Tissue‐specific Markers in Flow Cytometry of Urological Cancers: Cytokeratins in Bladder Carcinoma

Abstract

Thirty-eight transitional-cell carcinomas (TCC) were analyzed by flow cytometry (FCM) using propidium iodide for DNA analysis and antibodies to cytokeratin by indirect immunofluorescence. By means of two-dimensional FCM analysis, cytokeratin-positive tumor cells could be analysed separately from cytokeratin-negative stromal and inflammatory cells. This resulted in an 18% increase in sensitivity of FCM detection of aneuploidy (10/38 samples with one-parameter DNA analysis versus 15/38 samples with two-parameter DNA and cytokeratin analysis). In addition, S-phase could be determined in the 15 aneuploid samples by means of two-parameter analysis where this was not possible using only DNA content because of the overlap of diploid and aneuploid populations. FCM analysis allowed quantification of the percentage of tumor cells expressing cytokeratin 18 which has previously been shown to correlate quantitatively with higher grade, higher stage TCC. The quantitative measurement of tumor-cell expression of cytokeratin 18 by FCM analysis appears to provide additional information of potential prognostic value, independent of tumor-cell ploidy and proliferative fractions.

Application of Multiparameter DNA Content and Immunofluorescence Analysis in the Investigation of Human Neoplasia

“…The historical precedents for multiparam eter analysis of solid tumors came from stud ies of Braylan et al [3] which coupled analy sis of lymphoid cell surface antigens with DNA content measurements, and Darzynkiewicz et al [4] [1,5], endo metrium [6], and kidney [7], By coupling this approach with DNA analysis and gating on cytokeratin-positive cells, a more precise as sessment of proliferative activity and the presence of a rare DNA aneuploid popula tion among diploid neoplastic cells can be achieved. These cytoplasmic constituents, however, are lost for the most part in nuclear suspensions.…”

“…cytokeratin) in an effort to 'gate out' cytokeratin-negative host-reactive cells from carcinoma specimens [1], In principal this approach appears highly use ful for providing more precise measure ments of DNA content in that it allows for measurements based mainly upon the neo plastic cells in the tumor. One significant limitation to this method, however, arises from the fact that cell suspensions obtained from solid tumors often contain significant numbers of broken neoplastic cells (mainly nuclei) which can be falsely interpreted by FCM instrumentation as cytokeratin-nega tive cells.…”

Three parameter flow cytometric analysis for simultaneous detection of cytokeratin, proliferation associated antigens and DNA content

“…Using ethanol fixed cell suspensions, non epithelial cells could be excluded from the analysis by gating on cytokeratin positive epithelial cells. By application of such immunostaining procedures, low numbers of epithelial cells could be recovered for accurate DNA cell cycle analysis (7).…”

Correlation of DNA Ploidy Levels with Altered Cytokeratin Patterns in Rat Bladder Tumors

“…Previous studies that have attempted to associate CK expression with bladder cancer have utilized the anti body RGE-53 (anti-CK 18) [6,10,14,15]. These find ings indicate that CK 18 expression, as detected by RGE-53, is increased in high grade, aneuploid bladder tumors.…”

“…Ramaekers et al [6] con ducted a similar study using FITC-conjugated polyclonal anti-CK and monoclonal anti-CK 18 antibodies. Other studies [10,14,15] have correlated CK 18 expression with DNA ploidy analysis. The results of these studies indicate that CK 18 positively correlates with tumor grade.…”