Ki-67 proliferation indices were correlated with the mitotic counts of H&E (Pearson’s = 0.4479, = .0001) and PHH3 (Pearson’s = 0.4588, = .0001). and interobserver reproducibility. PHH3-specific mitotic thresholds should be adopted to avoid overgrading of meningioma when ancillary methods are employed. Keywords: meningioma, mitotic Encequidar mesylate count, PHH3, cutoff Encequidar mesylate Mitotic count in meningiomas, as in other primary central nervous system tumors, is a strong morphological parameter prognosticating recurrence and survival.1 Accordingly, the current World Health Organization (WHO) classification of meningiomas2 distinguishes 3 grades with increasing risk of local recurrence by means of mitotic thresholds as an objective grading Encequidar mesylate criterion:1 benign (WHO grade I) with <4 mitoses/10 high power field (HPF; 0.16 mm2),1,2 atypical (WHO grade II) with 4C19 mitoses/10 HPF, and anaplastic (WHO grade III) with 20 mitoses/10 HPF. In light of such a crucial diagnostic and prognostic role for mitoses, their assessment on hematoxylin and eosin (H&E)Cstained slides can be affected both by technical factors (crushing and staining artifacts, or mimickers such as apoptosis and karyorrhexis, especially in necrotic areas) and by the experience of the pathologist performing the count (for Encequidar mesylate the purpose of evaluating neuropathology specimens, choosing hot spot areas, and identifying true mitoses). The mitosis-specific antibody phosphohistone H3 (PHH3) (also known as serine-10 [Ser10])3 highlights the cell nucleus maximally during mitotic chromosome condensation in early prophase and negligibly at any other time (including apoptosis).4,5 PHH3 immunostaining proved to be a reliable tool with both diagnostic and prognostic implications, favoring mitotic count in melanoma,6C15 breast cancer,16C19 astrocytoma,20,21 lung neuroendocrine carcinoma,22 adrenocortical carcinoma,23 gastrointestinal stromal tumors,24 leiomyosarcoma,25,26 granular cell tumor,27 esophageal squamous cell carcinoma,28 ovarian29 and endometrial30 cancer, prostate31,32 and urothelial33 carcinoma, as well as in cytology materials of pancreatic34 and gastrointestinal35 neuroendocrine tumors and urothelial carcinoma.36 Four reports previously analyzed the usefulness of PHH3 in meningiomas. PHH3 facilitated a rapid reliable grading of meningiomas by focusing on the most mitotically active areas37,38 and showed a higher sensitivity in comparison with mitotic count on H&E-stained slides.39 A novel mitotic threshold of 6 mitoses/10 HPF slides was proposed as the most appropriate prognostic value for predicting recurrence-free survival using PHH3.39 Finally, a PHH3 mitotic index counted on 1000 tumor cells rather than per unit area on H&E sections was employed to identify 3 PHH3 subgroups, which were associated with recurrence-free survival.40 Concerning alternative techniques for evaluating the proliferative potential in meningiomas, a high Ki-67 index already proved useful in predicting tumor behavior.41C44 However, the use of a Ki-67 index is not officially incorporated in the current WHO classification, although deserving an indicative role in the diagnostic workup of meningiomas. Two crucial items of information have to be defined: (i) the level of diagnostic agreement among pathologists using PHH3 and (ii) the need for specific new cutoffs to equalize gradings referring to the AF-9 H&E WHO grade as the gold standard, and not based on the prognoses of the studied case series39 or on alternative mitotic index definitions.40 Based on the aforementioned, a reproducibility study was designed on a series of 70 meningiomas, aimed at confirming the diagnostic role of PHH3 in assessing mitotic count and at identifying appropriate PHH3-specific mitotic thresholds. We here show that (i) PHH3 staining is confirmed.