DNA Ploidy and Cell Cycle Analysis in Cancer Diagnosis and Prognosis

That nucleic acids can be measured spectrophotometrically in intact, fixed, or viable single cells was demonstrated in a series of publications by Caspersson in the 1930s and '40s, culminating in his now classic monograph [1]. In that paper, Caspersson demonstrated an increase in nucleic acid content in proliferating vs resting cells and malignant vs benign cells. However, although his measurements of ultraviolet absorption were specific for nucleic acids, the absorption maximums for DNA and RNA were too close to be distinguished. Probably, he was measuring differences in RNA, which are more striking than differences in DNA.

In the early 1950s, Mellors et al proposed a device for automated screening of Pap smears based on identifying malignant cells by their increased nucleic acid content; this device would use fluorescent nucleic acid stains rather than ultraviolet absorption [2]. Efforts to develop such an instrument eventually failed because of technical problems with light sources, automatic focusing, overlapped cells, staining variation, and data collection and analysis. To deal with these problems, which are inherent in a slide scanning system, Kamentsky et al developed the flow cytometer, and their 1965 description of the first instrument rekindled interest in automated quantitative cytology [3].

In those early years, skilled cytotechnologists were in short supply, and there was strong government support for the development of an automated device to screen Pap smears. Staining techniques and methodology for flow cytometry were developed first to measure DNA in intact cells and, subsequently, to measure other cellular constituents. DNA measurements eventually could be made with a precision of 2% or less, far better than had been possible with slide-based systems. The flow cytometer also was much faster and better able to measure large numbers of cells, thus providing needed statistical strength. During the ensuing years, the DNA content of literally thousands of tumors was studied. Dr. Ross summarizes the results of those studies.

DNA measurements have been most effective clinically in the detection and treatment monitoring of in situ and superficial bladder carcinomas but also are of value in predicting the behavior of other malignant tumors, including carcinomas of the prostate, breast, colon, and lung. Although DNA aneuploidy is not always associated with an unfavorable course, it is rarely a favorable predictor. Unfortunately, it is not an independent variable, but rather, correlates with tumor grade, stage, and other prognostic features.

More interesting than DNA ploidy are reports that proliferation-related parameters may have independent prognostic value. This was most firmly established by thymidine labeling of breast cancer4 but now includes studies of S-phase fraction calculated from the distribution of DNA measurements, expression of proliferation-related antigens (such as Ki-67, PCNA, and cyclins), measurements of the silver-staining nucleolar-organizing region, uptake of the thymidine analog bromodeoxyuridine, and others. Ideally, one would like to measure cell death as well as cell proliferation to assess tumor growth rate and perhaps treatment effect. Techniques to do so are now beginning to become available; these techniques identify cells undergoing apoptosis (programmed cell death) [5] and simultaneously identify proliferation and apoptosis [6]. Also of potential prognostic value are cell-surface growth-factor receptors, differentiation antigens, and, still to be studied, amplification or deletion of specific genes, messenger RNA, or protein products of the genes that control differentiation, proliferation, drug resistance, and other functions.

A Powerful New Measurement Tool

Some of these prognostic features probably are best measured on cells in cytology smears or histologic sections, in which the tumor cells can be specifically identified and admixed inflammatory or stromal cells are excluded. Until now, this has been difficult to do by flow cytometry, and measurements by slide scanning instruments have been imprecise. However, the newly developed laser scanning cytometer (CompuCyte Corp, Cambridge, Massachusetts) [7], which is under evaluation in our laboratory and others, does yield measurements on smears and histologic sections comparable to those of flow cytometry. We have measured DNA in combination with cyclins, bromodeoxyuridine label, apoptosis, estrogen and progesterone(Drug information on progesterone) expression, cDNA probes, and expression of various antigens. The precision of measurements is comparable to that of flow cytometry, and the cells or tissues to be measured can be selected visually or identified and classified visually on a cell-by-cell basis after measurements are made.

With this powerful new tool, we can move beyond population-based measurements by flow cytometry to a wider range of feature measurements on cells in visually selected populations, even in histologic sections containing mixtures of cell types. The measurements may include proliferation vs apoptosis or differentiation and amplification, deletion, or expression of specific genes by fluorescence in situ hybridization, as well as total DNA content and many other cell attributes. It appears that quantitative and analytical cytometry is about to take another giant step forward.