During the past 10 years, there has been considerable interest
in the application of new technologies to identify human malignancy
and predict disease outcome. Markers of cell proliferation and
the techniques of flow cytometry and image analysis for the determination
of DNA total content in human tumor cells have been at the forefront
of these new developments. This review will focus on the clinical
utility and potential value of cell cycle analysis and DNA ploidy
interpretation in the diagnosis of human tumors, their application
to cytologic diagnosis, and their capability for predicting disease
outcome in human neoplasia.
Human neoplasms actively synthesizing DNA replicate through a
process similar to that of normal cells known as the cell cycle.
Cells in the resting diploid state (G0) phase contain 7.14 picograms
of DNA and enter the cell cycle as gap 1 (G1) cells. During the
synthesis phase (S phase), cells increase their DNA content continuously
from 7.14 to 14.28 pg/cell until they reach the tetraploid state
with twice the diploid DNA content. The second gap (G2 phase)
refers to the tetraploid, premitotic fraction of cells that undergo
mitosis in the M phase to generate two diploid G0 cells, which
may reenter the cell cycle or persist in the resting state. A
DNA index of 1.0 corresponds to a 2N or 46 chromosome number characteristic
of G0 and G1 cells. The G2 and M cells feature a 2.0 DNA index
that corresponds to a 4N chromosome count of 92.
The distribution of a population of cells within the cell cycle
generates a pattern known as a histogram and represents DNA ploidy.
A DNA histogram is defined as diploid when the predominant or
G0/G1 peak is equal to the G0/G1 peak of a known diploid reference
cell population and the S and G2M phase contents are relatively
low (Figure 1). In normal tissues and most low-grade or slowly
proliferating neoplasms, approximately 85% of the cell population
forms the G0/G1 peak and 15% of the cells are in the S phase and
G2 and M phases.
DNA aneuploidy is defined as a DNA content of the G0/G1 peak of
a cell population that varies significantly from the mean peak
of the known diploid reference cell population. The DNA index
of an aneuploid cell population may rarely be < 1.0 (hypodiploid)
or > 1.0 (hyperdiploid). Aneuploid cell populations with a
DNA index near 2.0 must be differentiated from diploid cell populations
with significant G2M phases. Table 1 summarizes the terminology
used for DNA ploidy definitions.
Flow cytometry is a technique that features simultaneous measurement
of multiple characteristics of single cells stained with excitable
dyes moving in a fluid stream exposed to laser beam light. Computerized
analysis of light scattering and cell fluorescence produces data
analyzed by the on-board computer, resulting in the production
of a histogram. In addition to the well-described immunophenotyping
functions, when cells are stained with the dye propidium iodide,
fluorescence is proportional to the nuclear DNA content. Requiring
a cell suspension of individual cells, when solid human neoplasms
are analyzed by flow cytometry, the tissue must be disaggregated
by mechanical or enzymatic techniques.
Computer-based image analysis applies digital technology to quantitative
measurements performed on static cytopathologic and histopathologic
specimens. In contrast to flow cytometry, image analysis features
simultaneous morphologic assessment of cells measured for DNA
content by video imaging of nuclear optical density after Feulgen
staining. A comparison of the histogram generated from the computer
reconstruction of the digitized images of a population of cells
measured by image analysis with that determined from a flow of
similar cells through a flow cytometer is shown in Figure 1.
Technical Issues in DNA Ploidy Measurements
Various technical issues impact on the measurement of total DNA
content in human neoplasms. Specimen volume is important; image
analysis determination is available for as few as 100 cells, whereas
flow cytometry requires a minimum of 5,000 to 10,000 cells. Specimens
must be stored in a standard fashion and fixed in 10% neutral
buffered formalin, the optimal fixative for the DNA ploidy study.
As mentioned above, flow cytometry requires tissue disaggregation,
which is best performed by direct needle aspiration or mechanical
techniques on fresh tissue. Retrospective flow cytometric studies
utilizing enzymatic disaggregation of tissue can produce significant
errors in DNA ploidy measurement.
