Phosphohistone H3: implications for clinical practice and risk assessment in meningioma

Current World Health Organization (WHO) guidelines for meningioma grading are predominantly based on mitotic counts per 10 high-power microscope fields (HPF) (0.16 mm2) assessed on hematoxylin and eosin (H&E) stained slides.1 Based on specific morphological criteria, the routine H&E stain provides the initial platform for mitotic figure detection.2 Phosphorylation of histone H3 (pHH3) is a mitosis-specific event.3 Antibodies against pHH3 have been developed4 and used for mitotic figure detection and prognostic determination in various solid tumors including meningioma.5–11 Two imminent questions arise. (i) Does pHH3 immunohistochemistry add benefit to mitotic figure detection and count compared with H&E alone? (ii) Should this method replace the conventional H&E method for meningioma grading? Histone H3 is a protein with a role in preserving chromatin structure. Phosphorylation of histone H3 is mitosis specific, correlating with chromosome condensation.3,4,12 Histone H3 phosphorylation starts in the pericentromeric region in the premitotic, late G2 phase of the cell cycle, spreads throughout the condensed chromatin, and ends just before prophase. Histone H3 dephosphorylation starts in late anaphase and ends before chromosome decondensation in telophase.3,4 Importantly, pHH3 is not associated with apoptosis, which is a frequent mitosis mimicker.12 A highly specific antibody against the N-terminal pHH3 (Ser 10) is commercially available and suitable for use on formalin-fixed, paraffin-embedded tissues.4–6,13,14 On pHH3 immunohistochemistry, a mitosis call should only be made in the presence of positive staining associated with morphological features of chromatin condensation (Fig. 1). If observed, the finely stippled and/or strongly positive homogeneously stained nuclei should not be counted as mitoses because these are premitotic changes that represent spreading foci of histone H3 phosphorylation.4 Fig. 1. Meningioma with mitotic activity. Note the positively stained, condensed chromatin diagnostic of mitosis. All 4 mitoses depicted here are prophases (pHH3 [Ser10] immunohistochemistry [Antibody from Cell Signaling Technology, catalog#9701L, dilution 1:100], ... Several studies have investigated the role of pHH3 immunohistochemistry for mitotic figure detection in various solid tumors.6–11,13 The overall conclusions were that the anti-pHH3 (Ser10) antibody had increased sensitivity and resulted in faster mitosis detection time, decreased interobserver variability, and increased interobserver agreement compared with mitotic assessment on H&E. With H&E alone, several factors might preclude accurate mitotic figure identification: the prophase stage of mitosis is sometimes hard to detect,2,11 the presence of tissue folds, pyknotic nuclei, staining artifacts, crush artifacts, apoptotic nuclei, karyorrhexis, and lastly the pathologist's experience.11,13,14 In this issue of Neuro-Oncology, Duregon et al investigated 70 meningiomas for H&E and pHH3-positive mitoses counted per 10 HPF (0.16 mm2). They evaluated the intra- and interobserver reproducibility among 4 pathologists with different levels of experience in neuropathology and defined pHH3 mitotic cutoffs corresponding to current WHO-defined H&E mitotic cutoffs.14 They are the first to perform a thorough, long-awaited analysis of agreement for H&E and pHH-defined mitotic counts among different pathologists evaluating meningioma. Duregon et al found that HE however this has to be done with caution and on a case-by-case basis, having in mind that the mathematical model might change within different datasets. The interobserver agreement for pHH3 mitotic counts was high overall and increased when compared with the level of interobserver agreement for H&E counts. These findings were maintained following stratification by WHO grade.14 Regarding experience in pathology, there were significant differences (for both H&E and pHH3 counts), between the most experienced pathologist (dedicated to neuro-oncology diagnoses) and one of the two more junior pathologists. This shows that experience still plays an important role in assessing mitotic figures and therefore in grading meningiomas. After proving that the pHH3 mitotic count is an accurate tool for stratifying meningiomas into 3 groups, the authors identified the best pHH3 cutoffs corresponding to the current WHO H&E cutoffs. Based on maximization of the Youden index derived from 3-way receiver operating characteristic analysis, the pHH3 cutoffs chosen were 7 and 22 (corresponding to the 4 and 20 current WHO H&E mitotic cutoffs). The pHH3 cutoffs generated enhanced internal consistency and interobserver concordance compared with the WHO H&E mitotic cutoffs.14 A different study proposed a pHH3 mitotic cutoff of 6, corresponding to the WHO H&E mitotic cutoff of 4.16 In conclusion, despite possible technical issues, there are several advantages to using anti-pHH3 antibodies for mitotic figure detection in meningioma (eg, labeling of all mitotic stages; providing a more accurate call by combining staining positivity with morphological validation- chromatin condensation;3,12 decreased detection time and rapid count;7 improved intra- and interobserver reproducibility10,14), but expertise in pathology should still be accounted for. When used as a count-per-unit area, the cutoffs should be adjusted (ie, increased) to correct for antibody sensitivity.13,14,16 Two questions remain. First, what are the best pHH3 mitotic cutoffs for recurrence risk stratification in meningioma? Only 2 studies have proposed pHH3 cutoffs for counts per unit area.14,16 Although the lower cutoffs are somewhat similar (6 and 7), additional validation is needed to draw accurate conclusions. Moreover, only Duregon et al proposed an upper cutoff.14 Second, what would be more advantageous to report: a mitotic count per unit-area (eg, 10 HPF) or a mitotic index (number of pHH3-positive mitotic figures per 1000 tumor cells5)? Analogous to the Ki-67/MIB-1 proliferation marker, which is also reported as an index, pHH3-index reporting has the advantage of controlling for cellularity that can vary extensively within a single tumor. Different cutoffs apply (0–2, 3–4, ≥5) with the use of a mitotic index.5 In many institutions, assessment of the Ki-67/MIB-1 proliferation index in meningioma is routine, but carefully interpreted, because its values extend across WHO grades.17 Duregon et al reported lower correlations between MIB-1 index and both H&E and pHH3 mitotic counts compared with correlations between pHH3 and H&E mitotic counts.14 Interestingly, in multivariate models, MIB-1 index tends to lose its predictive power to Simpson grade and mitotic index.5 This evidence supports the use of pHH3 immunohistochemistry for mitotic figure detection and assessment of proliferation in addition to the routinely used MIB-1 index to improve prediction of meningioma recurrence. The evidence Duregon et al brings— that anti-pHH3 immunohistochemistry performs better compared with H&E regarding mitotic figure detection— is exciting because this inexpensive, highly accurate test refines risk stratification in meningioma. Additional studies will be necessary to validate these findings and characterize the full potential that pHH3 immunohistochemistry has for meningioma recurrence risk assessment in everyday clinical practice by defining the best cutoffs.