AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Editorial
Ki-67 Staining as a Means to Simplify
Analysis of Tumor Cell Proliferation
444
of cell proliferation. Sahin and colleagues demonstrate
that Ki-67 staining and the mitotic count correlate in some
but not all tumor types. Those S-phase cells labeling with
3
H-thymidine or bromodeoxyuridine form a subpopulation of Ki-67-staining cells. This yields labeling indices
smaller than Ki-67-staining values. Studies of various
tumors5'6 demonstrate a strong correlation between Ki67 staining and thymidine or bromodeoxyuridine-LI. The
correlation is equal to or better than that with mitotic
figures. There is no need to use these latter technically
complex and time-consuming tests outside the research
laboratory.
Many considerflowcytometry to be a rapid and reliable
method for assessment of tumor cell DNA ploidy and
proliferation. Its advantage is the ability to analyze large
numbers of cells or nuclei in a short period of time. This
is complemented by the use of dual-color analysis and
light scatter parameters to define better the proliferating
cells of interest.7 This analysis can be accomplished without direct pathologist intervention. For a routine pathology practice, its disadvantages appear to outweigh its advantages. The need for a single suspension of cells or nuclei
prevents direct assessment of tumor cell heterogeneity.
Analysis of DNA histograms is as much an art as a science.8 The "accuracy" of this analysis is influenced by
the amount of tissue necrosis and normal stromal components, the presence or absence of DNA aneuploid tumor
cells, as well as by instrument variables. The ability of Ki67 to identify directly proliferating tumor cells overcomes
several problems associated with DNA aneuploid tumors.
These include a significant overlap of DNA aneuploid
and DNA diploid peaks, the presence of only a small DNA
aneuploid peak, and the presence of multiple DNA aneuploid peaks. Despite these differences, Sahin and colleagues and others910 report a good correlation between
Ki-67 staining andflowcytometry results in carefully designed studies. Sahin and colleagues indicate that this correlation appears to be better in tumors with a high rather
than a low percentage of S phase cells or percentage of S
phase cells plus G2/M value. As the authors explain, this
discordance is due to dilution of tumor cells by stromal
cells and the variation in the length of Gl between tumor
cells with low and high proliferation (S phase) rates.
The third point to consider is that techniques to quantify cell proliferation lack precision and accuracy inde-
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There is an increasing demand by clinicians for pathologists to provide objective data that will help them
guide therapy and predict the prognosis of individual cancer patients. Recent reports indicate that measurement of
tumor cell proliferation yields such useful data.1'2 The
pathologist must choose among several methods used to
measure this parameter. These include counting mitotic
figures, measuring the labeling index after incorporation
of 3H-thymidine or bromodeoxyuridine, cell-cycle analysis by flow cytometry, and Ki-67 immunostaining of a
nuclear protein associated with cell proliferation. With
each technique having its advantages and disadvantages,
the final decision may well be based on which method is
the simplest to set up and interpret.
In a routine pathology practice, immunohistochemical
staining with Ki-67 monoclonal antibody is arguably the
best method to measure cell proliferation. Its staining of
frozen sections, smears, and imprints provides direct and
rapid identification of proliferating cells, without timeconsuming procedures or expensive equipment. Although
this equates to a cost-effective test easily established in
pathology laboratories already familiar with immunohistochemical procedures, there are several factors to consider
before deciding.
First, the methods to quantify cell proliferation do not
necessarily measure the same thing. The Ki-67 monoclonal antibody recognizes an antigenic epitope, associated
with distinct DNA-binding nuclear protein, that is destroyed in fixed, paraffin-embedded tissue.3 Cell staining
intensity is low in G1 and increases to peak levels in cells
in the G2 and M phases of the cell cycle. There is no
staining of quiescent (GO) cells.4 This pattern of cell staining identifies the growth fraction of the tumor. This contrasts with the other techniques. Counting mitotic figures
measures only a portion of those cells in the G2 phase
and all M-phase cells. Thymidine and bromodeoxyuridine-LI labeling indices directly measure those cells in the
S phase only. Flow cytometric analysis of DNA staining
quantifies cell proliferation as a function of cells in the S
phase alone or in combination with the G2/M phase of
the cell cycle.
Second, despite these differences, studies, such as that
by Sahin and colleagues (pages 517-524) in this issue of
the American Journal of Clinical Pathology demonstrate
that Ki-67 staining correlates well with the other measures
445
HITCHCOCK
The last point to consider is whether Ki-67 staining can
provide clinically useful information. Several reports indicate that it provides information useful to the clinician.
Sahin and colleagues demonstrate a good correlation between Ki-67 staining and the histopathologic features of
malignant conditions. The ability of Ki-67 staining to
measure cell proliferation in small samples has been combined with sequential in situ sampling of rectal carcinomas
to determine the effect of treatment.17 A review of the
literature indicates that it is the selection of the cut-off
point rather than the evaluation method that influences
the prognostic significance of Ki-67 staining. This also is
true for the other methods of measuring tumor cell proliferation. The level of Ki-67 staining has an exponential
pattern in most tumors. Thus the median rather than the
mean value is a more representative place to start determining the cut-off point. Ki-67 staining has been used to
determine the growth fraction in non-Hodgkin's lymphomas,1018 melanoma,19 non-small cell carcinoma of the
lung,16 and breast carcinomas.6-913'20 The level of expression parallels tumor grade and the clinical behavior of
these tumors.
All efforts must be made to standardize the methods
and instrumentation used to measure tumor cell proliferation. This is needed to minimize both interlaboratory
and intralaboratory variation in results, and must be accomplished before the true clinical significance of this
technology is known. Of the methods described, quantification of Ki-67 staining should be the simplest to standardize.
