activated fibroblasts occur in other situations, e.g., in Dupuytren's contracture (19) and in granu The infiltrating tumor cells were heterogeneously stained with both anti-keratin and anti-myosin sera. In the 18 infiltrating lation tissue (36). carcinomas studied, all contained keratin-positive cells, the There is disagreement concerning the distribution of keratins proportion varying from approximately 25% to greater than in the breast. This has arisen because laboratories have used 75% (Fig. 10). All of the tumors contained some myosin-posi- various sources of keratins for immunization purposes (5, 16, tive cells with some individual cells strongly stained. There was 18) and have tested the keratin antibodies on different species, no correlation between cytological appearance and antibody and tissues have been fixed and processed in different ways. staining with the anti-myosin and pre-keratin serum. In 17 out In our studies, anti-human keratin serum stains myoepithelial of the 18 tumors examined, the infiltrating myosin-positive cells cells in the rat and lining epithelial cells in human breast tissue. were randomly mixed in the tumor. In one tumor, however, The enhancement of staining for myoepithelial cells in human there was a minor population of strongly myosin-positive malig breast reported by Schlegel et al. (37) was seen in our studies nant cells which were spindle shaped and interposed at the only after prior treatment of the sections with trypsin. Recently, stromal-epithelial junction of the infiltrating islands of tumor Altmannsberger ef al. (5) have reported that fixation of breast cells (Fig. 11 ) suggestive of myoepithelial cell differentiation. tissues in alcohol gives satisfactory staining results which is The presence of myosin-positive or keratin-positive cells was contrary to our experience (44). Finally, changes in cellular not related to the degree of overt histological differentiation of staining patterns can be produced with antibodies raised in the tumor when assessed by the criteria of Bloom and Richard different species against the same immunogen (6). Reliable son (11). In one tumor which was a very well-differentiated comparisons can be made between results obtained in different adenocarcinoma, no peripheral tumor cells stained with anti- laboratories if antibodies are clearly defined in terms of their myosin serum, but the islands of well-differentiated acini were normal tissue distribution, the method of tissue fixation, and still surrounded by a clearly defined basement membrane. The the tissue-processing schedule used. relationship of basement membrane to the degree of differen In epitheliosis, both ultrastructural (3, 8, 26) and histochem- tiation is shown in Table 1. In 9 out of 18 infiltrating carcinomas, ical (8) techniques have suggested elements of both myoepi there were prominent myosin-positive benign proliferating spin thelial and epithelial differentiation with the conclusion that this condition may be due to a proliferation of "indeterminate" cells dle cells (Fig. 12). As with fibroadenomas, these cells failed to stain with antibodies against keratin (Fig. 13) even after prior (31). In our cases, myosin-positive cells formed a minor com trypsinization, suggesting that they were of stromal origin, ponent in the intraduct proliferation of epitheliosis, although in probably myofibroblasts. one case the majority of the cells in an area of solid epitheliosis were positively stained, supporting the potential role of an DISCUSSION indeterminate cell. In sclerosing adenosis, it has been sug gested that the lesion appears initially as an increase in num An immunocytochemical technique is described for the dem bers of acini and then develops through a series of phases in onstration of keratin, myosin, laminin, and type IV collagen in which the myoepithelial cells play an ever increasing role until paraffin-embedded biopsies of human breast. Using these an the epithelial elements become compressed and the myoepi tisera, it appears to be possible to distinguish epithelial, myo thelial cells take on a spindle cell morphology (8). Our results epithelial, and stromal cells and to delineate basement mem with anti-myosin staining are consistent with this interpretation. brane. The generation of cellular heterogeneity within breast lesions Myoepithelial cells constitute a large proportion of benign initially depends on the underlying developmental program of breast lesions, while in the majority of infiltrating carcinomas the normal breast. Our data would suggest that in infiltrating

