Identification of aneuploid cells in cytological specimens by combined in situ hybridization and immunocytochemistry.

The purpose of our study was the application of non-isotopic in situ hybridization with chromosome-specific repetitive DNA probes for the determination of cytogenetically aberrant cells in routine cytological materials, such as cervical smears and breast tumour aspirates. Hyperdiploid cells in fine needle aspirates (FNA) of breast tumours could be visualized by in situ hybridization with a chromosome 1-specific repetitive DNA probe. However, for the evaluation of a specific cell type in heterogeneous cell populations, i.e. cervical smears, a procedure combining immunocytochemistry and in situ hybridization can be required. Therefore, we developed a combination protocol using beta-galactosidase/ferri-ferrocyanide (blue-green) for immunocytochemistry and peroxidase/DAB (brown-black) for detection of the DNA probe. The described protocol enabled us to distinguish squamous epithelial cells within heterogeneous cell populations. By combining the chromosome 1 DNA probe with a specific cytokeratin marker it was possible to identify the chromosomal abnormal cells within cervical smears.

FB1, a monoclonal antibody reacting with a keratin 14 epitope, stains only a small subset of psoriatic basal keratinocytes.

A murine monoclonal antibody, FB1, reacted with the basal keratinocytes of human stratified epithelia. One-dimensional and two-dimensional immunoblotting assays, performed on keratins extracted from HaCat cells and normal human keratinocytes, showed that FB1 recognizes K14. When LL002, another K14 monoclonal antibody is added, the FB1 stained area in the 2D-immunoblot seems to cover a fraction of the LL002 spot. Immunohistochemical data obtained from studies on normal human tissues supported the K14 specificity of FB1, but when compared with two other monoclonal antibodies, LL002 and RCK107 reacting with K14, some differences appeared. These differences were mainly seen in sweat glands, hair follicles, psoriatic epidermis and salivary glands. In psoriatic epidermis, FB1 showed a heterogeneous pattern of staining of the basal cell compartment. Intense reactivity was only observed at the bottom of the rete ridges. Staining diminished and finally disappeared in the basal cells above the dermal papillae. This observation supports the view that an increased germinative cell population in psoriasis involves a partially differentiated amplifying compartment in which the number of cell divisions is increased.

Basal-cell keratins in cervical reserve cells and a comparison to their expression in cervical intraepithelial neoplasia.

Expression of keratins 5, 14 and 17 in endocervical subcolumnar reserve cells was detected by means of immunohistochemical studies using polypeptide specific monoclonal antibodies. These particular keratins that were found among others in basal cells could also be detected to a variable extent in metaplastic and dysplastic cervical lesions. In some cases of immature squamous metaplasia all three keratin subtypes were expressed throughout the full thickness of the epithelium. In contrast, in mature squamous metaplasia a compartmentalization of these keratins was observed. Mature squamous metaplastic epithelium showed a keratin distribution pattern comparable to ectocervical squamous epithelium, with the exception of keratin 17, which was only sporadically found in the basal layer of ectocervical epithelium and was always present in the basal cells of mature squamous metaplastic epithelium. During progression of cervical intraepithelial neoplasia a clear increase in the expression of keratin 17 was observed. However, also keratins 5 and 14 were expressed. Our results demonstrate that a considerable number of premalignant lesions of the uterine cervix express the same keratins as found in the progenitor reserve cells. Lesions that lack expression of keratin 17 may form a distinct group, which are regressive in nature and do not progress into cervical cancer.

Cytokeratin expression patterns in metastatic transitional cell carcinoma of the urinary tract. An immunohistochemical study comparing local tumor and autologous metastases.

The cytokeratin (CK) expression patterns of local, ie, primary or recurrent, high-grade-malignant transitional cell carcinomas (TCCs) of the human urinary tract and autologous lymphogenic and hematogenic metastases (n = 33) were compared. Special attention was paid to CK expression in the tumor invasion front and other areas where tumor-stroma interaction occurred to visualize cell populations with a metastatic phenotype. For this purpose, polypeptide-specific monoclonal antibodies to CKs 4, 7, 8, 10, 13, 14, 16, 17, 18, and 19 were used, employing the immunoperoxidase method. Results show that: 1) An increased expression of CK8 and CK18 is seen in the TCC tumor cells at the interface with peritumoral stroma in the tumor invasion front and with intratumoral stroma ('interface phenomenon'). Other than reflecting a quantitative change, this phenomenon might be explained by unmasking of CK8 and CK18 epitopes occurring in these regions. 2) Although in general the expression of CK13 in local TCC is decreased with increase of histopathologic parameters for progression, ie, grade and stage, an extensive proportion of CK13-positive tumor cells still can be found in some TCCs, even in metastases. 3) Morphologically recognizable types of aberrant differentiation in TCC, i.e., pseudosarcomatous or squamous differentiation and marked loss of differentiation, show altered expression of many of the CKs studied.

