Breast tumor diagnosis in cytologic aspirates using monoclonal antibodies to keratin 8 and 17.

OBJECTIVE: To determine the effect of double immunostaining in the differential diagnosis of the benign vs. malignant nature of breast lesions with fine needle aspiration results. STUDY DESIGN: The study group consisted of 26 samples aspirated from patients with borderline breast lesions. RESULTS: Direct immunostaining by means of monoclonal antibodies directed against individual intermediate filament proteins keratin 8 and 17 revealed that the percentage of K8+K17+ cells in material from patients with fibrocystic disease and fibroadenoma significantly exceeded that found in carcinoma specimens. The diagnoses were confirmed histologically. CONCLUSION: The quantitative method applied in the analysis of double-immunostained cytologic aspirates is recommended for the differential diagnosis of the benign vs. malignant nature of breast lesions in borderline cases.

Organotypic growth and differentiation of human mammary gland in sponge-gel matrix supported histoculture.

Blocks of breast tissue obtained during radical mastectomies from 23 patients with mammary gland carcinomas were used for cultivation in native-state, gel-supported histocultures. We show that the human mammary gland can be successfully maintained in this system so that normal epithelial breast structures proliferate and undergo differentiation for several weeks and a well-developed system of ducts and lobules is formed. Using antibodies to individual keratins 17 and 8 we have shown for the first time that ducts and alveoles developing in vitro undergo differentiation into the lining epithelium and myoepithelium in the same way as mammary gland epithelium in vivo. Growth of epithelial structures in vitro is also accompanied by the development of continuous basal membrane.

Organotypic growth and differentiation of human mammary gland in sponge-gel matrix supported histoculture.

Blocks of breast tissue obtained during radical mastectomies from 23 patients with mammary gland carcinomas were used for cultivation in native-state, gel-supported histocultures. We show that the human mammary gland can be successfully maintained in this system so that normal epithelial breast structures proliferate and undergo differentiation for several weeks and a well-developed system of ducts and lobules is formed. Using antibodies to individual keratins 17 and 8 we have shown for the first time that ducts and alveoles developing in vitro undergo differentiation into the lining epithelium and myoepithelium in the same way as mammary gland epithelium in vivo. Growth of epithelial structures in vitro is also accompanied by the development of continuous basal membrane.

Immunohistochemical localization of cytokeratin 17 in transitional cell carcinomas of the human urinary tract.

The expression of cytokeratin (CK) 17 was studied in 28 primary transitional cell carcinomas (TCCs) of the human urinary tract using CK 17-specific monoclonal antibody E3. While CK 17 was not detectable at all or only present in some areas of basal cells in normal--appearing urothelium, a certain subpopulation of cells of all G1 and G1/G2 TCCs examined (9 cases) stained positive for CK 17. These latter cells were either restricted to the basal compartment or located also in suprabasal layers exhibiting a decreasing intensity of immunoreactivity. CK 17 was seen in practically all cells in G2 and G2/G3 tumors (7 cases). In contrast, G3 TCCs and anaplastic carcinomas showed a highly variable CK 17 staining pattern ranging from completely negative to completely positive with several intermediate phenotypes. Our results indicate that CK 17 could be a useful marker for the progression of urinary tumors.

Myoepithelial and basement membrane antigens in benign and malignant human breast tumors.