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1982 American Association for Cancer Research. B. A. Gusterson et al. carcinomas phenotypic heterogeneity is marked but that dif 2. Ahmed. A. The myoepithelium in human breast fibroadenoma. J. Pathol., 114: 135-138, 1974. ferentiation is predominantly towards a cell with epithelial as 3. Ahmed, A. Atlas of the ultrastructure of human breast diseases. Edinburgh: opposed to myoepithelial characteristics. This could arise Churchill Livingstone, 1978. either by transformation of a normal epithelial cell or by trans 4. Albrechtsen, Ft., Nielsen, M., Wewer, U., Engvall, E.. and Ruoslahti. E. Basement membrane changes in breast cancer detected by immunohisto- formation of a stem cell with selection for a reduced capacity chemical staining for laminin. Cancer Res., 41: 5076-5081, 1981. to differentiate into myoepithelial cells. Analogies can perhaps 5. Altmannsberger, M., Osborn, M., Holscher, A., Schauer, A., and Weber, K. be drawn with carcinogen-induced rat mammary tumors where The distribution of keratin type intermediate filaments in human breast cancer. An immunohistological study. Virchows Arch. B Cell Pathol., 37: stem cells capable of both replicating and yielding myoepithe 277-284, 1981. lial cells have been described (10). The apparent phenotypic 6. Asch, B. B., Burstein, N. A., Vidrich, A., and Sun, T. T. Identification of mouse mammary epithelial cells by immunofluorescence with rabbit and heterogeneity of the carcinoma cells could then be generated guinea pig antikeratin antisera. Proc. Nati. Acad. Sei. U. S. A. 78: 5643- either by clonal and/or environmental variation in the control 5647, 1981. of gene expression or by retention of the last vestiges of the 7. Avrameas, S. Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochem- capability to differentiate to myoepithelial cells. These 2 expla istry, 6. 43-52, 1969. nations may not be mutually exclusive since clonal and/or 8. Azzopardi, J. G. Problems in breast pathology. Philadelphia: W. B. Saunders environmentally induced variation could still occur in the ves Co., 1979. 9. Barsky, S. H., Siegal, G. P., Jannotta, F., and Liotta, L. A. Loss of membrane tigial stem cell as it does in certain rat cell lines (34). Similar components by invasive tumors but not by their benign counterparts. Lab. considerations can also be applied when considering the his- Invest., 46: 7, 1982. 10. Bennett, D. C., Peachey, L. A., Durbin, H., and Rudland, P. S. A possible togenesis of human lung tumors (22). mammary stem cell line. Cell, 15: 283-298, 1978. Previous in vitro (25, 35) and in vivo (44) studies with 11. Bloom, H. J. G., and Richardson, W. W. Histological grading and prognosis antibodies to collagen IV and laminin have shown that the bulk in breast cancer. Br. J. Cancer, 11: 359-377, 1957. 12. Bussolati, G. Actin-rich (myoepithelial) cells in lobular carcinoma in situ of of the mammary basement membrane is laid down by the the breast. Virchows Arch. B Cell Pathol., 32: 165-176, 1980. myoepithelial cells in the young and adult rat. The thickening 13. Bussolati, G., Alfani, V., Weber, K., and Osborn, M. Immunocytochemical of the basement membrane in association with sclerosing ad- detection of actin on fixed and embedded tissues: its potential use in routine pathology. J. Histochem. Cytochem., 28. 169-173, 1980. enosis, observed here by immunolocalization techniques and 14. Bussolati, G., Botta, G., and Gugliotta, P. Actin-rich (myoepithelial) cells in by others using ultrastructural techniques (3, 8), suggests that ductal carcinoma-/n-s/fu of the breast. Virchows Arch. B Cell Pathol., 34: the myoepithelial cell is also the source of human breast 251-259, 1980. 15. Flotte, T. J.. Bell, D. A., and Greco, M. A. Tubular carcinoma and sclerosing basement membrane. The presence of basement membrane adenosis. Am. J. Surg. Pathol., 4: 75-77. 1980. proteins in Grade I carcinoma (Table 1) appears to contradict 16. Franke, W. W., Schmid, E., Freudenstein, C., Appelhans, B., Osborn, M., Weber, K., and Kennan, T. W. Intermediate-sized filaments of the prekeratin this hypothesis since fully mature myoepithelial cells were not type in myoepithelial cells. J. Cell Biol., 84: 633-654, 1980. seen in these tumors. However, epithelial cells in the mammary 17. Gabbiani, G., Csank-Brassert, J., Schneedberger, J. C., Kapanci, Y., Tren- glands of pre- and neonatal rats appear to be associated with chev, P., and Holborow, E. J. Contractile proteins in human cancer cells. Am. J. Pathol., 83. 456-467, 1976. the synthesis of the basement membrane prior to the appear 18. Gabbiani. G., Kapanci, Y., Barazzone, P., and Franke, W. W. Immunochem- ance of mature myoepithelial cells.3 Thus, either the better- ical identification of intermediate-sized filaments in human neoplastic cells. differentiated carcinomas consist of a class of developmental^ A diagnostic aid for the surgical pathologist. Am. J. Pathol., 104: 206-216, 1981. early breast epithelial cells or they retain some primitive myo- 19. Gabbiani, G., and Majno, G. Dupuytren's contracture: fibroblast contraction? epithelial-like cells with the capacity for making basement An ultrastructural study. Am. J. Pathol., 66: 131-146, 1972. 20. Goodman, Z. D., and Taxy, J. B. Fibroadenomas of the breast with prominent membrane proteins. smooth muscle. Am. J. Surg. Pathol., 5. 99-101, 1981. The use of antibodies to identify basement membrane pro 21. Gould, V. E., Jao, W., and Battifora, H. Ultrastructural analysis in the teins and myoepithelial cells has also been useful in studies of differential diagnosis of breast tumours. The significance of myoepithelial early tumor invasion. Earlier investigators using immunofluo- cells, basal lamina, intracytoplasmic lumina and secretory granules. Pathol. Res. Pract., Â»67:45-70, 1980. rescent antibodies to actin, noted defects in the myoepithelial 22. Gusterson. B., Mitchell, D., Warburton, M. J.. and Sloane, J. Immunohisto- cell layer in some in situ carcinomas (12, 14), and ultrastruc chemical localisation of keratin in human lung tumors. Virchows Arch. A Pathol. Anat. Histol. 394: 269-277, 1982. tural studies have suggested that these may be associated with 23. Hamperl, H. The myothelia (myoepithelial cells). Curr. Top. Pathol., 53. breaches in the basement membrane (21 ). More recent studies 161-220, 1970. using antibodies to laminin on frozen (39) and paraffin-embed 24. Liotta, L. A., Wicha, M. S., Foidart. J. M.. Rennard, S. l., Garbisa, S., and Kidwell, W. R. Hormonal requirements for basement membrane collagen ded (4, 9) sections have demonstrated that defects in the deposition by cultured rat mammary epithelium. Lab. Invest., 41: 511-518, basement membrane occur in association with local microin 1979. vasion. It has also been suggested that the presence of basal 25. Macartney, J. C., Roxburgh, J., and Curran, R. C. Intracellular filaments in human cancer cells. A histological study. J. Pathol., 129: 13-20. 1979. lamina when identified using the periodic acid-Schiff technique 26. Macartney, J. C., Trevithick, M. A., Kricka, L., and Curran, R. C. Identification is a useful differentiation feature in distinguishing sclerosing of myosin in human epithelial cancers with immunofluorescence. Lab. In vest., 41: 437-445, 1979. adenosis and infiltrating carcinoma (15). Thus, the loss of 27. Mackenzie, D. H. Smooth muscle in a fibro-adenoma. A fibro-adenoma of myoepithelial-like cell characteristics and of basement mem the breast with smooth muscle. J. Pathol. Bacteriol., 96: 231-232, 1968. brane proteins in the spectrum of disease states from the 28. Mukai, K., Schollmeyer, J. V., and Rosai, J. Immunohistochemical localiza tion of actin. Am. J. Surg. Pathol., 5: 91 -97, 1981. normal breast to the highly malignant carcinoma may reflect 29. Ontani. H., and Sasano. N. Myofibroblasts and myoepithelial cells in human the same underlying change in the cellular composition of the breast carcinoma. Virchows Arch. A Pathol. Anat. Histol., 385: 247-261, diseased lesions. 1980. 30. Owen, M. J., Auger, J., Barber, B. H., Edwards, A. J., Walsh, F. S.. and REFERENCES Crumpton, M. J. Actin may be present on the lymphocyte surface. Proc. Nati. Acad. Sei. U. S. A., 75: 4484-4488, 1978. 1. Ahmed, A. The myoepithelium in human breast carcinoma. J. Pathol.. 113: 31. Ozello, L. Ultrastructure of the human mammary gland. Pathol. Annu., 6: 1- 129-135. 1973. 58, 1971. 32. Puchtler, H., and Leblond, C. P. Histochemical analysis of cell membranes 3 M. J. Warburton, unpublished results. and associated structures as seen in the intestinal epithelium. Am J. Anat.,