The tissue section image analysis method has become a preferred
technique for small needle biopsies of such organs as the prostate
and breast. It should be emphasized that heterogeneity of DNA
content is common in many types of human neoplasia, and sampling
issues can be significant when searching for aneuploid populations
in a large neoplasm. The proper use of diploid controls and standards,
the expertise of the instrument operator, and the experience of
the histogram interpreter are all critical issues in creating
high standards of performance for DNA content analysis.
Flow Cytometry vs Image Analysis
Major reviews of DNA content analysis have highlighted the relative
advantages and disadvantages of both technologies [1-5]. These
comparative studies have highlighted an excellent overall performance
of both techniques, with approximately 95% of samples showing
similar diploid or aneuploid histograms when measured by either
image analysis or flow cytometry (Figure 2) . The selective
nature of the image analysis technique has led workers to conclude
that it is slightly more sensitive than flow cytometry . A
comparison of the two methods, including their relative advantages
and disadvantages for the determination of DNA content in human
neoplasms, is included in Table 2.
The earliest methods of estimating cell proliferation in human
neoplasia were based on mitosis counting. During the past 10 years,
various newer methods have been applied to determining the cell
proliferation rate, or percentage of cells actively synthesizing
DNA (Table 3).
In addition to determining DNA ploidy, flow cytometry and image
analysis also provide estimates of the percentage of cells in
the S phase by histogram evaluation and mathematic modeling. Although
flow cytometry is more accurate than image analysis for this purpose,
both techniques have serious drawbacks with regard to the accuracy
and reproducibility of S-phase determinations.
Mitotic figure counting is the easiest method to perform.
However, lack of reproducibility and standardization are important
problems with this method. Human tumors that currently feature
mitosis counting in the standard pathology report include breast
cancer, smooth muscle sarcoma, and malignant melanoma.
Tritiated Thymidine Labeling--This method directly measures
the S phase of proliferating cells but requires viable fresh tissue
incubated with radioactive thymidine and interpreted after autoradiography.
It also suffers from interobserver variation and is generally
cumbersome and rarely used clinically.
Bromodeoxyuridine assay uses a monoclonal antibody that
measures cells in S phase that incorporate the bromodeoxyuridine
thymidine analog. Although considered an accurate proliferation
marker when measured by routine immunohistochemistry or flow cytometric
technique, this nonradioactive method is not generally utilized
in most laboratories.
Ki-67 Immunostaining--The antibody Ki-67 was raised against
a Hodgkin's disease cell line and detects an antigen in the nucleus
associated with cell proliferation . Ki-67 immunostaining has
been applied to a wide variety of human neoplasms and has been
judged to be superior to bromodeoxyuridine assays and tritiated
thymidine uptake in the assessment of cell proliferation in human
neoplasms (Figure 3) .
The original Ki-67 antibody could be utilized only in fresh and
frozen tissues. The newer MIB-1 monoclonal antibody developed
through recombinant techniques is reactive to a selective part
of the Ki-67 antigen and can be utilized in archival formalin-fixed
paraffin-embedded specimens. Comparative studies indicate that
the MIB-1 marker accurately reflects an estimate of the S-phase
fraction . Currently, the MIB-1 antibody is considered the
most easy-to-read and widely applicable cell cycle marker available.
Proliferating cell nuclear antigen (PCNA), also known as
cyclin, is a nonhistome nuclear protein cofactor for DNA polymerase-delta.
Although this marker was originally believed to be an ideal cell
proliferation label that could be applied to archival specimens,
more recent studies suggest that it is less sensitive than Ki-67
 and subject to significantly variable results when specimens
are exposed to microwave antigen retrieval procedures .
In addition to this marker, immunoreagents for the detection of
cyclin A and cyclin D have recently become available. These may
prove to be of substantial interest as potential indicators of
DNA Polymerase-Alpha is a cell cycle-related enzyme detected
by monoclonal antibodies that requires fresh-frozen tissue sections
and may be relatively insensitive.
p105 detects a nuclear antigenic epitope involved with
RNA synthesis. In early studies, p105 has shown promise as a potential
marker of aggressive malignant lymphomas and solid tumors.
Nucleolar organizer regions (NORs) are loops of DNA that
encode ribosomal RNA production [13,14]. Nucleolar organizer region
staining is accomplished by a silver impregnation technique that
can be performed on paraffin sections; this technique has correlated
with cell proliferation in a wide variety of human neoplasms.
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