Flow cytometric analysis of DNA content and keratins by using CK7, CK8, CK18, CK19, and KL1 monoclonal antibodies in benign and malignant human breast tumors

Abstract

We have used a double‐labelling flow cytometry analysis of keratin (CK) and DNA in breast cancer. Five monoclonal anti‐keratin antibodies were tested: KL1 recognizing Mr 55,000–57,000 keratins, and “anti‐glandular epithelia,” LE41, RGE‐53, and LP2K specific for CK n° 7, 8, 18, and 19 of Moll's classification, respectively. Flow cytometric (DNA‐CK) analysis was performed on 10 benign and 19 malignant human breast tumors. All the benign tumors were diploid and 63% of the malignant tumors were aneuploid. This technique permits the analysis of DNA in the epithelial fraction alone. In aneuploid tumors, gating the DNA‐keratin‐positive population allowed accurate DNA analysis without interference due to debris background and non‐epithelial cells. Moreover, double‐labelling using the CK19 antibody gave a better identification of near‐diploid tumors. An enhancement of keratin expression in malignant tumors was observed with CK 19 (P < 0.001), KL1 (P < 0.01), CK 8 (P < 0.05), and CK18 (n.s.) compared to benign tumors. The comparison of keratin expression in aneuploid and diploid malignant tumors revealed reduced CK8, CK18, and CK19 in the former.

The prognostic significance of DNA flow cytometry in breast cancer: results from 881 patients treated in a single centre

“…Earlier reports from our group failed to demonstrate this relationship even in a univariate analysis (O'Reilly et al, 1990a-c), though all these studies had shown a trend for DNA aneuploidy to signify a poorer prognosis. Further, the results of the present study are in ine with the overview from the literature in that, while DNA ploidy is significntly ted to cinical outcome, the magnitude of the survival advantage of a DNA diploid tumour over an aneuploid one is small (Camplejohn and Macartney, 1992;Hedley et al, 1993 (Ferrero et al, 1990). Either method of removing normal cells from the analysis of SPF for DNA diploid tumours would be expected to improve further the prognostic power of this parameter.…”

Flow cytometric analysis of S-phase fraction in breast carcinomas using gating on cells containing cytokeratin

“…Furthermore, some breast cancers may have diminished expression of keratins. However, the monoclonal antibody CAM 5.2 seems to be a useful tool for selecting tumour cells, since the majority of breast tumours express the simple epithelial keratins 8 and 18 (Nagle et al, 1986;Ferrero et al, 1990;Wetzels et al, 1991).…”

“…The vast majority of normal and malignant mammary epithelial cells contain cytokeratins 7, 8, 18 and 19. The characteristics of cytokeratin in normal epithelia of the breast are mostly well preserved during malignant progression and, to some extent, even more pronounced in carcinomas (Osborn et al, 1983;Ferrero et al, 1990;Wetzels et al, 1991). A fluorescein isothiocyanate (FITC)-conjugated secondary antibody, together with a primary monoclonal antibody specific for cytokeratin, used with a suspension of cells with preserved antigenicity, allows flow cytometric sorting of the epithelial cell population (Zarbo et al, 1989;Visscher et al, 1990 1977 and 1990, were included in the study.…”

Cytokeratin expression in breast cancer: Phenotypic changes associated with disease progression

“…One of the main features of transition from a normal to cancerous state has been reputed to be an alteration in the cytoskeletal structure (22), and the relative level of CK within a cell during this transition may be an important marker of disease. Using quantitative multiparametric flow cytometry the degree of CK staining may allow the detection of subtle changes in CK expression between normal and abnormal cells (15). Use of pan CK antibodies, which label more than one type of CK at a time, coupled with single-color immunohistochemical detection may not fully quantify the individual changes in each CK type that may occur during tumor development and progression.…”