Serial cryostat sections of 160 human breast lesions and of 9 lymph-node metastases were studied by indirect immunofluorescence. We used monoclonal antibodies (MAbs) to lining-epithelium-specific keratin 8 and to myoepithelium-specific keratin 17 in combination with polyclonal and monoclonal antibodies to major basement membrane components, laminin, collagen type IV, entactin/nidogen, and large heparan sulfate proteoglycan (perlecan) core protein. Continuous basement membranes adjacent to a basal layer of keratin-17-positive myoepithelial cells were typical for normal, benign and in situ carcinomatous structures. In invasive and metastatic structures, always formed by keratin-8-positive tumor cells, basement membranes were found only rarely and with conspicuous fragmentations. This lack of basement membranes correlated with loss of myoepithelium identified by staining for keratin 17. In comedo structures of invasive ductal carcinomas and in papillary carcinomas, fibrovascular complexes with numerous blood vessels and deposition of basement membrane material were often seen in the stroma. Immunomorphological analysis of 41 cases of doubtful diagnosis at intra-operative biopsy was also performed. A combination of MAbs to keratins 8 and 17, and to basement membrane components, made it possible to distinguish between morphologically similar benign and malignant proliferations and to detect single-cell invasion of the stroma. This combination of antibodies may be recommended as an auxiliary immunomorphological tool for differential diagnosis of intra-operative breast biopsies in dubious cases.

Patterns of expression of keratin 17 in human epithelia: dependency on cell position.

By immunomorphology, using keratin 17-specific monoclonal antibody, it has been shown that this keratin is expressed only in the basal cells of a group of complex epithelia: glandular epithelium with myoepithelial component, transitional and pseudostratified epithelia. Immunolocalization of keratin 17 provides evidence that the expression of this keratin strongly depends on the cell position within epithelial structures. The topographical character of the keratin expression suggests that these proteins may be implicated in the generation of spatial organization of epithelial tissues.

Monoclonal antibody mapping of keratins 8 and 17 and of vimentin in normal human mammary gland, benign tumors, dysplasias and breast cancer.

The distribution of keratins 8 and 17 and of vimentin in 28 normal human mammary tissue samples, 16 benign tumors, 26 fibrocytic diseases and 52 malignant breast tumors have been studied using monoclonal antibodies HI, E3 and NT30, respectively. Three cell populations in normal mammary epithelium have been identified: luminal epithelium containing keratin 8, myoepithelium of the lobular structures positive for vimentin, and myoepithelium of extralobular ducts positive for keratin 17. In different kinds of benign tumor and dysplastic proliferation a mosaic of cells with all normal phenotypes has been observed. The majority of cells co-expressed keratins 8 and 17 or vimentin. In the overwhelming majority of carcinomas, cells did not contain myoepithelial markers (keratin 17 and vimentin) but expressed only keratin 8 specific to normal luminal epithelium.

Cell shape and organization of cytoskeleton and surface fibronectin in non-tumorigenic and tumorigenic rat liver cultures.

Morphological changes associated with neoplastic transformation of epithelial cells were studied in a series of IAR cell lines derived from rat liver. The series included three independently obtained, non-tumorigenic lines and five derived, tumorigenic lines. The morphology of cell surfaces was observed by scanning electron microscopy; the distribution of actin, tubulin and fibronectin was determined by indirect immunofluorescence. All the non-tumorigenic lines had a typical epithelioid morphology: isolated cells of these lines spread on the substratum had a discoid shape and contained circular, marginal bundles of microfilaments and microtubules. In denser areas, the cells formed monolayered sheets with characteristic marginal bundles of microfilaments near the free edges. Decreased spreading of isolated cells on the substratum was the characteristic feature that distinguished tumorigenic lines from their non-tumorigenic parent lines. In particular a decrease in the size of the ring-like, peripheral lamella and its disintegration into several discrete lamellar zones were often observed; as a result, the cell shape was altered from discoid to polygonal or elongated. The altered distribution of microfilament bundles and microtubules was characteristic in elongated cells; the pattern of the cytoskeletal elements of these cells resembled that of polarized fibroblasts. Complete disappearance of microfilament bundles was observed in cells of only one tumorigenic line. Various degrees of disorganization of monolayered cell sheets were observed in tumorigenic cultures, accompanied by an altered distribution of microfilament bundles. The alterations in the fibronectin-containing structures were more complex: there were often fewer fibronectin "spots' and fibrils at the lower surfaces of cells of tumorigenic cultures as compared with those of non-tumorigenic ones; there were more fibrils in dense cultures of certain lines but fewer in others. It is concluded that alterations in the ability to spread on the substratum and to form cell-cell contacts are common features of morphologically transformed fibroblastic and epithelial cultures. However, the actual changes in the cytoskeletal structures that accompany these alterations are different in transformed cultures of various tissue types.

alpha-fetoprotein-containing cells in the early stages of liver carcinogenesis induced by 3'-methyl-4-dimethyl-aminoazobenzene and 2-acetylaminofluorene.

