Mitotic Cell Count

One of the cheapest, yet most straightforward, means of analyzing cell proliferation is determination of the mitotic figure count, and many studies have attempted to standardize the counting of mitoses in surgical pathology.⁴ Similar approaches also have been made for central nervous system (CNS) neoplasms.⁵ Typically, the mitotic count is performed in a given area of the tissue specimen and is reported as the number of mitoses per 10 high-power (magnification) fields (HPFs). Usually, an HPF represents the image obtained with the use of a 40× objective and a 10× ocular piece, which yields 400× magnification of the area. The mitotic count is often calculated in 10 such HPFs and frequently repeated on other sections/slides to give a more realistic estimate. This method provides a fairly reproducible index and has been used successfully as a prognostic parameter for many CNS and non-CNS neoplasms. For example, the mitotic cell count has been very helpful in distinguishing grade I from grade II meningiomas.

There are technical and methodologic challenges with the simple method of mitotic count, such as the effects of delay in fixation, the duration of fixation, and the counting method. Thus, without an established standard for mitotic cell count, it is not surprising to find significant variations among studies of a given neoplastic entity, especially in those with low mitotic counts. Nevertheless, mitotic count has been the most commonly used method for assessing cell proliferation. It may be possible to develop algorithms to provide a much more objective assessment of mitotic count with the advent of virtual microscopy and whole-slide imaging, and this prospect may enhance the practical value of counting mitotic figures.

Immunohistochemical Markers of Cell Proliferation

The use of antibodies directed against the well-known elements of cell proliferation has steadily been replacing mitotic cell counting techniques over the past two decades. With the discovery of robust antibodies that are suitable for use in formalin-fixed, paraffin-embedded material, immunohistochemistry is poised to provide a more practical and less subjective assessment of proliferating cells. However, many of the available markers and methods still require significant refinement.

The most common and reliable immunohistochemical markers for cell proliferation are the Ki-67 antibodies, developed against the same-named protein. The name of the antigen is derived from the city of origin (Kiel) and the number of the antibody clone in a 96-well plate. The target protein is present exclusively in the nuclei of proliferating cells at all active phases of the cell cycle and is absent in cells in the G₀ phase. Despite abundant evidence connecting this molecule to cell proliferation, the specific function of the protein is still elusive.

Formalin-fixed, paraffin-embedded tissues provide the optimal material for Ki-67 immunohistochemistry. There are a number of well-characterized monoclonal antibodies against the Ki-67 protein that have different qualitative and quantitative staining characteristics. MIB-1 antibody appears to have higher sensitivity for detecting Ki-67 antigen than do the other antibodies available. These differences become important, especially when different studies are compared or when a specific cutoff value is sought.

One of the commonly used markers of cell proliferation is proliferating cell nuclear antigen (PCNA). PCNA is a protein associated with DNA polymerase delta and is involved in control of DNA replication through the enzyme’s availability during elongation of the leading strand. The more commonly used antibodies for PCNA have been developed with the use of frozen tissue, and the antibodies still perform better in fresh or frozen material. Many of the antibodies developed for use in paraffin-embedded tissue have been polyclonal, with poor specificity, and have found limited use in clinical practice.

Other less common markers used for the detection of proliferating cells include JC1, an antibody that recognizes a nuclear antigen in proliferating cells, and others such as antibodies against cell cycle checkpoint molecules, including cdc2 p34, cdc20, and CDK1. Most of these markers have been useful in research studies, whereas their clinical utility has been limited.

Another set of markers typically restricted to mitotic cells are antibodies such as MPM-2 and PHH3, which have been helpful in determining the mitotic rate.⁶ Phosphorylation of tyrosine 3 of histone H3 is highly conserved among many species, and PHH3 antibodies have been used successfully in paraffin-embedded tissue to detect mitotic cells.⁷ Although this marker has only recently been introduced and validation data are limited, it may find better clinical use in the future. PHH3 markers are distinctly different from those such as Ki-67 in that they do not label all cells in the cell cycle and highlight only mitoses.

