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Table of Contents    
Year : 2017  |  Volume : 65  |  Issue : 6  |  Page : 1236-1238

Meningiomas: Objective assessment of proliferative indices by immunohistochemistry and automated counting method

Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Rae Bareilly Road, Lucknow, Uttar Pradesh, India

Date of Web Publication10-Nov-2017

Correspondence Address:
Dr. Sushila Jaiswal
Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Rae Bareilly Road, Lucknow - 226 014, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0028-3886.217977

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How to cite this article:
Jaiswal S. Meningiomas: Objective assessment of proliferative indices by immunohistochemistry and automated counting method. Neurol India 2017;65:1236-8

How to cite this URL:
Jaiswal S. Meningiomas: Objective assessment of proliferative indices by immunohistochemistry and automated counting method. Neurol India [serial online] 2017 [cited 2021 Jul 25];65:1236-8. Available from:

World Health Organization (WHO) defines meningiomas as a group of mostly benign, slow growing neoplasms that are most likely derived from the meningothelial cells of the arachnoid layer. Although, these are benign neoplasms, the risk of recurrence does exist in all grades and histological subtypes of meningiomas. The most important factors predicting the recurrence of meningiomas are the WHO tumor grade, invasion of the surrounding brain tissues and extent of surgical resection. Perry et al., described mitotic index (MI) as the sum of mitotic figures (MFs) per 10 consecutive high-power fields (HPFs) in the area of the highest mitotic activity and reported that it is the most reliable histological predictor of the likelihood of meningioma recurrence. They also described mitotic thresholds, later adopted by WHO, as an objective grading criterion: benign (WHO grade I) meningiomas exhibit <4 mitoses per 10 HPF, atypical (WHO grade II) meningiomas exhibit 4 or more but fewer than 20 mitoses per HPF, and anaplastic (WHO grade III) meningiomas show a high mitotic activity with 20 or more mitoses per 10 HPF.[1]

A selection bias may occur while resorting to HPF assessment as a predictor of meningioma recurrence owing to the subjectivity that is required to determine the areas of highest mitotic activity, as well as to the heterogeneity of mitotic activity in different areas of the tumor. These two factors limit the reliability and reproducibility of meningioma classification based on MI in hematoxylin and eosin (H and E) stained slides. Moreover, condensed chromatin of apoptotic cells; crushed, distorted or pyknotic nuclei; and, nuclear debris mimic MFs. Hence, sometimes it becomes difficult to distinguish MFs from these MF mimickers. Due to these limitation in its interpretation, various other objective methods are being sought that can better define the cellular proliferation. It should also be noted that different methods to quantify cell proliferation do not necessarily give the same result. Counting MFs on H and E stained slides measures cells only in the later part of G2 phase and all M-phase cells [Figure 1]. It does not represent the actual proliferation of the cell.
Figure 1: Schematic diagram; (a) Normal cell cycle with G0, G1, S, G2 and M phases; (b) H and E stain counts proliferating cells in the late G2 and M phase (shaded area); (c) Ki 67 stain counts proliferating cells in all the phases, except G0 and early G1 (shaded area). Staining is low in G1 and progressively increases from S to G2 and M phase; (d) PHH3 stain counts cell proliferation in late G2 and M phase (shaded area)

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Gerdes et al., first reported the Ki 67 antibody. The name of the antibody was derived from the city of origin (Kiel, Germany) and the number of the original clones in the well plate. In routine pathology practice, immunohistochemicaI staining of Ki 67 is most commonly used in identification and quantification of MFs, and its role as a cell proliferation marker in many central nervous system (CNS) neoplasms including meningiomas is well established.[2],[3]

Ki-67 is a labile, non-histone nuclear protein, expressed in G1, S, G2 and M phases of the cell cycle; it rapidly gets catabolized at the end of M phase and is not identifiable in the G0 and early G1 cells. Cell staining intensity is low in the G1 phase and increases to peak levels in the G2 and M phases of the cell cycle [Figure 1]. There is no staining of quiescent (G0) cells. Ki 67 has the advantage over H and E stain in that the immune-labelled nuclei can be highlighted and more easily counted when compared to the MFs in the H and E stain. It also overcomes the problems of MF mimickers related to H and E stain. However, it has limitations in the form of lack of methodological standardization, inter-laboratory variability in staining and counting methods as well as in having a validated cut-off level. It may not truly represent the actual proliferation of the cells since only a subset of the cells expressing Ki-67 in fact will go through mitosis.[4]

Hendzel et al., first described phosphohistone H3 (PHH3) as a mitosis-specific marker. Several studies have shown that PHH3, determined by immunohistochemistry (IHC), is a reliable method for the mitosis counting in different types of central nervous system (CNS) tumors, including meningiomas. It has a low inter-observer variability, and a high inter-observer agreement compared with the mitotic assessment on H and E staining. PHH3 detects the phosphorylated histone H3 which is not seen during apoptosis. Therefore, it does not stain apoptotic cells, which act as a MF mimicker, making it difficult to distinguish apoptosis from mitosis on the H and E stain.[5]

PHH3 specifically detects the phosphorylated core protein histone H3, which starts in the late G2 phase cells and then spreads progressively throughout the condensed chromatin during the M phase [Figure 1]. Histologically, MFs are maximally condensed chromatin in the M phase only. This chromatin condensation begins during the S phase and reaches its maximum during the M phase. Finely-speckled or intensely-stained nuclei, when stained with PHH3 in the absence of condensed chromatin, represent the late G2 phase (just before the mitotic phase), and not the actual mitotic phase (M-phase). Hence, positively stained nuclei in the absence of chromatin condensation should not be counted as mitosis in PHH3 staining. That is why PHH3 has to be meticulously interpreted by an experienced pathologist in combination with both H and E and PHH3 for a more objective and unambiguous identification of MFs. Although considered more sensitive than other proliferative markers, PHH3 sometimes fails to stain clear mitoses, which are well delineated by the H and E stain. The other drawback of PHH3 is background staining especially in areas with abundant vessels, making it difficult to count MFs.