The morphology of rat liver cells containing alpha-feto-protein (AFP) during early stages of carcinogenesis induced by 3'methyl-4-dimethylaminoazobenzene and 2-acetylaminofluorene was investigated. Indirect immunofluorescence was used to detect AFP. AFP was not found in the cells of hyperplastic nodules but was present in the cells located in areas of transitional cell proliferation. A large proportion of the AFP-positive cells formed gland-like structures. The cells containing AFP were at various levels of differentiation according to morphological criteria. Poorly differentiated, small, basophilic cells were predominant amont the AFP-positive population. The most highly differentiated AFP-positive cells had the morphology of hepatocytes.

Serum dependence of expression of the transformed phenotype: experiments with subline of mouse L fibroblasts adapted to growth in serum-free medium.

Characteristics of LSF cells grown in serum-containing and serum-free medium were compared. LSF is a subline of the L-strain of mouse transformed fibroblasts adapted to continuous growth is serum-free medium. Proliferation of LSF cells in monolayer on solid substratum was almost similar in serum-containing and in serum-free media. However, several other characters were found to be altered by the addition of serum to the serum-free medium: ability of cells to form colonies in semi-solid medium increased considerably; agglutinability of cells by Concanavalin A increased; uptake of 2-deoxy-D-glucose by the cells increased considerably; ability of cells to metabolize benzo (a) pyrene was inhibited; cell morphology was altered and, in particular, the cells became less spread on the substratum and density of microvilli on the cell surface increased. All these changes induced by serum were reversed by transfer of the cells back into serum-free medium. Thus, addition of serum increased the expression of a number of cellular traits characteristic of transformed phenotype, while in serum-free medium partial phenotypic reversion of transformation was observed. A possible role of serum in the expression of the transformed phenotype is discussed. It is pointed out that cell lines adapted to growth in serum-free medium provide an experimental system convenient for analysis of the effects of different serum components on the cell phenotype.

Contact inhibition of movement in the cultures of transformed cells.

Results of cell-cell collisions were studied with the aid of time-lapse microcinematography in primary cultures of normal mouse-embryo fibroblast-like cells and in cultures of transformed mouse cells of two types: (a) primary fibroblast-like cells transformed by Moloney mouse sarcoma virus; (b) neoplastic fibroblasts of the CIM strain. Collisions of normal fibroblast-like cells and CIM cells in mixed cultures were also analyzed. Classification of the results of collisions was based on observation of the movements of the active cell edge during the first hour after the moment when this edge had contacted another cell. Three types of collision results were detected: halt of the active edge, overlapping, and underlapping. The relative number of overlappings was not higher and that of halts not lower in the cultures of transformed cells as compared with those of normal cells. Analysis of the collisions of normal fibroblasts with transformed cells gave similar results. Thus, the altered morphology of the cultures of these transformed cells cannot be explained by loss of contact inhibition of movement leading to increased ability of cells to move over the surfaces of other cells after collision.

Initiation of DNA synthesis in cell cultures by colcemid.

Mitotic inhibitors (colcemid, colchicine, and vinblastine) initiate DNA synthesis in dense stationary cultures of mouse embryo fibroblast-like cells. This initiation is not due to any changes in local cell population density. Relationships between the activation of proliferation and other changes of interphase fibroblast-like cells produced by the mitotic inhibitors (disappearance of cytoplasmic microtubules and activation of movements of cell surface) are discussed.