Distribution patterns of type VII collagen in normal and malignant human tissues.

The distribution of basement membrane type VII collagen was detected immunohistochemically and compared in normal human organs and their neoplastic derivatives using monoclonal antibody LH7.2. In normal tissues, type VII collagen was found to be restricted to the basement membrane surrounding or underlying combined epithelia, such as those lining breast, prostate, and bronchus, which are composed of a basal and luminal cell layer, and stratified epithelia, such as larynx, esophagus, trachea, vagina, ectocervix, and epidermis. No type VII collagen was found in the "simple' epithelia lining the major part of the gastrointestinal tract (GI) tract, such as liver, stomach, and intestine, or around blood vessels, muscle, and nerve fibers, which are surrounded, however, by a basement membrane containing type IV collagen and laminin. When tested in benign and malignant local tumors, antibody LH7.2 showed staining patterns partly similar to those observed in the corresponding normal tissues. This resulted in a well-circumscribed positive reaction around ducts in carcinomas in situ of the breast, in benign prostate tumors, in pleomorphic adenomas, and in a negative reaction in tumors of the GI tract. Furthermore type VII collagen was predominantly seen in carcinomas with a squamous differentiation, such as squamous carcinomas of the lung, head and neck, vulva, and vagina. These results indicate that the presence of type VII collagen in malignant tumors is correlated with (squamous) differentiation rather than with the origin of the tumor. With tumor progression, an increased presence of type VII collagen, as compared with normal urinary bladder, was found in infiltrating transitional cell carcinomas. Thus, although in general invasive and metastatic tumors do not express extensively type VII collagen, exceptions to this rule exist in bladder cancer, squamous carcinomas of the lung, tumors of the head and neck region, female genital tract tumors, and in some adenocarcinomas of the breast.

Anti-GBM nephritis in the mouse: role of granulocytes in the heterologous phase.

The role of polymorphonuclear granulocytes (PMNs) was studied in a model of anti-GBM nephritis in mice, in which PMN depletion was induced by total body irradiation of 7.5 Gy. Both in complement-normal B10.D2 new and in C5-deficient B10.D2 old mice, PMN depletion completely prevented the albuminuria occurring after injection of low doses of anti-GBM serum, and severely depressed the albuminuria after injection of high doses. In immunofluorescence, glomerular deposition of antibody and C3 was similar to that in control mice. The glomerular influx of PMNs in both the complement-normal and C5-deficient controls was inhibited to 10% or less of control values. Fibrin deposition or necrosis did not occur. Injection of F(ab')2 fragments of the anti-GBM antibody in non-irradiated mice caused only limited PMN influx and reduced the albuminuria to physiological levels, although the binding of 125I labeled F(ab')2 fragments to the glomeruli was as high as 82% of that of the complete antibody. We conclude that the albuminuria in this model is Fc-dependent and largely, if not completely, dependent on the influx of PMNs in the glomeruli. Among the many experimental models of anti-GBM nephritis, this is the first one in which the heterologous phase is complement-independent but PMN-dependent.

Use of monoclonal antibodies to keratin 7 in the differential diagnosis of adenocarcinomas.

