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

Abstract

Summary We investigated distant recurrence and S-phase fraction (SPF), estimated by flow cytometry with and without selection of the epithelial cell population, in 201 stage II breast carcinomas. The tumour tissue was disintegrated mechanically by scissors and one part of the cell suspension was treated with a detergenttrypsin method for single-parameter analysis, and the other part, for immunological selection of epithelial cells, was incubated with a monoclonal antibody (CAM 5.2) Kallioniemi et al., 1987;Clark et al., 1989;Stal et al., 1989;Lewis, 1990). Aneuploidy and high SPF are generally associated with early distant recurrence and decreased survival time, while diploidy and low SPF correlate with good prognosis.Estimation of SPF in carcinomas using single-parameter flow cytometry is complicated by the content of inflammatory, stromal and normal epithelial cells in the tumour. The risk of underestimating SPF depends on the proportion of diluting host cells (Wingren et al., 1992) and is highly variable from tissue to tissue and within the same type of tissue. It is, thus, impossible to introduce correction factors because of this sample heterogeneity. The contamination of DNA diploid cancer cells by non-neoplastic cells results in an overlap in the diploid region of the histogram and increases the risk of falsely low SPF values. The dilution of aneuploid tumours with host cells decreases the ability to detect minor populations and may introduce artifacts into the calculation of SPF. Cancer tissue with a low proportion of DNA tetraploid cells compared with diploid cells may be misinterpreted as a DNA diploid tumour. Furthermore, overlap of the tetraploid stemline by diploid G2/M cells makes the assessment of SPF unreliable in some cases.Although these potential pitfalls are numerous, they may be solved using immunocytochemical technology. However, tumour-specific markers are not available for flow cytometric selection of cancer cells; epithelial cells normally express cytokeratins in a tissue-specific fashion, which may be used for identification (Moll et al., 1982). 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. The patients' median age was 57 years and the median follow-up time was 59 months. Seventy per cent of the tumours had oestrogen receptor levels greater than 0.1 fmol per Lg of DNA and 35% were 20 mm or less in diameter. Twenty per cent of the patients wer...

S‐phase fraction after gating on epithelial cells predicts recurrence in node‐negative breast cancer

“…In 2 earlier reports we have demonstrated that SPF from cytokeratingated cell populations compared with SPF estimated with single-parameter flow cytometry improves the prediction of tumor recurrence (Wingren et aL, 1993(Wingren et aL, , 1994. The results from the present study, confined to 209 breast-cancer patients with no nodal involvement, confirm earlier findings.…”

“…As reported earlier (Wingren et al, 1994), SPF values in aneuploid tumors are lower with the cytokeratin method than A difference in the proportion of cytokeratin-positive cells was found between DNA-diploid and DNA-aneuploid tumors and, in general, the proportion of aneuploid cells increased after gating in comparison with the diploid population (data not shown), which reduced the interference from the overlap of the diploid cells. However, in a sub-group of breast tumors, SPF could not be calculated due to the small number of cytokeratin-positive cells.…”

“…As we have demonstrated in 2 earlier studies, SPF from cytokeratin-containing cells improved the prediction of recurrence in a population dominated by stage-I1 breast cancer (Wingren et al, 1993(Wingren et al, , 1994. The aim of the present study was to compare the ability of SPF to predict recurrence in node-negative breast cancer measured on unselected cells by a routine method and SPF estimated on cytokeratin-gated cells, respectively.…”

One-tube triple staining method for flow cytometric analysis of DNA ploidy and phenotypic heterogeneity of human solid tumors using single laser excitation

“…This was also found by others using mechanical dissociation (42,43,45). The DNA-aneuploid SPF of both mixed Miillerian tumors decreased after gating on the vimentin-positive fractions.…”

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

“…To date, the main use of CK antibodies, especially in the field of flow cytometry, has been as a marker to allow accurate gating of the CK-positive population within a tumor, removing nonepithelial cell noise (15). This has enabled the detection of important, weakly expressed antigens present on tumors (7,8), such as the estrogen receptor (ER) (6), and the enhancement of DNA analysis and S-phase measurements (19,34).…”