Markers that have been used in the past include fluorescence-labeled antibodies, special stains such as the silver stain for argyrophilic nucleolar organizer regions (AgNOR), and bromodeoxyuridine labeling.⁸ Although these markers have been useful for research applications in the past, they are of little practical value today.

Interpretation of Proliferation Markers

Whether one uses sections stained routinely with hematoxylin and eosin or those stained immunohistochemically, quantification of the proliferation rate requires a standardized evaluation method. The current methods are inherently semiquantitative because variables such as staining intensity and the number and type of cells counted are subject to marked variation. Determination of mitotic count is further confounded by the variability of the visual field in a given microscope. Typically, 400× HPFs vary from 0.18 to 0.25 mm², and this affects the total number of cells present in the HPF of a given microscope. Therefore, it is necessary to state the specific area of the HPF when reporting the mitotic count. This information is frequently omitted from manuscripts, a failure that is often attributable to lack of input from a pathologist.

Evaluation and reporting of immunohistochemical markers of proliferation are highly variable among different studies. The most common reporting method is the percentage of positively staining cells among a total of 1000 cells of interest. The percentage of positive cells is referred to as the labeling index. In general, antibodies against nuclear proteins are better suited for calculating a labeling index. This is typically done by counting tumor cells only. Yet other cell types may also be inadvertently counted, depending on the experience of the observer and the complexity of the tissue. One such source of variability is positive staining in cells of activated lymphocyte or macrophage lineage within tumors, which may be counted in the positive column.

Three significant challenges that can further complicate the current methods of evaluation should be emphasized. First, immunohistochemical stains may vary from one batch to another, and unless all stains are performed on the same day with the same controls, they are likely to show variation. This is critical when the labeling indices from different laboratories are compared. Second, it is important to determine a visual threshold of staining intensity above which the cells would be scored as positive. Unless such a practice is performed with actual optical measurements of staining intensity, the results are influenced by individual observation bias. Third, because the observer often seeks the area with the highest staining intensity and almost all tumors show marked regional variation in the distribution of positive cells, delineation of the area of interest is critical for reproducibility of the measurement. Furthermore, the results could vary depending on how many cells are counted, even if the same area were selected. Figure 99-1A demonstrates a graphic example of the influence of counting a different number of cells starting from the area of highest staining. The percentages in the figure represent the labeling indices if all cells within the respective circles are to be counted. As in this example, increasing the total number of cells counted may alter the labeling index. Figure 99-1B and C also highlight the fact that by counting only the area of highest intensity, it is possible to get a similar labeling index from two slides that actually have marked variation in overall labeling.

FIGURE 99-1 A, Graphic representation of potential variations in the Ki-67 labeling index based on the total number of cells counted. Each percentage represents the ratio of positive nuclei (black dots) to the total number of cells (black and white dots) within a given circle. Because the counting begins in an area of maximal staining, including a larger number of cells in the count is likely to alter the labeling index number considerably. B and C, Graphic example of the caveat in counting a single area of maximalm staining to obtain the Ki-67 labeling index. When counting the maximal area of staining (red circles), both examples would give a similar labeling index, although Example 1 has a much lower overall index than Example 2 does.

Although such challenges appear difficult to resolve, improved digital quantification and immunohistochemical analytic methods with increased reliability and standardization are emerging.⁹ We should expect to see much better validity and reliability for methods that take advantage of such advances.

Pilocytic and Pilomyxoid Astrocytomas

Pilocytic astrocytoma (PA) and its more aggressive variant pilomyxoid astrocytoma (PMA) are indolent, circumscribed tumors that are more common in children and young adults. PMA has been recognized as a variant with a higher rate of recurrence and dissemination, but its prognosis is still much more favorable than that of infiltrating gliomas. Nevertheless, markers of proliferation have not been useful in distinguishing between PA and PMA.

Numerous studies on proliferation markers for PA have been published, and the results are variable. Currently, no consensus exists on the typical proliferation rate of PA or its prognostic significance. A cutoff value for mitotic rate in either PA or PMA is not available.