Monoclonal antibodies (MAbs) to specific keratin subtypes were prepared and characterized by immunoblotting and immunohistochemical assays on human cell cultures and normal and malignant human tissues. Chain-specific MAbs to keratin 7 (RCK 105, OV-TL 12/30) and keratin 18 (RGE 53, RCK 106, CK18-2), as well as broadly cross-reacting keratin MAbs (RCK 102, OV-TL 12/5) could be shown to react with different types of human epithelial tissues and were therefore tested for their usefulness in the differential diagnosis of carcinomas. The two broad-spectrum antibodies stained virtually all of the more than 350 carcinomas tested, especially when combined, and distinguished them from most nonepithelial tumors. The keratin 18 MAbs distinguished adenocarcinomas (which are keratin 18 positive) from most squamous cell carcinomas (which are generally keratin 18 negative). The MAbs to keratin 7 could be shown to recognize specific subtypes of adenocarcinoma and could, for example, distinguish between ovarian carcinomas (keratin 7 positive) and carcinomas of the gastrointestinal tract (keratin 7 negative), or between transitional cell carcinomas (keratin 7 positive) and prostate cancer (keratin 7 negative). In general, malignancies showed the expected keratin reactivity pattern as concluded from the keratin pattern of its cell of origin or its type of differentiation. The use of an extended series of malignancies did, however, also illustrate that exceptions to this rule exist. For example, certain antibodies to keratin 18 stained tumor areas in squamous cell carcinomas of the lung. Also a certain percentage of tumors, which generally showed no keratin 7 expression, were positive with RCK 105 or OV-TL 12/30. On the other hand, a certain percentage of tumors, which were generally positive for keratin 7, did not show a staining reaction with these MAbs. Furthermore subtle differences between reactivity patterns of different MAbs recognizing the same keratin protein were observed, both in the normal and malignant human tissues, indicating that specific keratin epitopes may be masked in certain tissues and that unmasking of such epitopes can occur with malignant progression. This phenomenon may be of some use in a further subtyping of carcinomas, especially those of the gastrointestinal tract. Despite these exceptional staining patterns, the keratin MAbs described above have proved to be useful tools in the characterization of epithelial tumors in routine histopathology and cytopathology, in which they add to a more refined diagnosis of (adeno)carcinomas.

Changing patterns of keratin expression during progression of cervical intraepithelial neoplasia.

The expression of keratins in normal cervical epithelia, metaplastic epithelium, and cervical intraepithelial neoplasia (CIN) grades I, II, and III is investigated with a panel of keratin polypeptide-specific monoclonal antibodies. This approach allowed the detection of individual keratins 4, 7, 8, 10, 13, 14, 18, and 19 at the single-cell level. By using an antibody recognizing keratins 5 and 8 (RCK 102) and two antibodies specific for keratin 8 (CAM 5.2 and M 20), it was also possible to derive information on the distribution of keratin 5. Our results show that during immature squamous metaplasia there is an acquisition of keratins typical of squamous epithelium, ie, keratins 4, 5, 13, and 14. This process continues during further differentiation to mature squamous metaplasia. In premalignant lesions the expression pattern of the progenitor reserve cells and immature squamous metaplastic epithelium is partly conserved. However, in most cases an induction in the expression of the keratins 4, 13, and 14 was observed. Furthermore, CIN III shows a more extensive expression of keratins typical of simple epithelia, ie, keratins 8 and 18, as compared to CIN I and CIN II.

Distribution of cytokeratin polypeptides in human transitional cell carcinomas, with special emphasis on changing expression patterns during tumor progression.

The expression of cytokeratin (CK) polypeptides was studied in 59 transitional cell carcinomas (TCC) of the urinary tract of different grade and stage. Using a panel of 14 polypeptide-specific monoclonal CK-antibodies we identified immunohistochemically 8 different CKs separately, ie, CKs 4, 7, 8, 10, 13, 14, 18, and 19, while in immunoblotting studies CK5 expression was detected indirectly by using the antibody RCK102, recognizing CK5 + 8. In low-grade TCCs (G1-G2), the CK distribution was comparable to that in normal urothelium, however with a variable expression of CK13 in the different tumors and a uniform distribution of CK7. In higher-grade TCCs (G3), a decrease in CK13 expression was observed, particularly in the areas of muscle invasion. Furthermore, the appearance and increasing expression of CK14 (not present in normal urothelium or G1 TCCs) with higher grade and stage was striking. With tumor progression changes in epitope configurations of CK8 and CK18 were detected, as concluded from immunohistochemical assays with the panel of monoclonal antibodies for each of these two CKs. In extreme cases this resulted in differential staining patterns of the invasive and noninvasive components within one tumor. In 7 of 32 G3 TCCs, some of which showed areas with evident squamous differentiation, a decrease in the expression of CK7 and/or CK8 was seen. We conclude that tumor progression in TCCs is associated with discrete changes of CK expression, which can be detected using monoclonal antibodies.