Olar et al., described PHH3 MI, that is, the number of PHH3-positive mitoses per 1000 cells in the highest mitotically active area. It has the advantage of controlling for cellularity that can vary extensively within a single tumor.[6] Some studies have also proposed mitotic count cut offs for PHH3 per unit area, its lower values being 6 and 7 PHH3 mitoses/10 HPF and the higher values being 22 PHH3 mitoses/10 HPF, corresponding to the MF count of 4/10 HPF and 20/10 HPF in H and E respectively, for recurrence risk-stratification in meningiomas. Several studies have reported a higher sensitivity of PHH3 for MF detection than is present with Ki 67 and H and E staining. Hence, sometimes, there is change in the tumor grade from a lower to higher grade, if the same WHO criteria (H and E) is applied to PHH3 staining. That is why, the criteria of current WHO guidelines for mitotic activity are not appropriate for PHH3-stained sections. That is why the criteria present in the current WHO guidelines for assessing the mitotic activity are not appropriate for PHH3-stained sections.

In this issue of Neurology India, Chavali et al., have retrospectively evaluated 32 intracranial meningiomas diagnosed during the period of one year. They studied mitosis on H and E stained slides as well as immunohistochemistry using Ki 67 and PHH3 antibodies. The Ki 67 analysis was performed by both manual counting as well as computer-based automated counting using Immunoratio (IR) software. This is probably the first Indian study investigating these markers in meningioma. The study included both pediatric and adult patients, with their age ranging from 10 to 75 years. There were 22 WHO grade I and 10 WHO grade II meningiomas. They have also summarized the merits and demerits of various proliferation markers used in meningiomas.[7]

The authors found significant correlation between the tumor grade and the value of Ki 67 (both using the manual and the IR software method) and PHH3. Ki 67 was much easier and quicker to obtain using the IR software method compared to the manual counting; however, the time required for the assessment of each procedure was not evaluated. There was also a significant correlation between Ki-67 manual counting and Ki-67 IR values. The authors also revealed that the PHH3 staining was easy to identify and was higher in the grade II meningiomas as compared to the grade I tumors, and that this difference was found to be statistically significant. Some cells with clear mitoses failed to stain with PHH3. However, there was no change in the grade of the meningioma based on the PHH3 index. Statistically significant correlation was noted between the mitotic count in each tumor grade and PHH3 values. It was also noted by the authors that the automated method for Ki 67 labelling index had the advantages of being faster, objective, accurate, reproducible, and easy to use. In addition, there was a good intra- and inter-observer agreement.[7]

Brain invasion is also a predictor of recurrence in meningiomas. The median proliferation activity (MFs, Ki 67 and PHH3) within the subset of brain-invasive meningiomas was found to be higher as compared to that found in the WHO grade I meningiomas (without brain invasion), while the PHH3 staining did not provide any additional information regarding the brain invasive focus over the H and E stain.[8]

Proliferative markers do not have any role in some histomorphological variants of meningiomas such as clear cell, chordoid or papillary meningiomas, because these subtypes are more aggressive by nature, and, for them, tumor grading is not dependent on mitosis.

Although the sample size was small, the findings of the study by Chavali et al., are concordant with those described in literature. They emphasized the usefulness of PHH3 antibody and the IR software method for automated counting of Ki-67 labeled nuclei for MF detection in meningiomas. The authors concluded that both these techniques have distinct advantages over, as well as, correlated well with the existing techniques and hence, can be used routinely for recurrence risk- stratification in meningiomas. Further prospective studies with larger cohorts of all grades of meningiomas are necessary to validate these findings. These studies will be useful to characterize the full potential that PHH3 immunohistochemistry and IR software exhibit for meningioma recurrence-risk assessment in routine clinical practice.

  References Top

Perry A, Stafford SL, Scheithauer BW, Suman VJ, Lohse CM. Meningioma grading: An analysis of histological parameters. Am J Surg Pathol 1997;21:1455-65.  Back to cited text no. 1
Gerdes J, Schwab U, Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with human nuclear antigen associated with cell proliferation. Int J Cancer 1983;31:13-20.  Back to cited text no. 2
Hitchcock CL. Ki-67 staining as a means to simplify analysis of tumor cell proliferation. Am J Clin Path 1991;96:444-6.  Back to cited text no. 3
Scholzen T, Gerdes J. The Ki-67 protein: From the known and the unknown. J Cell Physiol 2000;182:311-22.  Back to cited text no. 4
Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, et al. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 1997;106:348-60.  Back to cited text no. 5
Olar A, Wani KM, Sulman EP, Mansouri A, Zadeh G, Wilson CD, et al. Mitotic index is an independent predictor of recurrence-free survival in meningioma. Brain Pathol 2015;25:266-75.  Back to cited text no. 6
Chavali P, Uppin MS, Uppin SG, Challa S. Meningiomas: Objective assessment of proliferative indices by immunohistochemistry and automated counting method. Neurol India 2017;65:1345-9.  Back to cited text no. 7
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Backer-Grøndahl T, Moen BH, Arnli MB, Torseth K, Torp SH. Immunohistochemical characterization of brain-invasive meningiomas. Int J Clin Exp Pathol 2014:15:7206-19.  Back to cited text no. 8


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