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102: 1-31, 1958. neoplastic transformation of human breast tissue as monitored by dissolution 33. Pulley, L. T. Ultrastructural and histochemical demonstration of myoepithe- of basement membrane components. An immunoperoxidase study. Invasion lium in the normal canine mammary gland. Am. J. Vet. Res., 34: 1505- Metastasis, 1: 54-70, 1981. 1512. 1973. 40. Sieinski, W., Dorsett, B., and loachim. H. L. Identification of prekeratin by 34. Rudland, P. S., Warburton, M. J., Monaghan, P., and Ritter, M A. Thy-1 immunofluorescence staining in the differential diagnosis of tumours. Hum. antigen on normal and neoplastic rat mammary tissue. Changes in location Pathol.. 72. 452-458, 1981. and amount of antigen during differentiation of cultured stem cells. J. Nati. 41. Sun, T. T., and Green, H. Keratin filaments of cultured human epidermal Cancer Inst., 68: 799-811, 1982. cells. J. Biol. Chem., 253. 2053-2060, 1978. 35. Russo, J., and Wells, P. Ultrastructural localization of adenosine triphospha- 42. Timpl, R., Rohde. H., Robey, P. G., Rennard, S. I., Foidart, J. M., and Martin. tase activity in resting mammary gland. J. Histochem. Cytochem., 25. 135- G. R. Lamininâ€”a glycoprotein from basement membranes. J. Biol. Chem., 148. 1977. 254:9933-9937, 1979. 36. Ryan, G. B., Cliff, W. J., Gabbiani, G., Irle, C., Montandon. D., Statkovs, P. 43. Warburton, M. J.. Head, L. P., and Rudland. P. S. Redistribution of fibro- R., and Manjo, G. Myofibroblasts in human granulation tissue. Hum. Pathol., nectin and cytoskeletal proteins during the differentiation of rat mammary 5. 55-67, 1974. tumour cells in vitro. Exp. Cell Res., 132: 57-66, 1981. 37. Schlegel, R., Banks-Schlegel, S., and Pinkus, G. S. Immunohistochemical 44. Warburton, M. J., Mitchell, D., Ormerod, E. J., and Rudland, P. S. Distribution localization of keratin in normal human tissues. Lab. Invest., 42: 91-95, of myoepithelial cells and basement membrane proteins in the resting, 1980. pregnant, lactating and involuting rat mammary gland. J. Histochem. Cyto 38. Schurch, W.. Seemayer, T. A., and Lagace. R. Stromal myofibroblasts in chem., 30. 667-676. 1982. primary invasive and metastatic carcinomas. Virchows Arch. Path. Anat. 45. Warburton, M. J., Ormerod, E. J., Monaghan, P., Ferns. S.. and Rudland, P. Physiol. Klin. Med., 391: 125-139, 1981. S. Characterization of a myoepithelial cell line derived from a neonatal rat 39. Siegal, G. P., Barsky, S. H.. Terranova. V. P.. and Liotta, L. A Stages of mammary gland. J. Cell Biol., 97. 827-836, 1981.