CHARLES L. HITCHCOCK M.D.,
Ph.D.
Department of Cellular Pathology
Armed Forces Institute of Pathology
Washington, D.C.
REFERENCES
1. Merkel DE, McGuire WL. Ploidy, proliferative activity and prognosis. DNAflowcytometry of solid tumors. Cancer 1990;65:11941205.
2. Meyer JS. Cell kinetics in selection and stratification of patients for
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3. McGurrin JF, Doria Ml, Dawson PJ, et al. Assessment of tumor
cell kinetics by immunohistochemistry in carcinomas of the breast.
Cancer 1987;59:1744-1750.
4. Gerdes J, Lemke H, Baisch H, et al. Cell cycle analysis of a cell
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5. Sasaki K, Matsumura K, Tsuji T, Shinozaki F, Takahashi M. Relationship between labeling indices of Ki-67 and BrdUrd in human
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6. Kamel OW, Franklin WA, Ringus JC, Meyer JS. Thymidine labeling
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7. Zarbo RJ, Visscher DW, Crissman JD. Two-color multiparametric
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Vol. 96 No. 4
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pendent of the laboratory or methodology. Ki-67 staining
methodology is less rigorous than the others, and thus a
better candidate for standardization. Good-to-excellent
staining intensity is obtained from frozen sections, smears,
or touch imprints fixed with acetone or 1% paraformaldehyde alone or in combination with methanol." Prolonged storage of frozen tissue or fixed slides does not
affect staining.1213 Detection varies with the threshold of
sensitivity of the antibody used. Cells with relatively low
Ki-67 content could appear negative if the immunocytochemical system is titered to detect cells with high levels.
Therefore, all antibody dilutions should be determined
using a checkerboard titration technique. The peroxidaseanti-peroxidase and alkaline phosphatase-anti-alkaline
phosphatase staining techniques yield equivalent results.14
Dual staining with another antibody-chromogen technique can also be used for those difficult cases in which
stromal and tumor cells cannot be distinguished
clearly.1213
Ki-67 staining, as well as counting mitoticfiguresand
3
H-thymidine- and bromodeoxyuridine-labeled cells, suffer from a lack of standardized sampling of microscopic
fields and expression of results. In their article, Sahin and
colleagues note that Ki-67 staining is heterogenous in most
tumors; oftentimes restricted to specific areas. This requires a pathologist to undertake a systematic approach
to sampling the microscopic slide. This is time consuming
and costly and is a significant disadvantage of this technique. Because all staining of nuclei is considered positive,
interpretation of Ki-67 staining does not vary with the
level of observer experience as noted for counting mitotic
figures.15 There is no standard method for sampling the
slide. Care must be taken to insure that selection of fields
does not bias evaluation of the tumor cells. The pathologist
can randomly sample areas or select areas with the highest
labeling density. There appears to be a good correlation
of results from either approach as long as enough fields
are sampled. Wintzer and co-workers13 recommended that
at least 10 high-power fields be sampled and report that
sampling of 40 high-power fields yields statistically representative results regardless of the total number of tumor
cells counted per section. The influence of section planes
on their results was small. The number of positive cells
can be expressed relative to the number of high-power
fields, per number of tumor cells, or per square millimeter. The use of an ocular reticule overcomes the problem of variation in the area of a single high-power field
among different microscopes, and permits counting of
the number of stained cells per square millimeter. Quantification of Ki-67 staining can be adapted easily to image
analysis techniques. The results are associated with total
nuclear area staining, which is not the same as the percentage of positive nuclei.1016
446
AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Editorial
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Porschan R, Lohe B, Hengels K-J, Borchard F. Assessment of cell
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Slymen DJ, Miller TP, Lippman SM, et al. Immunobiologic factors
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Brown RW, Allred DC, Clark GM, Tandon AK, McGuire WL.
Prognostic significance and clinical-pathologic correlations of cellcycle kinetics measured by Ki-67 immunocytochemistry in axillary node-negative carcinoma of the breast. Abstract presented
to the 13th Ann San Antonio Breast Cancer Symposium, 1990.
A.J.C.P. • October 1991
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8. Frierson HF. The need for improvement inflowcytometric analysis
of ploidy and S-phase fraction. Editorial. Am J Clin Pathol
1991;95:439-441.
9. Isola JJ, Helin HJ, Hella MJ, Kallioniemi O. Evaluation of cell
proliferation in breast carcinoma. Comparison of Ki-67 immunohistochemical study, DNAflowcytometric analysis, and mitotic
count. Cancer 1990;65:1180-1184.
10. Schwartz BR, Pinkus G, Bacus S, Toder M, Weinberg DS. Cell proliferation in non-Hodgkin's lymphomas. Digital image analysis
of Ki-67 antibody staining. Am J Pathol 1989;134:327-336.
11. van Dierendonck JH, Wijsman JH, Keijzer R, van de Velde CJH,
Cornelisse CJ. Cell-cycle-related staining patterns of anti-proliferating cell nuclear antigen monoclonal antibodies. Comparison
with BrdUrd labeling and Ki-67 staining. Am J Pathol 1991,138:
1165-1172.
12. Porschan R, Kriegel A, Langan C, et al. Assessment of proliferative
activity in carcinomas of the human alimentary tract by Ki-67
immunostaining. Int J Cancer 1991;47:686-691.
13. Wintzer H-O, Zipfel I, Schulte-Monting J, Hellerich U, von Kieist
S. Ki-67 immunostaining in human breast tumors and its relationship to prognosis. Cancer 1991;67:421-429.
14. Hofmann-Wellenhof R, Smolle J, Kerl H. The influence of staining
procedures on the assessment of cell proliferation as defined by
447
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