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Fig. 1. Photomicrograph of a lactiferous sinus stained for myosin. The peripheral myoepithelial cells are clearly delineated, x 250. Fig. 2. Photomicrograph of a segmental duct stained for keratins indicating the strong positivity of the lining epithelial cells and weaker staining of the myoepithelial cells, x 530. Fig. 3. Photomicrograph of the same duct as shown in Fig. 2 but at a deeper level stained for keratins after trypsinization. Both the lining epithelial cells and the myoepithelial cells are strongly positive, x 530. Fig. 4. Photomicrograph of a portion of a lobule stained for laminin, demonstrating the basement membrane adjacent to the myoepithelial cells and the darkly stained stromal blood vessels, x 350.

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7 Figs. 5 and 6. Photomicrographs of an area of sclerosing adenosis. Sections were cut through the same area but at a slightly different level and stained for keratins (Fig. 5) and myosin (Fig. 6). There is strong cytoplasmic staining of the few residual distorted acini and weak staining of the intervening spindle cells with the anti-keratin antisera. These spindle cells are strongly stained with the anti-myosin sera (Fig. 6). X 250. Fig. 7. Photomicrograph of an area of sclerosing adenosis stained for laminin. The laminin is very diffuse and strongly positive, masking the myoepithelial cells and outlining residual distorted acinar structures, x 530. Fig. 8. Photomicrograph of an area of intraduct carcinoma stained for myosin. The myoepithelial cells are very prominent and give a "saw-toothed" appearance, x 280.

4768 CANCER RESEARCH VOL. 42

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Fig. 9. Photomicrograph of an area of intraduct carcinoma stained for myosin demonstrating loss of myoepithelial cells in some areas, x 125. Fig. 10. Photomicrograph of an infiltrating ductal carcinoma stained for keratin. The majority of the tumor cells are stained, and a few cells are very strongly positive, x 280. Fig. 11. Photomicrograph of an infiltrating ductal carcinoma stained for myosin. Strongly positive malignant spindle cells are identifiable at the stromal-tumor interface suggesting areas of myoepithelial differentiation. X 280.

NOVEMBER 1982 4769

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Figs. 12 and 13. Photomicrographs of an infiltrating ductal carcinoma stained for myosin (Fig. 12) and keratin (Fig. 13). The tumor cells are weakly positive with myosin and the stromal spindle cells are strongly positive. The same region stained for keratins fails to stain the stromal cells, but focal tumor cells are strongly positive, x 280.

4770 CANCER RESEARCH VOL. 42

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1982 American Association for Cancer Research. Distribution of Myoepithelial Cells and Basement Membrane Proteins in the Normal Breast and in Benign and Malignant Breast Diseases

Barry A. Gusterson, Michael J. Warburton, Diana Mitchell, et al.

Cancer Res 1982;42:4763-4770.

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